The Interactive Fly
Zygotically transcribed genes

Initiation of Translation

Translation Initiation
Dever, T. E. and Green, R. (2012). The Elongation, Termination, and Recycling Phases of Translation in Eukaryotes. Cold Spring Harb Perspect Biol 4(7):a013706 PubMed ID: 22751155

Model of the eukaryotic translation elongation pathway. Model of the eukaryotic translation elongation pathway. In this model the large ribosomal subunit is drawn transparent to visualize tRNAs, factors, and mRNA binding to the decoding center at the interface between the large and small subunits and tRNAs interacting with the peptidyl transferase center in the large subunit. Starting at the top, an eEF1A-GTP-aminoacyl-tRNA ternary complex binds the aminoacyl-tRNA to the 80S ribosome with the anticodon loop of the tRNA in contact with the mRNA in the A site of the small subunit. Following release of eEF1A-GDP, the aminoacyl-tRNA is accommodated into the A site, and the eEF1A-GDP is recycled to eEF1A-GTP by the exchange factor eEF1B. Peptide bond formation is accompanied by transition of the A- and P-site tRNAs into hybrid states with the acceptors ends of the tRNAs moving to the P and E sites, respectively. Binding of eEF2-GTP promotes translocation of the tRNAs into the canonical P and E sites, and is followed by release of eEF2-GDP, which unlike eEF1A does not require an exchange factor. The ribosome is now ready for the next cycle of elongation with release of the deacylated tRNA from the E site and binding of the appropriate eEF1A-GTP-aminoacyl-tRNA to the A site. Throughout, GTP is depicted as a green ball and GDP as a red ball; also, the positions of the mRNA, tRNAs, and factors are drawn for clarity and are not meant to specify their exact places on the ribosome (Dever, 2012).

  • RNA binding proteins and proteins involved in post-transcriptional regulation
  • A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing: Regulation of silencing by Ago1
  • mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes
  • Patterning of the Drosophila oocyte by a sequential translation repression program involving the d4EHP and Belle translational repressors
  • The interactions of GW182 proteins with PABP and deadenylases are required for both translational repression and degradation of miRNA targets
  • Regulators of alternative polyadenylation operate at the transition from mitosis to meiosis
  • Diverse cell-specific patterns of alternative polyadenylation in Drosophila
  • A CAF40-binding motif facilitates recruitment of the CCR4-NOT complex to mRNAs targeted by Drosophila Roquin
  • Mechanism of translation control of the alternative Drosophila melanogaster Voltage Dependent Anion-selective Channel 1 mRNAs
  • A regulatory response to ribosomal protein mutations controls translation, growth, and cell competition
  • Modulating eIF6 levels unveils the role of translation in ecdysone biosynthesis during Drosophila development
  • Differential requirement for translation initiation factor pathways during ecdysone-dependent neuronal remodeling in Drosophila
  • Establishment of 5'-3' interactions in mRNA independent of a continuous ribose-phosphate backbone
  • Canonical nucleators are dispensable for stress granule assembly in intestinal progenitors
  • Premature termination codon readthrough in Drosophila varies in a developmental and tissue-specific manner
  • mRNA decapping is an evolutionarily conserved modulator of neuroendocrine signaling that controls development and ageing
  • Loss of the RNA trimethylguanosine cap is compatible with nuclear accumulation of spliceosomal snRNAs but not pre-mRNA splicing or snRNA processing during animal development
  • Developmental regulation of canonical and small ORF translation from mRNAs
  • Variants in SCAF4 Cause a Neurodevelopmental Disorder and Are Associated with Impaired mRNA Processing
  • Nucleation by rRNA dictates the precision of nucleolus assembly
  • tRNA Fragments Populations Analysis in Mutants Affecting tRNAs Processing and tRNA Methylation
  • Mutations in Drosophila tRNA processing factors cause phenotypes similar to Pontocerebellar Hypoplasia
  • Structural and Genetic Determinants of Convergence in the Drosophila tRNA Structure-Function Map
  • Effects of codon usage on gene expression are promoter context dependent
  • Distinct responses to rare codons in select Drosophila tissues
  • Tissue-Specific Ribosome Profiling in Drosophila
  • Cell competition removes segmental aneuploid cells from Drosophila imaginal disc-derived tissues based on ribosomal protein gene dose
  • A neural m(6)A/Ythdf pathway is required for learning and memory in Drosophila
  • Ribosome heterogeneity in Drosophila melanogaster gonads through paralog-switching
  • Unconventional translation initiation factor EIF2A is required for Drosophila spermatogenesis
  • Somatic genetic rescue of a germline ribosome assembly defect
  • The DEAD-Box Protein Rok1 Coordinates Ribosomal RNA Processing in Association with Rrp5 in Drosophila
  • The mRNA decapping protein 2 (DCP2) is a major regulator of developmental events in Drosophila-insights from expression paradigms
  • Ribbon boosts ribosomal protein gene expression to coordinate organ form and function
  • Puromycin Labeling Coupled with Proximity Ligation Assays to Define Sites of mRNA Translation in Drosophila Embryos and Human Cells
  • The Drosophila ribosome protein S5 paralog RpS5b promotes germ cell and follicle cell differentiation during oogenesis
  • The Disease-Associated Proteins Drosophila Nab2 and Ataxin-2 Interact with Shared RNAs and Coregulate Neuronal Morphology
  • The Transcriptional Response to Oxidative Stress is Independent of Stress-Granule Formation
  • Modulation of RNA stability regulates gene expression in two opposite ways: through buffering of RNA levels upon global perturbations and by supporting adapted differential expression
  • Non-canonical initiation factors modulate repeat-associated non-AUG translation
  • A Novel Mistranslating tRNA Model in Drosophila melanogaster has Diverse, Sexually Dimorphic Effects
  • CMTr mediated 2'-O-ribose methylation status of cap-adjacent nucleotides across animals
  • eIF3j inhibits translation of a subset of circular RNAs in eukaryotic cells
  • Serotonergic neuron ribosomal proteins regulate the neuroendocrine control of Drosophila development
  • The Drosophila hnRNP F/H homolog Glorund recruits dFMRP to inhibit nanos translation elongation
  • Extended disordered regions of ribosome-associated NAC proteins paralogs belong only to the germline in Drosophila melanogaster
  • Population-scale long-read sequencing uncovers transposable elements associated with gene expression variation and adaptive signatures in Drosophila
  • A translation control module coordinates germline stem cell differentiation with ribosome biogenesis during Drosophila oogenesis
  • The transcription factor Xrp1 orchestrates both reduced translation and cell competition upon defective ribosome assembly or function

  • Factors involved in translation initiation and repression

    helicase that unwinds RNA - translational repression - regulates expression of transgenes - essential for male germline stem cell maintenance and
    division - suppresses the effects of ectopic Wg signaling - regulates ecdysone-triggered transcriptional cascades - regulates
    the temporal pattern of Notch signaling in Drosophila follicle cells, regulates chromosome segregation

    bicoid stability factor (common alternative name: Lrpprc1, Leucine-rich pentatricopeptide repeat domain-containing protein 1
    functions in mRNA stability and post-transcriptional control of gene expression - has multiple roles in gene expression in
    mitochondria - reader of the N6-methyladenosine (m6A) modification of RNA - required for progression through oogenesis and viability.

    Eukaryotic initiation factor 1A (eIF1A),
    essential for transfer of the initiator Met-tRNAf to 40 S ribosomal subunits in the absence of mRNA to form the 40 S preinitiation complex

    eukaryotic translation Initiation Factor 2α (2α)
    a GTP-binding protein responsible for bringing the initiator tRNA to the P-site of the pre-initiation complex

    eukaryotic translation Initiation Factor 2β (2β)
    a GTP-binding protein responsible for bringing the initiator tRNA to the P-site of the pre-initiation complex

    Eukaryotic initiation factor 3 p40 subunit (eIF-3p40)
    Component of the eukaryotic translation initiation factor 3 (eIF-3) complex, which is involved in protein synthesis and,
    together with other initiation factors, stimulates binding of mRNA and methionyl-tRNAi to the 40S ribosome

    eIF-3p66 (eIF-3p66)
    Component of the eukaryotic translation initiation factor 3 (eIF-3) complex, which is involved in protein synthesis and,
    together with other initiation factors, stimulates binding of mRNA and methionyl-tRNAi to the 40S ribosome

    Eukaryotic initiation factor 4B (eIF-4B)
    RNA recognition motif protein

    Eukaryotic initiation factor 4E
    binds to the mRNA 5' cap thus controlling a crucial step in translation initiation - required for cell growth -
    promotes dedifferentiation of neuroblasts back to a stem cell-like state thus functioning as an oncogene -
    a target of Ago2 in translational repression - functions as a splice factor for msl-2 and Sxl pre-mRNAs

    eIF4E-Homologous Protein
    Eukaryotic initiation factor 4E - a cap binding protein that inhibits hunchback, caudal and bicoid mRNA translation

    eukaryotic translation initiation factor 2 subunit alpha
    mediates the binding of tRNAiMet to the ribosome in a GTP-dependent manner - eIF2 is a heterotrimer
    consisting of an alpha, a beta, and a gamma subunit - activation of the integrated stress response
    signaling includes endoplasmic reticulum stress, and amino acid deprivation, sensed by
    specialized kinases PERK and GCN2 that converge on phosphorylation of eIF2α

    eukaryotic translation initiation factor 4A
    dead-box RNA helicase - eIF3 complex and the helicase eIF4A are required for dendritic pruning - inactivates TORC1 in response to amino acid
    starvation - resistance to eIF4A inhibition in conferred by the insulin receptor cellular IRES - controls germline stem cell self-renewal by
    directly inhibiting BAM function in the ovary - negative regulator of Dpp/BMP signalling that mediates SMAD degradation

    eukaryotic translation initiation factor 4G (eIF4G)
    eukaryotic initiation factor 4E binding - RNA 7-methylguanosine cap binding - RNA binding - a scaffolding protein
    that interacts with eIF3, as well as the other members of the eIF4F complex - binds to Poly(A)-binding protein,
    which binds the poly(A) tail, circularizing and activating the bound mRNA.

    eIF6 (eIF6)
    Binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit to form the 80S initiation complex
    in the cytoplasm. May also be involved in ribosome biogenesis

    4E-BP (preferred name: Thor)
    messenger RNA 5' cap binding protein - regulates translation during environmental stress

    protein kinase that promotes avoidance of the essential amino acid-deficient diet - blocks translation initiation
    through eIF2a phosphorylation - required for infection-induced host translational blockage

    Nuclear polyadenosine RNA-binding 2
    poly(A) RNA binding protein - functions in cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP - patterns axon projection in the developing brain

    pancreatic eIF-2α kinase (common alternative name: Perk)
    phosphorylates and inhibits the translation initiation factor 2 α, control of intestinal stem cell proliferation,
    homeostatic regeneration, unfolded protein response of the ER, endoplasmic reticulum stress

    polyA-binding protein
    RNA-binding protein involved in translational regulation and nonsense-mediated mRNA decay

    Thor (common alternative name: d4E-BP)
    messenger RNA 5' cap binding protein - regulates translation during environmental stress

    A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing: Regulation of silencing by Ago1

    In eukaryotic cells degradation of bulk mRNA in the 5' to 3' direction requires the consecutive action of the decapping complex (consisting of DCP1 and DCP2) and the 5' to 3' exonuclease XRN1 (Pacman). These enzymes are found in discrete cytoplasmic foci known as P-bodies or GW-bodies (because of the accumulation of the GW182 antigen). Proteins acting in other post-transcriptional processes have also been localized to P-bodies. These include SMG5, SMG7, and UPF1, which function in nonsense-mediated mRNA decay (NMD), and the Argonaute proteins, which are essential for RNA interference (RNAi) and the micro-RNA (miRNA) pathway. In addition, XRN1 is required for degradation of mRNAs targeted by NMD and RNAi. To investigate a possible interplay between P-bodies and these post-transcriptional, processes P-body or essential pathway components were depleted from Drosophila cells and the effects of these depletions were analyzed on the expression of reporter constructs, allowing specific monitoring of NMD, RNAi, or miRNA function. The RNA-binding protein GW182 and the DCP1:DCP2 decapping complex are required for miRNA-mediated gene silencing, uncovering a crucial role for P-body components in the miRNA pathway. This analysis also revealed that inhibition of one pathway by depletion of its key effectors does not prevent the functioning of the other pathways, suggesting a lack of interdependence in Drosophila (Rehwinkel, 2005).

    In eukaryotic cells, bulk messenger RNA (mRNA) is degraded via two alternative pathways, each of which is initiated by the removal of the poly(A) tail by deadenylases. Following this first step, mRNAs can be degraded from their 3' ends by the exosome, a multimeric complex of 3' to 5' exonucleases. Alternatively, after deadenylation, the cap structure is removed by the DCP1:DCP2 decapping complex, and the mRNA is degraded by the major cytoplasmic 5' to 3' exonuclease XRN1 (Rehwinkel, 2005).

    Proteins required for 5' to 3' mRNA degradation (e.g., DCP1, DCP2, and XRN1) colocalize in specialized cytoplasmic bodies or mRNA decay foci, also known as mRNA processing bodies (P-bodies) or GW-bodies, because of the accumulation of the RNA binding protein GW182 in these bodies. Additional components of P-bodies in yeast and/or human cells include the deadenylase Ccr4, the cap binding protein eIF4E and its binding partner eIF4E-transporter (eIF4E-T), auxiliary decay factors such as the LSm1-7 complex, Pat1p/Mtr1p, and the putative RNA helicase Dhh1/rck/p54. Among these, human GW182, eIF4E-T, and Dhh1 are required for P-body formation, while the decapping enzymes and XRN1 are dispensable. In addition, mRNA decay intermediates, microRNA (miRNA) targets, and miRNAs have been localized to P-bodies, suggesting that these bodies are sites where translationally silenced mRNAs are stored before undergoing decay (Rehwinkel, 2005 and references therein).

    Recently, proteins involved in other post-transcriptional processes have been localized to P-bodies in human cells. These include the proteins SMG5, SMG7, and UPF1 involved in the nonsense-mediated mRNA decay (NMD) pathway and the Argonaute (AGO) proteins that play essential roles in RNA silencing. Moreover, XRN1 is recruited by both the NMD and the RNA interference (RNAi) machineries to degrade targeted mRNAs, suggesting a possible link between NMD, RNAi, and P-bodies. NMD is an mRNA quality control (or surveillance) mechanism that degrades aberrant mRNAs having premature translation termination codons (PTCs), thereby preventing the synthesis of truncated and potentially harmful proteins. Core components of the NMD machinery include the proteins UPF1, UPF2, and UPF3, which form a complex whose function in NMD is conserved. The activity of UPF1 is regulated in multicellular organisms by additional proteins (i.e., SMG1, SMG5, SMG6, and SMG7) that are also required for NMD in all organisms in which orthologs have been characterized (Rehwinkel, 2005 and references therein).

    In yeast and human cells, a major decay pathway for NMD substrates involves decapping and 5' to 3' degradation by XRN1. Although degradation of nonsense transcripts in Drosophila is initiated by endonucleolytic cleavage near the PTC, the resulting 3' decay intermediate is also degraded by XRN1. A molecular link between the NMD machinery and the decay enzymes localized in P-bodies is provided by SMG7 in human cells. Indeed, when overexpressed, human SMG7 localizes in P-bodies and recruits both UPF1 and SMG5 to these bodies, suggesting that NMD factors may reside at least transiently in P-bodies. RNA silencing pathways are evolutionarily conserved mechanisms that elicit decay or translational repression of mRNAs selected on the basis of complementarity with small interfering RNAs (siRNAs) or miRNAs, respectively. siRNAs are fully complementary to their targets and elicit mRNA degradation via the RNAi pathway. Animal miRNAs are only partially complementary to their targets and do not generally elicit decay, but repress translation instead (Rehwinkel, 2005 and references therein).

    To perform their function, the siRNAs and miRNAs associate with the AGO proteins to form multimeric RNA-induced silencing complexes (RISC). Drosophila AGO1 mediates miRNA function, while AGO2 catalyzes the endonucleoytic cleavage of siRNA targets within the region complementary to the siRNA. Following this initial cleavage, the resulting 5' mRNA fragment is degraded by the exosome, while the 3' fragment is degraded by XRN1. The localization of AGO proteins in P-bodies in human cells provides a possible link between these bodies and silencing pathways (Rehwinkel, 2005 and references therein).

    The NMD, the siRNA, and the miRNA pathways are therefore interlinked by the use of common decay enzymes and/or the coexistence of components of these pathways in P-bodies, suggesting a possible interdependence between these post-transcriptional mechanisms. Evidence for a link between NMD and RNAi has been reported in Caenorhabditis elegans where UPF1, SMG5, and SMG6 are required for persistence of RNAi, though not to initiate silencing. In contrast, UPF2, UPF3, and SMG1, which are also essential for NMD, are not required to maintain silencing, suggesting that UPF1, SMG5, and SMG6 may have evolved specialized functions in RNAi (Rehwinkel, 2005 and references therein).

    This study investigates the interplay between NMD, RNAi, and the miRNA pathway using the Drosophila Schneider cell line 2 (S2 cells) expressing reporters allowing the monitoring of NMD, RNAi, or miRNA function. To this end, factors involved in NMD (UPF1, UPF2, UPF3, SMG1, SMG5, and SMG6), RNAi (AGO2), or the miRNA pathway (AGO1) were depleted and the effect on the expression of the reporters analyzed. These proteins showed a high degree of functional specificity. To determine the role of P-body components in these pathways the DCP1:DCP2 decapping complex, the decapping coactivators LSm1 and LSm3, the 5' to 3' exonuclease XRN1, GW182, and the Drosophila protein CG32016, which shares limited sequence homology with human eIF4E-T, were depleted. The results uncovered a crucial role for GW182 and the DCP1:DCP2 decapping complex in the miRNA pathway (Rehwinkel, 2005).

    Components of the NMD, RNAi, and miRNA pathways exhibit functional specificity in Drosophila To investigate a potential role of components of RNA silencing pathways or of P-body components in NMD, use was made of cell lines expressing wild-type or PTC-containing reporter constructs in which the coding regions of the bacterial chloramphenicol acetyl transferase (CAT) or the Drosophila alcohol dehydrogenase (adh) genes were placed downstream of inducible or constitutive promoters. The PTCs were inserted at codon 72 and 83 of the CAT and adh open reading frames, respectively. P-body components and proteins involved in NMD, RNAi, or the miRNA pathway were depleted by treating the cells with double-stranded RNAs (dsRNAs) specific for the different factors. A dsRNA that targets green fluorescent protein (GFP) served as a control. The steady-state levels of the wild-type and PTC-containing mRNAs were analyzed by Northern blot and normalized to those of the endogenous rp49 mRNA (encoding ribosomal protein L32) (Rehwinkel, 2005).

    Relative to the expression levels of the wild-type mRNAs, the levels of the corresponding PTC-containing transcripts are reduced because these transcripts are rapidly degraded via the NMD pathway. Depletion of UPF1 inhibits NMD, so the levels of the PTC-containing mRNAs are restored. Depletion of AGO1 or AGO2, both singly and in combination, does not interfere with the NMD pathway, although these depletions do inhibit siRNA- or miRNA-mediated gene silencing. The levels of the CAT wild-type transcript were not affected by the depletions. Similar results were obtained with the NMD reporter based on the adh gene. Together, these results indicate that inhibition of RNAi or of the miRNA pathway does not interfere with NMD. XRN1 is the only P-body component known to be required for degradation of decay intermediates arising from mRNAs undergoing NMD in Drosophila. Nevertheless, in cells depleted of XRN1 the NMD pathway is not inhibited, and only the 3' decay intermediate generated by endonucleolytic cleavage of the mRNA accumulates (Rehwinkel, 2005).

    In contrast to XRN1, none of the P-body components tested, including GW182 and the DCP1:DCP2 decapping complex, affected NMD or the accumulation of the 3' decay intermediate. The lack of a significant effect of the depletion of the DCP1:DCP2 complex was confirmed using the adh reporter. The decapping enzymes are certainly involved in NMD in yeast and human cells because the major decay pathway for NMD substrates is initiated by decapping in these organisms (for review, see Conti, 2005). Thus, it is possible that the requirement for P-body components and/or P-body integrity in NMD varies across species (Rehwinkel, 2005).

    Two different approaches were used to investigate the RNAi pathway. In one approach, a cell line constitutively expressing the wild-type Drosophila adh gene was treated with a dsRNA complementary to a central region of ~300 nucleotides (nt) of adh mRNA (adh dsRNA). This dsRNA elicits decay of the adh mRNA via the RNAi pathway. Cells were treated with dsRNAs targeting various factors in the presence or absence of adh dsRNA. The steady-state levels of the adh mRNA were analyzed by Northern blot and normalized to those of the rp49 mRNA. In cells treated with GFP dsRNA, the normalized levels of the adh transcript were reduced to 4% after addition of adh dsRNA, relative to the levels detected in the absence of adh dsRNA. In cells depleted of AGO2, a sixfold increase of adh mRNA levels was observed despite the presence of adh dsRNA. In contrast, when AGO1 was depleted, adh dsRNA could still trigger a reduction of adh mRNA levels, though a slight increase in transcript levels was observed. Similarly, depletion of UPF1 did not prevent silencing of adh expression by adh dsRNA. These results indicate that UPF1 is not required for RNAi in Drosophila. Additional NMD components (i.e., UPF2, UPF3, SMG1, SMG5, and SMG6) have been identified, but no SMG7 ortholog has been identified in Drosophila. No significant change was observed in the efficacy of RNAi under the conditions in which NMD was inhibited (Rehwinkel, 2005).

    Similarly to the results reported for the NMD pathway, depletion of XRN1 leads to the accumulation of the 3' decay intermediate generated by endonucleolytic cleavage by RISC, while depletion of the DCP1:DCP2 decapping complex does not prevent RNAi or the degradation the 3' decay intermediate. In contrast, depletion of GW182 leads to a modest increase in the adh mRNA level in the presence of adh dsRNA, suggesting that this protein could influence the efficiency of RNAi (Rehwinkel, 2005).

    In a second approach, RNAi was triggered by an siRNA instead of a long dsRNA, to uncouple RISC activity from processing of dsRNAs. To this end, S2 cells were transiently transfected with a plasmid expressing firefly luciferase (F-Luc) and an siRNA targeting the luciferase coding sequence (F-Luc siRNA) or a control siRNA. A plasmid encoding Renilla luciferase (RLuc) was included to normalize for transfection efficiencies. Cotransfection of the F-Luc reporter with the F-Luc siRNA led to a 50-fold inhibition of firefly luciferase activity relative to the activity measured when the control siRNA was cotransfected, indicating that F-Luc siRNA effectively silences firefly luciferase expression (Rehwinkel, 2005).

    The results obtained with the luciferase reporter correlate well with those obtained with adh mRNA, in spite of differences between the methods used to detect changes in reporter levels (RNA levels vs. protein levels), and the nature of the RNA trigger (long dsRNA vs. siRNA). Indeed, depletion of AGO2 impaired silencing of firefly luciferase expression by the F-Luc siRNA, leading to an eightfold increase in firefly luciferase activity relative to the activity of the Renilla control. Depletion of AGO1 led to a twofold increase of firefly luciferase activity (Rehwinkel, 2005).

    The observation that depletion of AGO2, but not AGO1, significantly inhibits RNAi is in agreement with previous reports showing that only AGO2-containing RISC is able to catalyze mRNA cleavage triggered by siRNAs. The results together with these observations indicate that Drosophila AGO1 and AGO2 are not redundant (Rehwinkel, 2005).

    Depletion of GW182 or the DCP1:DCP2 complex led to a 1.5- to twofold increase of the firefly luciferase activity, although RNAi was not abolished. These results together with those obtained with the adh reporter suggest that GW182 and the DCP1:DCP2 complex are not absolutely required for RNAi but may modulate siRNA function (Rehwinkel, 2005).

    Finally, depletion of core NMD components does not inhibit the silencing of firefly luciferase expression by F-Luc siRNA. The results are consistent with results from C. elegans showing that NMD per se is not required for the establishment of silencing (Rehwinkel, 2005).

    To investigate the miRNA pathway firefly luciferase reporters were generated in which the coding region of firefly luciferase is flanked by the 3' UTRs of the Drosophila genes CG10011 or Vha68-1. These genes were identified as miRNA targets in a genome-wide analysis of mRNAs regulated by AGO1. The 3' UTR of CG10011 mRNA contains two binding sites for miR-12, while the 3' UTR of Vha68-1 has two binding sites for miR-9b. Expression of the firefly luciferase construct fused to the 3' UTR of CG10011 (F-Luc-CG10011) was strongly reduced by cotransfection of a plasmid expressing the primary (pri) miR-12 transcript, but not pri-miR-9. Conversely, expression of the firefly luciferase reporter fused to the 3' UTR of Vha68-1 (FLuc-Vha68-1) was inhibited by cotransfection of pri-miR-9b, but not of primiR-12 (Rehwinkel, 2005).

    Silencing of luciferase expression by the cognate miRNAs was prevented in cells depleted of AGO1. Indeed, despite the presence of the transfected miRNAs, in cells depleted of AGO1 an 11-fold and a 16-fold increase of firefly luciferase expression was observed from the FLuc- CG10011 and F-Luc-Vha68-1 reporters, respectively. Notably, the firefly luciferase activity measured in AGO1-depleted cells in the presence of the transfected miRNAs was at least twofold higher than the activity measured in control cells in the absence of exogenously added miRNAs. Since endogenous miR-9b and miR-12 are expressed in S2 cells, these results suggest that depletion of AGO1 also suppresses silencing mediated by the endogenous miRNAs. Depletion of AGO2 does not suppress the effect of coexpressing the reporters with the cognate miRNAs. These results provide additional evidence supporting the conclusion that the siRNA and miRNA pathways are not interdependent (Rehwinkel, 2005).

    miRNA-mediated silencing of firefly luciferase expression was not affected by depletion of UPF1 or by the additional NMD factors (i.e., UPF2, UPF3, SMG1, SMG5, and SMG6). Thus, the individual NMD factors and NMD per se are not required for miRNA function. Unexpectedly, although the efficiency of NMD and RNAi was unaffected or only modestly affected in cells depleted of GW182 or the DCP1:DCP2 complex, miRNA-mediated silencing of firefly luciferase expression was effectively relieved in these cells. In the presence of cognate miRNAs, depletion of GW182 resulted in a sixfold increase of firefly luciferase expression. Therefore, despite the presence of transfected miRNAs, firefly luciferase activity in GW182-depleted cells was similar to that measured in controls cells in the absence of transfected miRNAs. Codepletion of DCP1 and DCP2 led to a fourfold increase of firefly luciferase expression. Finally, depletion of CG32016 resulted in a twofold increase of firefy luciferase activity, but only for the F-Luc-Vha68-1 reporter, suggesting that this effect may not be significant (Rehwinkel, 2005).

    To investigate whether depletion of GW182 affects RISC activity directly, as opposed to interfering with miRNA processing, use was made of a tethering assay. This assay involves the expression of a lN-fusion of AGO1 that binds with high affinity to five BoxB sites (5-BoxB) in the 3' UTR of a firefly luciferase reporter mRNA. When AGO1 is tethered to this reporter transcript, luciferase expression is inhibited relative to the activity measured in cells expressing the lN-peptide alone. The inhibition was partially relieved in cells depleted of GW182 but not of AGO2. It is concluded that GW182 and the decapping DCP1: DCP2 complex play a critical role in the effector step of the miRNA pathway. These results are in agreement with the observation that Argonaute proteins localize to P-bodies and interact with DCP1 and DCP2 independently of RNA or of P-body integrity (Rehwinkel, 2005).

    Thus, despite convergence in P-bodies, NMD, RNAi, and the miRNA pathway are not interdependent in Drosophila. This conclusion is based on the observation that the inhibition of one pathway by depleting key effectors may slightly interfere with, but does not significantly inhibit, the functioning of the other pathways. The lack of interdependence between RNAi and the miRNA pathway is further supported by the observation that knockouts of AGO1 or AGO2 in Drosophila have different phenotypes. Nevertheless, cross-talk between the RNAi and the miRNA pathways may still occur at the initiation step, since Dicer-1 plays a role in RISC assembly (Rehwinkel, 2005).

    Biochemical and genetic approaches in several organisms have led to the identification of essential components of the miRNA pathway. These include AGO1 and the enzymes required for miRNA processing, such as Drosha and Dicer-1 and their respective cofactors, Pasha and Loqs. However, the mechanisms by which miRNAs inhibit protein expression without affecting mRNA levels are not completely understood. Recent evidence suggests that translation initiation is inhibited and that the targeted mRNAs are stored in P-bodies, where they are maintained in a silenced state either by associating with proteins that prevent translation or possibly by removal of the cap structure. This study identified the P-body components GW182 and the DCP1:DCP2 decapping complex as proteins required for the miRNA pathway. The precise molecular mechanism by which these proteins participate in this pathway remains to be established. These proteins may have an indirect role in the miRNA pathway by affecting P-body integrity. Alternatively, these proteins may play a direct role in this pathway by escorting miRNA targets to P-bodies or facilitating mRNP remodeling steps required for the silencing of these targets. Consistent with a direct role for the DCP1:DCP2 decapping complex, and thus for the cap structure, in miRNA function is the observation that mRNAs translated via a cap-independent mechanism are not subject to miRNA-mediated silencing. In conclusion, the results uncover an important role for the P-body components, GW182 and the DCP1:DCP2 complex, in miRNA-mediated gene silencing (Rehwinkel, 2005).

    mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes

    MicroRNAs (miRNAs) silence the expression of target genes post-transcriptionally. Their function is mediated by the Argonaute proteins (AGOs), which colocalize to P-bodies with mRNA degradation enzymes. Mammalian P-bodies are also marked by the RNA-binding protein GW182, which interacts with the AGOs and is required for miRNA function. Depletion of Drosophila GW182 (Gawky), leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of the essential Drosophila miRNA effector AGO1, indicating that GW182 functions in the miRNA pathway. When GW182 is bound to a reporter transcript, it silences its expression, bypassing the requirement for AGO1. Silencing by GW182 is effected by changes in protein expression and mRNA stability. Similarly, miRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay, and both mechanisms require GW182. mRNA degradation, but not translational repression, by GW182 or miRNAs is inhibited in cells depleted of CAF1 and NOT1, components of a deadenylase complex, or the DCP1:DCP2 decapping protein complex. The N-terminal GW repeats of GW182 interact with the PIWI domain of AGO1. These findings indicate that GW182 links the miRNA pathway to mRNA degradation by interacting with AGO1 and promoting decay of at least a subset of miRNA targets (Behm-Ansmant, 2006).

    To accomplish their regulatory function miRNAs associate with the Argonaute proteins to form RNA-induced silencing complexes (RISCs), which elicit decay or translational repression of complementary mRNA targets. In plants, miRNAs are often fully complementary to their targets, and elicit mRNA decay. In contrast, animal miRNAs are only partially complementary to their targets, and silence gene expression by mechanisms that remain elusive. Recent studies have shown that miRNAs silence gene expression by inhibiting translation initiation at an early stage involving the cap structure; mRNAs translated via cap-independent mechanisms escape miRNA-mediated silencing. Other studies have suggested that translation inhibition occurs after initiation, based on the observation that miRNAs and some targets remain associated with polysomes. In addition, animal miRNAs can also induce significant degradation of mRNA targets despite imperfect mRNA-miRNA base-pairing (Behm-Ansmant, 2006 and references therein).

    The existence of a link between the miRNA pathway and mRNA decay is supported by the observation that mammalian Argonaute proteins (AGO1-AGO4), miRNAs, and miRNA targets colocalize to cytoplasmic foci known as P-bodies. These mRNA processing bodies are discrete cytoplasmic domains where proteins required for bulk mRNA degradation in the 5'-to-3' direction accumulate (e.g., the decapping DCP1:DCP2 complex and the 5'-to-3' exonuclease XRN1). Additional components of P-bodies in yeast and/or human cells include the CCR4:NOT deadenylase complex, auxiliary decapping factors (e.g., the LSm1-7 complex and Pat1p/Mtr1p), the cap-binding protein eIF4E, and the RNA helicase Dhh1/Me31B involved in translational repression. In metazoa, P-bodies are also marked by the presence of GW182, a protein with glycine-tryptophan repeats (GW repeats) required for P-body integrity (Behm-Ansmant, 2006 and references therein).

    The presence of Argonaute proteins, miRNAs, and miRNA targets in P-bodies has led to a model in which translationally silenced mRNAs are sequestered to these bodies, where they may undergo decay. At present, it is unclear whether the localization in P-bodies is the cause or consequence of the translational repression, though several lines of evidence point to a direct role for P-body components in miRNA-mediated gene silencing. (1) DCP1, GW182, and its paralog TNRC6B associate with AGO1 and AGO2 in human cells; (2) depletion of GW182 in human cells impairs both miRNA function and mRNA decay triggered by complementary short interfering RNAs (siRNAs). Similarly, miRNA function is impaired in Drosophila Schneider cells (S2 cells) depleted of GW182 or the decapping DCP1:DCP2 complex (Rehwinkel, 2005). (3) The Caenorhabditis elegans protein AIN-1, which is related to GW182, is required for gene regulation by at least a subset of miRNAs (Behm-Ansmant, 2006 and references therein).

    In Drosophila, siRNA-guided endonucleolytic cleavage of mRNAs (RNA interference [RNAi]) is mediated by AGO2, while gene silencing by miRNAs is mediated by AGO1. That siRNAs and miRNAs enter separate pathways in Drosophila is further supported by the observation that depletion of GW182 inhibits miRNA-mediated, but not siRNA-mediated gene silencing (Rehwinkel, 2005). The precise role of GW182 in the miRNA pathway is unknown. GW182 could have an indirect role by affecting P-body integrity. Alternatively, it could be more directly involved, localizing miRNA targets to P-bodies or facilitating the mRNP remodeling steps required for the silencing and/or decay of these targets (Behm-Ansmant, 2006 and references therein).

    This study investigates the role of Drosophila GW182 in the miRNA pathway. Depletion of GW182 leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of AGO1, indicating that GW182 is a genuine component of the miRNA pathway. In cells in which miRNA-mediated gene silencing is suppressed by depletion of AGO1, GW182 can still silence the expression of bound mRNAs, suggesting that GW182 acts downstream of AGO1. It is further shown that GW182 triggers silencing of bound transcripts by inhibiting protein expression and promoting mRNA decay via a deadenylation and decapping mechanism. Finally, evidence is provided that mRNA degradation by miRNAs requires GW182, the CCR4:NOT deadenylase, and the DCP1:DCP2 decapping complexes. Together with the observation that GW182 interacts with AGO1, these results indicate that binding of GW182 to miRNA targets induces silencing and can trigger mRNA degradation, providing an explanation for the observed changes in mRNA levels, at least for a subset of animal miRNA targets (Behm-Ansmant, 2006).

    These results indicate that GW182 is a genuine component of RNA silencing pathways, associating with the Argonaute proteins and with components of the mRNA decay machinery and, providing a molecular link between RNA silencing and mRNA degradation. Depletion of GW182 or AGO1 from Drosophila cells leads to correlated changes in mRNA expression profiles, indicating that these proteins act in the same pathway. Transcripts commonly up-regulated by AGO1 and GW182 are enriched in predicted and validated miRNA targets. These results, together with the observation that GW182 associates with AGO1, identify GW182 as a component of the miRNA pathway (Behm-Ansmant, 2006).

    GW182 belongs to a protein family with GW repeats, a central UBA domain, and a C-terminal RRM. Multiple sequence alignment of all proteins possessing these domains revealed that there are three paralogs (TNRC6A/GW182, TNRC6B, and TNRC6C) in vertebrates, a single ortholog in insects, and no orthologs in worms or fungi. At present, it is unclear whether the vertebrate paralogs have redundant functions, but both GW182 and TNRC6B have been shown to associate with human AGO1 and AGO2 (Behm-Ansmant, 2006).

    In Drosophila, GW182 interacts with AGO1 in vivo and in vitro. No stable interaction with AGO2 was detected under the same conditions, suggesting that AGO2 may act independently of GW182. This is consistent with the observation that depletion of GW182 does not affect siRNA-guided mRNA cleavage or RNAi, which is mediated exclusively by AGO2 in Drosophila. Nevertheless, since AGO2 also regulates the expression levels of a subset of miRNA targets (Rehwinkel, 2006), the lack of interaction with GW182 raises the question of whether this regulation occurs by a similar or different mechanism from that mediated by AGO1. Further studies are needed to elucidate the mechanism by which Drosophila AGO2 regulates the expression of a subset of miRNA targets (Behm-Ansmant, 2006).

    The N-terminal GW repeat region of GW182 encompasses two highly conserved motifs (I and II) and is expanded in vertebrates. This region is shorter in insects and bears similarity to the GW-like regions in the C. elegans protein AIN-1, involved in the miRNA pathway. However, AIN-1 does not contain UBA, Q-rich, or RRM domains. This lack of common domain architecture suggests that AIN-1 represents a functional analog. Nevertheless, the observation that C. elegans AIN-1 also localizes to P-bodies and interacts with AGO1 (i.e., worm ALG-1), and the finding that the N-terminal GW repeats of Drosophila GW182 interact with the PIWI domain of AGO1, suggest a conserved role for these repeats in mediating the interaction with Argonaute proteins. It would be of interest to determine the molecular basis of the specific interaction between the N-terminal GW repeats of GW182 and the PIWI domain of AGOs, and whether this interaction affects the catalytical activity of the domain (Behm-Ansmant, 2006).

    Apart from the interaction with AGO1, the N-terminal repeats and the UBA and Q-rich domains contribute to the localization of GW182 in P-bodies, which is in turn required for P-body integrity. This suggests that GW182 may act as a molecular scaffold bringing together AGO1-containing RISCs and mRNA decay enzymes, possibly nucleating the assembly of P-bodies. Understanding the precise role of the various GW182 domains in the interaction with mRNA decay enzymes and AGO1 as well as in P-body integrity awaits further biochemical characterization (Behm-Ansmant, 2006).

    Tethering GW182 to a reporter transcript silences its expression, bypassing the requirement for AGO1. Silencing by GW182 occurs by two distinct mechanisms: repression of protein expression, and mRNA degradation. It remains to be elucidated how GW182 represses translation. mRNA degradation by GW182 is inhibited in cells depleted of CAF1, NOT1, or the DCP1:DCP2 complex, indicating that GW182 promotes mRNA deadenylation and decapping. Thus, binding of GW182 appears to be a point of no return, which marks transcripts as targets for degradation (Behm-Ansmant, 2006).

    More studies are needed to determine whether decapping triggered by GW182 requires prior deadenylation or whether these two events occur independently. The observation that mRNA levels are fully restored in cells depleted of DCP1:DCP2, suggests that deadenylation followed by 3'-to-5' exonucleolytic degradation is unlikely to represent a major pathway by which these mRNAs are degraded. Future studies should also reveal the identity of the nuclease(s) acting downstream of the decapping enzymes (Behm-Ansmant, 2006).

    Previous studies indicate that miRNAs can reduce the levels of the targeted transcripts, and not just the expression of the translated protein. Consistently, transcripts up-regulated in cells depleted of AGO1 or GW182 are enriched in predicted and validated miRNA targets. In this paper further evidence is provided indicating that miRNAs silence gene expression by two mechanisms: one mechanism involving translational silencing, and one involving mRNA degradation. The contribution of these mechanisms to miRNA-mediated gene silencing appears to differ for each miRNA:target pair. Indeed, of the three reporters analyzed, Nerfin is silenced mainly at the translational level, silencing of the CG10011 reporter can be attributed to mRNA degradation, while Vha68-1 is regulated both at the translational and mRNA levels. Regardless of the extent of the contribution of these two mechanisms to silencing, both require AGO1 and GW182, because the levels of the mRNA reporter and luciferase activity are restored in cells depleted of any of these two proteins (Behm-Ansmant, 2006).

    In contrast, although the levels of the mRNA reporter are restored in cells depleted of CAF1 or NOT1, translational repression is not fully relieved, indicating that deadenylation is required for mRNA decay, but not for translational silencing by miRNAs. In agreement with this, two reports published while this manuscript was in preparation have shown that miRNAs trigger accelerated deadenylation of their targets (Giraldez, 2006; Wu, 2006). This study extends these observations further by demonstrating: (1) deadenylation is mediated by the CCR4:NOT complex; (2) decapping is also required for miRNA target degradation, and (3) both deadenylation and decapping triggered by miRNAs requires GW182 (Behm-Ansmant, 2006).

    Based on the results presented in this study and the observations that GW182 associates with AGO1 and is required for miRNA-mediated gene silencing, the following model is proposed: AGO1-containing RISCs binds to mRNA targets by means of base-pairing interactions with miRNAs; AGO1 may then recruit GW182, which marks the transcripts as targets for decay via a deadenylation and decapping mechanism (Behm-Ansmant, 2006).

    A question that remains open is whether miRNA-mediated translational repression is the cause of mRNA degradation or whether these represent two independent mechanism by which miRNAs silence gene expression as proposed by Wu (2006). Indeed, changes in mRNA levels are not observed for all miRNA targets (Rehwinkel, 2006), suggesting that inhibition of translation is not always followed by mRNA decay. Conversely, depletion of CAF1 or NOT1 prevents mRNA decay but does not relieve translational silencing, suggesting that these two processes are independent (Behm-Ansmant, 2006).

    An important finding is that miRNAs elicit degradation to different extents. One possible explanation is that the extent of degradation depends on the stability of the miRNA:mRNA duplexes. Also, the extent of degradation might depend on the particular set of proteins associated with a given target. For instance, some targets may assemble with a set of proteins that antagonize degradation. Finally, GW182 might interact only with a subset of AGO1-containing RISCs, as suggested for AIN-1. A major challenge will be to identify the specific features of miRNA targets and/or RISC complexes that lead to regulation of gene expression at the level of translation or at the level of mRNA stability (Behm-Ansmant, 2006).

    Patterning of the Drosophila oocyte by a sequential translation repression program involving the d4EHP and Belle translational repressors

    During Drosophila development, translational control plays a crucial role in regulating gene expression, and is particularly important during pre-patterning of the maturing oocyte. A critical step in translation initiation is the binding of the eukaryotic translation initiation factor 4E (eIF4E) to the mRNA cap structure, which ultimately leads to recruitment of the ribosome. d4EHP is a translational repressor that prevents translation initiation by out-competing eIF4E on the cap structure for a subset of mRNAs. However, only two examples of mRNAs subject to d4EHP translation repression in Drosophila are known. This study shows that the belle (bel) mRNA is translationally repressed by the d4EHP protein in the Drosophila ovary. Consistent with this regulation, d4EHP overexpression in the ovary phenocopies the bel mutant. Evidence is provided that the Bel protein binds to eIF4E and may itself function as a translation repressor protein, with bruno as a potential target for Bel repression in the oocyte. Bruno is known to repress the mRNA of the key oocyte axis determinant oskar (osk) during oogenesis, and this study found that an increase in the level of Bruno protein in bel mutant ovaries is associated with a reduction in Osk protein. Overall, these data suggest that a translational regulatory network exists in which consecutive translational repression events act to correctly pattern the Drosophila oocyte (Yarunin, 2011).

    The interactions of GW182 proteins with PABP and deadenylases are required for both translational repression and degradation of miRNA targets

    Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation. Silencing requires association of miRNAs with an Argonaute protein and a GW182 family protein. In turn, GW182 proteins interact with poly(A)-binding protein (PABP) and the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These interactions are required for the deadenylation and decay of miRNA targets. Recent studies have indicated that miRNAs repress translation before inducing target deadenylation and decay; however, whether translational repression and deadenylation are coupled or represent independent repressive mechanisms is unclear. Another remaining question is whether translational repression also requires GW182 proteins to interact with both PABP and deadenylases. To address these questions, this study characterized the interaction of Drosophila melanogaster GW182 with deadenylases and defined the minimal requirements for a functional GW182 protein. Functional assays in D. melanogaster and human cells indicate that miRNA-mediated translational repression and degradation are mechanistically linked and are triggered through the interactions of GW182 proteins with PABP and deadenylases (Huntzinger, 2013).

    Recent studies indicate that translational repression of miRNA targets precedes deadenylation and decay. This study shows that these two functional outcomes of miRNA regulation are linked and both require the interaction of GW182 proteins with PABP and deadenylases (Huntzinger, 2013).

    The interaction of GW182 proteins with PABP has been well documented using biochemical and structural studies, and the PAM2 motif is highly conserved among vertebrate and insect GW182 proteins. Despite conservation, the study of the role of PABP in silencing in different systems has led to conflicting conclusions. For example, several studies have reported that the PABP–GW182 interaction is important for silencing in Drosophila and human cells and in cell-free systems that recapitulate silencing. Furthermore, PABP depletion prevented miRNA-mediated deadenylation in cell-free extracts from mouse Krebs-2 ascites cells, and mutations in the PAM2 motif of TNRC6C reduced the rate of deadenylation in tethering assays. In addition, a study in Drosophila cell-free extracts wherein silencing is mediated through endogenous preloaded miRISCs indicated that PABP stimulates silencing by facilitating the association of miRISC complexes with mRNA targets. It was also shown that on miRISC binding, PABP progressively dissociated from the mRNA target, in the absence of deadenylation (Huntzinger, 2013).

    In contrast to the studies mentioned above, studies in zebrafish embryos and in a Drosophila cell-free assay wherein miRISCs are loaded with exogenously supplemented miRNA duplexes indicate that PABP is dispensable for miRNA-mediated silencing. Intriguingly, efficient silencing in zebrafish embryos required the GW182 PAM2 motif. Moreover, the observation that multiple and non-overlapping fragments of Drosophila GW182 (including N-term fragments that do not interact with PABP) silenced mRNA reporters in tethering assays was interpreted as evidence that the interaction of GW182 proteins with PABP is not required for silencing. This study shows that unlike in tethering assays, N-term fragments of GW182 fail to restore the silencing of a majority of the reporters tested in complementation assays. Thus, tethering assays bypass the requirement for PABP binding, and may not faithfully recapitulate silencing. Furthermore, the observation that PABP dissociates from the poly(A) tail of miRNA targets in the absence of deadenylationprovides one explanation for the occurrence of silencing in extracts in which PABP has been depleted or displaced from the poly(A) tail using an excess of Paip2 (Huntzinger, 2013).

    In summary, these results confirm and further extend previous observations that a single amino acid substitution in the PAM2 motif of human TNRC6 proteins abolishes PABP binding and impairs silencing activity, despite the interaction of this mutant with deadenylases. Furthermore, Drosophila GW182 N-term protein fragments that bind deadenylases, but not PABP, failed to complement the silencing of eight of the nine reporters tested, although they are active in tethering assays. These results provide evidence for a role of PABP in silencing in human and Drosophila cells. However, it is possible that PABP becomes dispensable for silencing depending on cellular conditions or the nature of the specific mRNA target, as shown, for example, for the F-Luc-Nerfin-1 reporter when silencing is mediated by miR-9b (Huntzinger, 2013).

    The SDs of human TNRC6 proteins directly interact with CNOT1 through tryptophan-containing motifs in the M1, M2 and C-term regions of the S. This study shows that these motifs contribute additively to CNOT1 binding and silencing activity in human cells. Indeed, when at least two motifs are simultaneously mutated, CNOT1 binding is strongly reduced and silencing activity impaired (Huntzinger, 2013).

    The interaction between GW182 and deadenylases is conserved in Drosophila; however, in contrast to human SDs, the Drosophila SD is not sufficient for NOT1 binding. This study shows that in addition to the SD, the Q-rich region is required for full NOT1 binding activity. Thus, although Drosophila GW182 has lost the CIM-2 motif, this protein has acquired additional motifs that can interact with NOT1. This study also shows that in contrast to the human proteins, Drosophila GW182 can interact with NOT2 and PAN3 via N-term sequences. Consequently, Drosophila GW182 can recruit deadenylases in multiple ways. Considering that (1) NOT1 interacts with NOT2, (2) the PAN2–PAN3 complex interacts with PABP and (3) the CCR4–NOT and PAN2–PAN3 complexes form a larger multiprotein complex in vivo, the current observations indicate a high degree of connectivity and redundancy within the GW182 interaction network, which could explain why mutations in individual motifs do not abolish partner binding or silencing activity, but a combination of two or more mutations is required to abrogate binding and silencing activity (Huntzinger, 2013).

    In addition, the ability of Drosophila GW182 N-term fragments to bind deadenylases also explains why these fragments are potent triggers of translational repression and mRNA degradation in tethering assays, whereas the corresponding fragments of the human proteins exhibit only residual activity. As discussed previously, despite their activity in tethering assays, Drosophila GW182 N-term fragments failed to complement the silencing of several of the reporters tested. The reason for the different activities of these fragments in tethering and complementation assays remains unknown (Huntzinger, 2013).

    This study has demonstrated that silencing (i.e. translational repression and target degradation) requires the interaction between GW182 proteins and both PABP and deadenylases. Several lines of evidence support this conclusion. First, the TNRC6C SD, which is sufficient for PABP and deadenylase binding, rescues silencing when fused to a minimal ABD. Similarly, the minimal fragment of Drosophila GW182 that rescues silencing comprises the Q+SD region, which also binds both deadenylases and PABP. Second, the Drosophila GW182 N-term fragments that bind deadenylases but not PABP are generally inactive in complementation assays. Third, mutations that specifically disrupt TNRC6 binding to PABP or deadenylase impair silencing, and mutations that disrupt deadenylase binding exhibit a stronger deleterious effect. Silencing activity is abolished when these mutations are combined. Finally, silencing is inhibited in human cells overexpressing the CNOT1 Mid domain together with a catalytically inactive CNOT7 mutant. In combination with the previously published data, these results indicate that silencing minimally requires an AGO, a GW182 protein, PABP and deadenylases, thus defining the minimal interaction network required for silencing. The findings do not rule out that additional interactions are potentially required to achieve maximal repression, depending on the cellular context or the mRNA target. For example, the P-GL motif is highly conserved and important for silencing in zebrafish embryos. This motif may mediate interactions with additional partners (Huntzinger, 2013).

    The finding that deadenylase complexes, in particular, are required for miRNA-mediated translational repression has broad implications regarding post-transcriptional mRNA regulation. Indeed, in addition to the GW182 proteins, various sequence-specific mRNA-binding proteins, such as Nanos, Bicaudal-C and Pumilio, recruit the CCR4–NOT complex to their mRNA targets. Furthermore, the direct tethering of the subunits of the CCR4–NOT complex represses the translation of mRNA reporters lacking a poly(A) tail, suggesting that the CCR4–NOT complex promotes translational repression in the absence of deadenylation. Therefore, elucidating the mechanism by which the CCR4–NOT complex regulates the fates of mRNA targets promises to increase understanding of the mechanism underlying repression by miRNAs and diverse sequence-specific RNA-binding proteins (Huntzinger, 2013).

    Regulators of alternative polyadenylation operate at the transition from mitosis to meiosis

    In the sexually reproductive organisms, gametes are produced by meiosis following a limited mitotic amplification. However, the intrinsic program switching cells from mitotic to meiotic cycle is unclear. Alternative polyadenylation (APA) is a highly conserved means of gene regulation and is achieved by the RNA 3'-processing machinery to generate diverse 3'UTR profiles. In Drosophila spermatogenesis, this study observed distinct profiles of transcriptome-wide 3'UTR between mitotic and meiotic cells. In mutant germ cells stuck in mitosis, 3'UTRs of hundreds of genes were consistently shifted. Remarkably, altering the levels of multiple 3'-processing factors disrupted germline's progression to meiosis, indicative of APA's active role in this transition. An RNA-binding protein (RBP) Tut could directly bind 3'UTRs of 3'-processing factors whose expressions were repressed in the presence of Tut-containing complex. Further, this RBP complex could execute the repression post-transcriptionally by recruiting CCR4/Twin of deadenylation complex. Thus, it is proposed that an RBP complex regulates the dynamic APA profile to promote the mitosis-to-meiosis transition (Shan, 2017).

    Diverse cell-specific patterns of alternative polyadenylation in Drosophila

    Most genes in higher eukaryotes express isoforms with distinct 3' untranslated regions (3' UTRs), generated by alternative polyadenylation (APA). Since 3' UTRs are predominant locations of post-transcriptional regulation, APA can render such programs conditional, and can also alter protein sequences via alternative last exon (ALE) isoforms. Previous work used 3'-sequencing from diverse Drosophila samples to define multiple tissue-specific APA landscapes. This study exploited comprehensive single nucleus RNA-sequencing data (Fly Cell Atlas) to elucidate cell-type expression of 3' UTRs across >250 adult Drosophila cell types. The cellular bases of multiple tissue-specific APA/ALE programs were revealed, such as 3' UTR lengthening in differentiated neurons and 3' UTR shortening in spermatocytes and spermatids. Dynamic 3' UTR patterns were traced across cell lineages, including in the male germline, and new APA patterns were discovered in the intestinal stem cell lineage. Finally, expression of RNA binding proteins (RBPs), miRNAs and global levels of cleavage and polyadenylation (CPA) factors were correllated in several cell types that exhibit characteristic APA landscapes, yielding candidate regulators of transcriptome complexity. These analyses provide a comprehensive foundation for future investigations of mechanisms and biological impacts of alternative 3' isoforms across the major cell types of this widely-studied model organism (Lee, 2022).

    A CAF40-binding motif facilitates recruitment of the CCR4-NOT complex to mRNAs targeted by Drosophila Roquin

    Human (Hs) Roquin1 and Roquin2 are RNA-binding proteins that promote mRNA target degradation through the recruitment of the CCR4-NOT deadenylase complex and are implicated in the prevention of autoimmunity. Roquin1 recruits CCR4-NOT via a C-terminal region that is not conserved in Roquin2 or in invertebrate Roquin. This study shows that Roquin2 and Drosophila melanogaster (Dm) Roquin also interact with the CCR4-NOT complex through their C-terminal regions. The C-terminal region of Dm Roquin contains multiple motifs that mediate CCR4-NOT binding. One motif binds to the CAF40 subunit of the CCR4-NOT complex. The crystal structure of the Dm Roquin CAF40-binding motif (CBM) bound to CAF40 reveals that the CBM adopts an α-helical conformation upon binding to a conserved surface of CAF40. Thus, despite the lack of sequence conservation, the C-terminal regions of Roquin proteins act as an effector domain that represses the expression of mRNA targets via recruitment of the CCR4-NOT complex (Sgromo, 2017).

    Mechanism of translation control of the alternative Drosophila melanogaster Voltage Dependent Anion-selective Channel 1 mRNAs

    The eukaryotic porin, also called the Voltage Dependent Anion-selective Channel (VDAC), is the main pore-forming protein of the outer mitochondrial membrane. In Drosophila melanogaster, a cluster of genes evolutionarily linked to VDAC is present on chromosome 2L. The main VDAC isoform, called VDAC1 (Porin1), is expressed from the first gene of the cluster. The porin1 gene produces two splice variants, 1A-VDAC and 1B-VDAC, with the same coding sequence but different 5' untranslated regions (UTRs). The influence of the two 5' UTRs, 1A-5' UTR and 1B-5' UTR, was studied on transcription and translation of VDAC1 mRNAs. In porin-less yeast cells, transformation with a construct carrying 1A-VDAC results in the expression of the corresponding protein and in complementation of a defective cell phenotype, whereas the 1B-VDAC sequence actively represses VDAC expression. Identical results were obtained using constructs containing the two 5' UTRs upstream of the GFP reporter. A short region of 15 nucleotides in the 1B-5' UTR should be able to pair with an exposed helix of 18S ribosomal RNA (rRNA), and this interaction could be involved in the translational repression. These data suggest that contacts between the 5' UTR and 18S rRNA sequences could modulate the translation of Drosophila 1B-VDAC mRNA. The evolutionary significance of this finding is discussed (Leggio, 2018).

    This work focused on the regulation of expression of VDAC1 in D. melanogaster. In this species, the porin1 gene produces two alternative transcripts named 1A-VDAC and 1B-VDAC, containing an identical coding sequence but two completely different 5' UTRs. To gain further insights into the biological function of these two alternative splicing forms of VDAC, they were introduced into a VDAC-lacking system, an established S. cerevisiae strain where the porin1 gene was inactivated (δpor1 strain). The advantage of the yeast cell is its viability (under fermentative conditions), whereas D. melanogaster cells cannot survive the deletion of the VDAC1 gene (Leggio, 2018).

    In δpor1 yeast, the heterologous 1A-5' UTR directed transcription and translation of VDAC and of GFP used as a reporter; in contrast, the 1B-5' UTR directed the transcription but not the translation of the VDAC or the reporter gene. These results confirm that only the 1A-VDAC, but not the 1B-VDAC, is able to complement the growth defect of the δpor1 yeast cells. Similar data were obtained in Drosophila cells by using a luciferase reporter gene downstream of the 1A- or 1B-5' UTR. These results suggest that the 1B-5' UTR affects VDAC expression by inhibiting protein translation. Furthermore, the results suggest that this mechanism is independent of the coding region cloned downstream of the 5'-UTR (Leggio, 2018).

    This study aimed to understand the mechanism responsible for the negative influence of the 1B-5' UTR on the translation of the coding sequences fused downstream. Gene expression in eukaryotic cells is regulated at multiple levels, including mRNA translation. Such control allows rapid changes in protein concentrations and, thus, it is used to maintain cellular homeostasis. Most translation regulation is exerted at the very first stage, when the AUG start codon is identified after the 5' UTR ribosome scanning. Consequently, any occurrence that prevents or inhibits the ability of the ribosome to scan the 5' UTR reduces the efficiency of translation initiation. Mechanisms that produce this effect are well known. Therefore, some of these were assayed, such as the presence of uORFs or stable secondary structures and the association with regulatory RBPs (Leggio, 2018).

    The possibility was ruled out that the small upstream ORF (uORF) located in the 1B sequence is involved in translational control. Bioinformatic analysis suggested that no putative strong secondary structure in the untranslated region of 1B-VDAC mRNA should be involved in the inhibition of translation. In addition, bioinformatic predictive analysis of RBPs showed that there is no known RBP specific for the 1B-5' UTR, within the limitations of computational tools. Moreover, the possible involvement of miRNAs was not considered, because the 3' UTR of 1B-VDAC is included in the corresponding 3' UTR of 1A-VDAC, which is longer. Therefore, because a regulatory mechanism involving a miRNA action targeted to this region of 1B-VDAC mRNA could not be specific for the 1B-mRNA, this mechanism was ruled out (Leggio, 2018).

    Using a mutagenesis scanning approach, the 16-31 nucleotide region of the 1B-5' UTR sequence was identified as responsible in yeast for the inhibitory effect on translation. The defect in the growth of the δpor1 yeast strain was indeed complemented when the strain was transformed with 1B(Δ16-31)-VDAC mutant, underlining that its removal is sufficient to re-establish the translation. It was also verified that the 16-31 sequence works similarly in Drosophila, although the translation inhibition must rely also on others factors. Therefore, by MS analysis of the proteins bound to an RNA oligo containing the 10-34 sequence of the 1B-5' UTR, proteins were sought that were directly or indirectly involved in the translation control. In particular, eIF4A, eIF5a and Asc1 were recognized. eIF4A is a RNA helicase working in the first stage of translation as a subunit of the cap-binding complex eIF4F, which unwinds the RNA secondary structures in the 5' UTR. Asc1/RACK1 associates with the 40S subunit close to the mRNA exit channel, where it interacts with eIF4E of eIF4F51. Asc1/RACK1 is involved in the control of the translation of housekeeping genes and, in general, represses gene expression. It is known that RACK1 loss-of-function mutations cause early developmental lethality in the mouse and the fly, like VDAC knockout organisms. Moreover, in yeast, loss of ASC1 reduces translation of mitochondrial r-proteins and, like for lack of VDAC1, causes cells to be unable to use non-fermentable carbon sources, demonstrating a direct control of ASC1 on mitochondria functionality. Interestingly, RACK1 has many interaction partners, ranging from kinases and signalling proteins to membrane-bound receptors and ion channels. Thus, under stress conditions, RACK1 can function as a signalling hub of newly synthesised proteins (Leggio, 2018).

    From this viewpoint, it can be hypothesised that in yeast the 16-31 sequence might prevents eIF4A function, maybe trapping eIF4A in an inactive conformation. In Drosophila, 1B-VDAC translation could be repressed at the starting point by the coordinated action of more molecules, probably recruited in situ by RACK1. Gus1, which together with Arc1, is known to form a protein complex operating in the control of translation, was identified. In addition, the presence of two different heat-shock proteins (Hsp12 and Hsp76) in this pool of interacting proteins should indicate their recruitment after stress conditions (Leggio, 2018).

    The ability of the 1A- and 1B-5' UTR sequences to contact protein-free domains of 18S rRNA, the only rRNA in the 40S subunit, was also tested. Because 18S rRNA mutations impair the integrity of the scanning-competent pre-initiation complex and/or its joining together with the 60S subunit, the translation initiation rate might be reduced by strong and long-range interactions between the protein-free domains of 18S rRNA and the 5' UTR(s) of the incoming mRNA. It has already been demonstrated in eukaryotes that gene expression regulation at the level of translation may occur thanks to specific interactions between mRNAs and rRNA domains. In particular, a highly specific sequence complementarity between 18S rRNA and the 5' UTRs of mRNAs across species has been predicted; this complementarity may modulate the scanning processivity of the 40S subunit through the 5' UTR of mRNAs, which could even stall the initiating PICs in the case of long-range interactions (Leggio, 2018).

    In particular, by prediction analysis of RNA:RNA interactions between yeast 18S rRNA and the two alternative D. melanogaster VDAC mRNAs (1A-VDAC and 1B-VDAC), it was found that, in yeast as in D. melanogaster, almost the whole 1B-5'UTR sequence is able to strongly interact with a long sequence of 18S rRNA. In contrast, the 1B(Δ16-31)-5'UTR sequence can only weakly interact with a short sequence of rRNA in the 40S subunit, thus showing a behaviour similar to that 1A-5' UTR. These results underline the relevance of 1B-5' UTR and, in particular in yeast, of its 16-31 sequence for the mechanism of translation control. Interestingly, it was also found that some regions of the rRNA sequence involved in the interaction with the 1B-5' UTR fold in solvent-exposed domains, and some of them are turned towards the mRNA path of the ribosome 40S subunit. Therefore, these rRNA domains should be able to contact the 5' UTR in the incoming 1B-VDAC mRNA, producing a stop in the ribosome scanning. It is noteworthy that a sequence of about 35 nucleotides can be allocated inside the ribosomal mRNA path of PIC and that it was found that almost the whole 1B-5' UTR sequence, (2-116 nucleotides), may potentially interact with three 18S rRNA helices (helix 35, helix 36 and a portion of the helix 34) arranged near the mRNA path at the neck of 40S. In addition, the large helix 33, together with parts of helix 31 and helix 32, being arranged at the beck of the 40S subunit, could easily interact with the 1B-5' UTR. In this way, the 1B-VDAC mRNA translation rate would be negatively controlled by its 5' UTR sequence through the collective action of several interactions with 18S rRNA, the result of which would be a strong delay in ribosome scanning of 1B-VDAC. Probably, this effect in Drosophila could also be the result of additional interactions with fly-specific proteins, ribosomal or not. In any case, it is extremely relevant that the sequences encompassing these rRNA helices are highly conserved between S. cerevisiae and D. melanogaster; this indicates that the mechanism described in the mixed yeast-fly system is likely to act in D. melanogaster (Leggio, 2018).

    VDAC is an essential but dangerous protein. Its function as a pro-apoptotic factor is well known and therefore it is essential for the cell to implement a suitable control of VDAC protein level. Also, specific conditions of cell growth involving high energy demand are known to induce up-regulation of VDAC associated with the requirement of mitochondrial biogenesis. Furthermore, these events must be coordinated with the expression of the other mitochondrial proteins, codified by the nuclear genome and from mitochondrial DNA. Therefore, it is conceivable to suppose the presence in the cell of a 'sentry' molecule able to sense, directly or indirectly, the amount of this crucial protein. It was demonstrated that in Drosophila the level of 1B-VDAC transcript is highly increased as a result of overexpression of 1A-VDAC mRNA. When the level of the 1B-VDAC transcript was increased by its overexpression, the endogenous 1A-VDAC mRNA level was meaningfully reduced. Importantly, the results show that the unproductive 1B-VDAC mRNA is able to respond to 1A-VDAC transcript levels, and thus it might work as a molecule signalling the need for activation of mitochondrial biogenesis. This hypothetical role of 1B-VDAC mRNA is supported by its interaction with Asc1/RACK1. Asc1/RACK1 responds to multiple signals, and might act to coordinate the expression of other mitochondrial proteins and thus affect cell respiration (Leggio, 2018).

    In addition, the assignment of this important role to 1B-VDAC mRNA might lead to an understanding of why the evolution of the Drosophila genus proceeded towards the acquisition of an alternative 5' UTR with specific features (Leggio, 2018).

    In conclusion, these results extend earlier reports and provide further evidence that in D. melanogaster the 1A-VDAC transcript is responsible for protein expression, while the alternative 1B-VDAC mRNA is not active in this respect. Moreover, this work showd that a specific mechanism could be responsible for the translation inhibition of the alternative D. melanogaster 1B-VDAC1 transcript (Leggio, 2018).

    A regulatory response to ribosomal protein mutations controls translation, growth, and cell competition

    Ribosomes perform protein synthesis but are also involved in signaling processes, the full extent of which are still being uncovered. This study reports that phenotypes of mutating ribosomal proteins (Rps) are largely due to signaling. Using Drosophila, this study discovered that a bZip-domain protein, Xrp1, becomes elevated in Rp mutant cells. Xrp1 reduces translation and growth, delays development, is responsible for gene expression changes, and causes the cell competition of Rp heterozygous cells from genetic mosaics. Without Xrp1, even cells homozygously deleted for Rp genes persist and grow. Xrp1 induction in Rp mutant cells depends on a particular Rp with regulatory effects, RpS12, and precedes overall changes in translation. Thus, effects of Rp mutations, even the reductions in translation and growth, depend on signaling through the Xrp1 pathway and are not simply consequences of reduced ribosome production limiting protein synthesis. One benefit of this system may be to eliminate Rp-mutant cells by cell competition (Lee, 2018).

    Ribosomes are the essential protein synthesis machines of the cell. Large and small subunits (LSU and SSU), 40S and 60S in eukaryotic cells, form an 80S complex together with mRNA and perform translation in the cytoplasm. Each ribosome subunit is a ribonucleoprotein complex containing one (SSU) or three (LSU) non-coding rRNA molecules and a battery of ribosomal proteins (Rps) and is assembled in the nucleolus for export to the cytoplasm. Rps can contribute to folding and assembly of the ribosomal subunits as well as their function in translation. Most Rps are essential, and cells homozygous for their mutations die, while heterozygous Rp mutants that lack one copy of the gene are abnormal in both humans and in Drosophila (Lee, 2018).

    To what extent do the defects in Rp mutants reflect deficient translation, and to what extent do they reflect signaling pathways that monitor ribosome status? Aspects of Diamond-Blackfan Anemia, the ribosomopathy that occurs in humans heterozygous for mutations in a number of Rp genes, are thought to reflect chronic p53 signaling, activated by accumulation of a ribosome assembly intermediate and nucleolar stress. On the other hand, Diamond-Blackfan Anemia is also characterized by short stature and delayed maturation as well as skeletal defects, and it has sometimes been treated with L-leucine to stimulate protein synthesis. Reduced protein synthesis has been measured in both Drosophila embryos and in mouse fibroblasts and hematopoietic cells from heterozygous, Rp+/- genotypes (Lee, 2018).

    This study made use of Drosophila to investigate the effects of Rp mutations further. Drosophila that are haploinsufficient for any of 66 of the 79 Rp genes exhibit a common phenotype, first recognized a century ago (the 'Minute' phenotype), which includes a reduction in the size and thickness of bristles on the adult body ('Minute' bristles) and a developmental delay associated with reduced translation and growth rate. Unlike the bristle structures, most mutant cells are of normal size, as are mutant flies themselves, suggesting that the extended growth period is sufficient to compensate for reduced cellular growth. In fact, mutant organs can be larger than normal, depending on the particular balance of growth between organs (Lee, 2018).

    In Drosophila, and possibly in mammals, Rp+/- genotypes are subject to 'cell competition' in genetic mosaics. If growing imaginal discs (progenitor cells that grow in an undifferentiated state in the larva to give rise to the adult tissues) contain both wild-type and Rp+/- cells, the latter are progressively lost during growth. Conversely, wild-type cells growing in Rp+/- backgrounds come to dominate developmental compartments at the expense of the Rp+/- cells. Both competitive situations are associated with selective apoptosis of Rp+/- cells in proximity to wild-type, which is responsible for the loss of Rp+/- clones. There are other genotypes that can be competed from genetic mosaics, but neither is it clear that the mechanisms are the same nor whether deficits in translation or growth are required. There are also examples of 'super-competitor' genotypes that can eliminate nearby wild-type cells, even though wild-type cells should have normal ribosomes. In the mouse embryo, cells expressing more Myc or less p53 are super-competitors (Lee, 2018).

    Cell competition is also seen in mammalian cell co-cultures, in many cases eliminating hyperplastic or preneoplastic cells. Such cells can also be eliminated from mosaics with otherwise normal tissues in vivo (Lee, 2018).

    The current studies reported originated in a genetic screen designed to identify new components of cell competition. This led to isolation of a mutation affecting a basic leucine zipper (bZip)-domain protein gene, Xrp1 (Lee, 2016). Xrp1 was previously known as a putative transcription factor induced by p53 following X-irradiation of Drosophila and implicated in genome maintenance, although no point mutant alleles had been studied previously. Xrp1 was also characterized as a component of the protein complex that binds to the P element transposon in Drosophila and found to contribute to P element transposition (Franci, 2016). This study reports a major role for Xrp1 in multiple features of Rp mutants. Xrp1 expression is elevated in mutant cells by a signal from the ribosome and controls cellular translation rate and growth in addition to cellular competitiveness and almost the entire gene expression signature of Rp+/- cells. Xrp1 is even responsible for eliminating cells homozygously mutant for essential Rp genes that are deficient for new ribosome biogenesis. It is concluded that Xrp1 controls a cellular stress pathway that monitors Rps, regulates multiple cellular properties, and acts upstream of the major defects in global translation, which are in fact only indirectly related to the initial mutation of an Rp gene (Lee, 2018).

    Xrp1, which behaved like a master-regulator of responses to Rp mutations. Even the acute lethality of Rp-/- cells depended on Xrp1. The only aspect of the Rp+/- phenotype that appeared largely independent of Xpr1 was the reduced size of the bristles, which was only slightly restored by Xrp1 mutations. Bristle size might depend on ribosome function directly or on a different regulatory gene that replaces Xrp1 in bristle precursors (Lee, 2018).

    Rp/+ genotypes express a phenotype of reduced translation, slow cellular growth rate, and reduced competitiveness in comparison to wild-type cells. All of these effects of the Rp+/- genotypes depended on the bZip-domain protein Xrp1. The signal to increase Xrp1 transcription depended on the RpS12 protein, which appears to signal the existence of a ribosomal defect or ribosomal protein imbalance (Kale, 2018). Xrp1 was responsible for reducing the bulk translation rate in Rp+/- cells, and this must include a reduction in the translational activity of mature ribosomes. Reduced translation was likely responsible for the slow growth of Rp+/- cells, although Xrp1 might also affect growth independently of translation. Xrp1 also controlled the competitiveness of Rp+/- cells in mosaics with wild-type cells, which provides a possibility for eliminating Rp+/- cells in favor of non-mutant replacements. It is hypothesized that one or more target genes control competitiveness, either in response to Xrp1 itself, or indirectly in response to the changes in translation or growth rates. By utilizing cell competition, a decision could be made to eliminate defective cells only where better cells were available to replace them (Lee, 2018).

    A null allele of Xrp1 was isolated in a screen for mutations preventing cell competition. Xrp1 transcription and Xrp1 protein were found to be selectively elevated in Rp+/- cells and to be required cell-autonomously to render these cells less competitive than wild-type cells. Later it was found that Xrp1 also acted to reduce the growth rate of Rp+/- cells. In the absence of Xrp1, or even when Xrp1 gene dose was reduced to one copy, Rp+/- cells grew more like wild-type cells. Xrp1 contributed substantially to the developmental delay of Rp+/- animals, which without Xrp1 could reach adulthood only slightly later than wild-type animals, despite lacking one copy of essential Rp genes (Lee, 2018).

    Xrp1 probably reduces growth by reducing the overall translation rate. Rp+/- cells had lower translation rates than wild-type cells, but it was Xrp1, not haploinsufficiency for an important Rp gene, that reduced translation rate, because the difference from wild-type disappeared when Xrp1 was mutated simultaneously. Although ribosome numbers have not been counted directly, the proportion of rRNAs was not reduced in most Rp+/- genotypes, suggesting that an Xrp1-dependent reduction in translational activity per ribosome may occur. A mutation in RpL27A was the exception that did appear to reduce LSU number, but this was not rescued in RpL27A+/-Xrp1+/- discs and so was not responsible for the Xrp1-dependent growth inhibition. The persistence of Rp-/- mutant clones in the absence of Xrp1 also suggests changes in ribosome activity. Rp-/- cells should be deficient in synthesizing new ribosomes, so the prolonged survival of Rp-/-Xrp1-/- clones cannot easily be explained through restored ribosome biogenesis (Lee, 2018).

    These findings suggest Xrp1 influences the rate of translation by cytoplasmic ribosomes, but they do not exclude additional roles in ribosome biogenesis. For example, late third instar wing discs from wild-type, RpS18+/-, and RpS18+/-Xrp1+/- larvae contained indistinguishable ribosome numbers, but these genotypes developed at different rates. Based on the time taken for adults to emerge, it is estimated that the ribosomes in late third instar wing discs had accumulated over ~80 hr, ~115 hr, and ~100 hr of larval life, respectively, so this is consistent with different rates of ribosome biogenesis generating similar absolute numbers of ribosomes over different durations of larval development. A reduced rate of ribosome biogenesis was reported previously in the mouse RpL24Bst/+mutant (Lee, 2018).

    Regarding the overall rate of organismal development, it is well known that progress through the insect life cycle is controlled, in part, through systemic signals ultimately controlling ecdysone levels. This is not the primary means that Xrp1 affects imaginal disc growth because this occurs cell-autonomously. Additional non-autonomous effects of Xrp1 on organismal development are not ruled out. For example, Dilp8, a secreted factor that regulates organismal growth, undergoes Xrp1-dependent upregulation in Rp+/- wing discs (Lee, 2018).

    Hundreds of genes show altered mRNA levels in Rp+/- wing discs, but it has not been clear how these changes arise. This study now reports that >80% of altered mRNA levels were Xrp1-dependent. Some of these genes might be indirect targets of Xrp1; for example, Xrp1-dependent changes in overall translation rate may change gene transcription through a variety of mechanisms. The Xrp1-dependent changes include oxidative stress responses, which are reported to make cells less competitive, and also DNA repair genes (Lee, 2018).

    How is Xrp1 induced by Rp mutations? Xrp1 is a transcriptional target of p53 in the response to irradiation, but p53 is not required for the elimination of Rp+/- cells by cell competition (Kale, 2015). Accordingly, this study showed that p53 was not required to elevate Xrp1 in Rp+/- wing discs. Reduced overall translation was unlikely to induce Xrp1 because Xrp1 was actually responsible for this. Perhaps more subtle changes in the translation of specific mRNAs occur first and induce Xrp1 expression. Another possibility is that a signal is sent when ribosome assembly is altered, for example, through an accumulated assembly intermediate. This study reports that a particular Rp, RpS12, that was already recognized as a gene required for cell competition (Kale, 2018), was required to elevate Xrp1 transcription. Although the molecular mechanism by which RpS12 can affect transcription is not yet known, this demonstrates that a link between a particular Rp and the Xrp1 gene triggers most of the response that occurs to mutations in other Rp genes, upstream of overall changes in bulk translation rates, which are a later consequence of the Xrp1 pathway. All the DNA repair gene expression in Rp+/- wing discs was also downstream of Xrp1 and may reflect Xrp1's other role in the response to irradiation (Brodsky, 2004, Akdemir, 2007). How DNA repair genes contribute to aspects of the Rp+/- phenotype remains to be determined (Lee, 2018).

    The importance of Xrp1 extends to homozygous Rp mutant cells. Remarkably, even Rp-/- cells survived and underwent limited growth if Xrp1 was completely removed. It is not believed that imaginal disc cells can grow and divide without ribosomes and protein synthesis, but since ribosome turnover occurs very slowly, Rp-/- recombinant cells probably retain most of the ribosome complement from the Rp+/- mother cell at first. They would be deficient in replenishing their ribosome complement, however, which would dilute with further growth and cell division. Previous studies indicate that when ribosome activity diminishes below a critical threshold, Rp-/- cells undergo apoptosis (Kale, 2015). The absence of Xrp1 allowed Rp-/- cells to survive longer, by allowing more translation by the remaining ribosomes and possibly by preventing competitive elimination of Rp-/- cells by Rp+/- cells (Lee, 2018).

    Despite the unquestioned importance of Rps in ribosome structure and function, the current results indicate that the effects of Rp mutations in Drosophila are largely due to a regulatory response. Even the reduced translation in Rp mutant genotypes, which has also been observed in other organisms, is downstream of Xrp1 and does not play a primary role as a sensor of Rp mutations. Even when Rp genes are homozygously mutated, which seemingly should affect overall translation very quickly, Xrp1 normally kills the Rp-/-cells before such effects become evident. Rp+/- cells can also be killed by Xrp1, but indirectly, by cell competition, when wild-type cells are nearby (Lee, 2018).

    Mutations that reduce translation or ribosome biogenesis by other routes are phenotypically distinct from Rp mutants. For example, mutations in the myc gene homolog or in components of the Drosophila TOR pathway lead to smaller flies, unlike Rp+/- flies. In mammals as well, mutations that affect ribosome biogenesis independently of Rp genes lead to human disease, but the symptoms of such ribosomopathies differ from Diamond-Blackfan Anemia. These differences may occur because translation is affected indirectly in mutations of Rp genes and is not the primary trigger for all the cellular responses leading to pathology (Lee, 2018).

    Although it may at first seem surprising that mutations in Rps that are so directly involved in translation affect the cell through another mechanism, perhaps it is advantageous to mount such a coordinated response, for example to enable cell competition. Much as it is adaptive to eliminate cells with damaged DNA through apoptosis, perhaps cell competition is a mechanism to eliminate one or a few cells with defective ribosomes in favor of other, more normal cells (Lee, 2018).

    Modulating eIF6 levels unveils the role of translation in ecdysone biosynthesis during Drosophila development

    During development, ribosome biogenesis and translation reach peak activities, due to impetuous cell proliferation. Current models predict that protein synthesis elevation is controlled by transcription factors and signalling pathways. Developmental models addressing translation factors overexpression effects are lacking. Eukaryotic Initiation Factor 6 (eIF6) is necessary for ribosome biogenesis and efficient translation. eIF6 is a single gene, conserved from yeasts to mammals, suggesting a tight regulation need. This study generated a Drosophila melanogaster model of eIF6 upregulation, leading to a boost in general translation and the shut-down of the ecdysone biosynthetic pathway. Indeed, translation modulation in S2 cells showed that translational rate and ecdysone biosynthesis are inversely correlated. In vivo, eIF6-driven alterations delayed Programmed Cell Death (PCD), resulting in aberrant phenotypes, partially rescued by ecdysone administration. These data show that eIF6 triggers a translation program with far-reaching effects on metabolism and development, stressing the driving and central role of translation (Russo, 2019).

    Differential requirement for translation initiation factor pathways during ecdysone-dependent neuronal remodeling in Drosophila

    Dendrite pruning of Drosophila sensory neurons during metamorphosis is induced by the steroid hormone ecdysone through a transcriptional program. In addition, ecdysone activates the eukaryotic initiation factor 4E-binding protein (4E-BP) to inhibit cap-dependent translation initiation. To uncover how efficient translation of ecdysone targets is achieved under these conditions, the requirements for translation initiation factors during dendrite pruning were assessed. The canonical cap-binding complex eIF4F was found to be dispensable for dendrite pruning, but the eIF3 complex and the helicase eIF4A are required, indicating that differential translation initiation mechanisms are operating during dendrite pruning. eIF4A and eIF3 are stringently required for translation of the ecdysone target Mical, and this depends on the 5' UTR of Mical mRNA. Functional analyses indicate that eIF4A regulates eIF3-mRNA interactions in a helicase-dependent manner. It is proposed that an eIF3-eIF4A-dependent alternative initiation pathway bypasses 4E-BP to ensure adequate translation of ecdysone-induced genes (Rode, 2018).

    Pruning, the developmentally controlled degeneration of synapses and neurites without loss of the parent neuron, is an important mechanism used to specify neuronal connections or to remove developmental intermediates. In holometabolous insects like Drosophila, the nervous system is remodeled during metamorphosis in response to the steroid hormone ecdysone. In the peripheral nervous system (PNS), the sensory class IV dendritic arborization (c4da) neurons completely prune their long and branched larval dendrites at the onset of the pupal phase, while their axons stay intact. C4da neuron dendrite pruning involves the specific destabilization of the dendritic cytoskeleton and plasma membrane and phagocytosis of severed dendrites by surrounding epidermal cells (Rode, 2018).

    Ecdysone induces c4da neuron dendrite pruning through the hormone receptors EcR-B1 and ultraspiracle (Usp), which activate the transcription of pruning genes. Among these are headcase, a pruning gene of unknown function, and SOX14, an HMG box transcription factor that activates transcription of MICAL, encoding an actin-severing enzyme. Regulation of MICAL expression also involves the ubiquitin-proteasome system at a posttranscriptional level (Rode, 2018).

    In addition to transcriptional activation of target genes, several lines of evidence suggest that ecdysone also regulates global translation rates through activation of the translation inhibitor eukaryotic initiation factor 4E-binding protein (4E-BP). In the Drosophila fat body, this occurs transcriptionally through FOXO, while in c4 da neurons, ecdysone inhibits the insulin and Target of Rapamycin (TOR) pathway to activate 4E-BP posttranslationally (Rode, 2018).

    4E-BP inhibits translation initiation, the rate-limiting step of protein synthesis, by sequestering the cap-binding protein eIF4E. During canonical translation initiation, eIF4E binds to the 7-methylguanosine (m7Gppp) cap of eukaryotic mRNAs and then forms the so-called eIF4F complex by recruiting eIF4G, an adaptor that binds the 43S preinitiation complex (PIC), containing the 40S small ribosomal subunit, and the helicase eIF4A, which is thought to resolve hairpin structures in the 5' UTRs of mRNAs. This enables the 43S complex to scan 5' UTRs for the initiation codon, where it is joined by the large ribosomal subunit and translation can start. While eIF4A's role has been mainly linked to 5' UTR hairpins, it can also stimulate translation of mRNAs with unstructured 5' UTRs. Moreover, eIF4A is more abundant than eIF4E, suggesting that it has functions beyond the eIF4F complex (Rode, 2018).

    Activated 4E-BP binds to eIF4E and prevents eIF4F assembly, thus inhibiting ribosome recruitment to mRNAs and globally dampening translation rates under stress or during development. Interestingly, 4E-BP affects translation of some mRNAs more than others. To explain this, eIF4E-independent translation initiation mechanisms have been proposed. One such mechanism could depend on internal ribosome entry sites (IRESs) that bypass the requirement for the m7Gppp cap. For example, the mRNAs of the Drosophila cell death factors reaper and hid may contain IRES sequences in their 5' UTRs that allow them to be translated under stress (Rode, 2018).

    Alternative cap recognition mechanisms have also been proposed under conditions of high 4E-BP activity. In particular, the initiation factor eIF3, a 13-subunit complex, could provide a mechanism for eIF4E-independent initiation (Lee, 2015; Lee, 2016). It binds to the small ribosomal subunit as part of the 43S PIC, and it is thought to act downstream of eIF4G in mRNA recruitment. However, eIF3 dependence varies between mRNAs, and eIF3 can even suppress translation of some targets (Lee, 2015). Importantly, it was recently shown that translation of some 4E-BP-resistant mRNAs depends on an eIF3-based cap recognition activity in the eIF3d subunit that is stimulated by hairpin motifs in the 5' UTR (Lee, 2016). Other eIF3 subunits have also been shown to interact with the cap (Rode, 2018).

    Given that ecdysone inhibits eIF4E-dependent translation, this study asked whether there are mechanisms that ensure the translation of ecdysone target mRNAs. To this end, the requirements for translation initiation factors during c4da neuron dendrite pruning was assessed. The canonical eIF4F components eIF4E and eIF4G were found not to be required for c4da neuron dendrite pruning, while the helicase eIF4A and the eIF3 complex are. Both eIF4A and eIF3 are required for Mical expression, and this specificity is conferred by the 5' UTR of Mical mRNA. Further biochemical analyses suggest that eIF4A regulates the interaction between eIF3 and the Mical 5' UTR. It is proposed that eIF4A/eIF3 constitute a 4E-BP bypass mechanism that ensures the adequate translation of ecdysone-induced genes in c4da neurons (Rode, 2018).

    Developmental control of translation rate is required under various conditions. One well-characterized regulatory mechanism is through 4E-BP, which inhibits assembly of the cap-binding eIF4F complex. Despite the obvious need for global translation control during development, it is also clear that there must be exceptions to such regulation. Several lines of evidence suggest that global, eIF4E-dependent translation is downregulated by ecdysone during the pupal phase and that this is important for c4da neuron dendrite pruning. How downregulation of eIF4E-dependent translation contributes to dendrite pruning is not clear. TOR activity (and hence eIF4E-dependent translation) is associated with neurite regrowth after pruning in a Drosophila model for neuronal remodeling, and beta-actin mRNA was identified as a 4E-BP target in vertebrate neurons. General suppression of eIF4E-dependent translation may, therefore, serve to prevent precocious neurite growth or neurite stabilization through increased actin polymerization (Rode, 2018).

    Despite the need for translation downregulation during dendrite pruning, ecdysone-induced mRNAs must still be efficiently translated. This study found that c4 da neuron dendrite pruning does not depend on the eIF4F subunits eIF4E and eIF4G, but instead on eIF3 and eIF4A. In keeping with a specific effect on dendrite-pruning genes, Mical mRNA was identified as the crucial target for eIF3 and eIF4A. The data suggest that this specificity is encoded in the 5' UTR of Mical mRNA, as a UAS-GFP reporter containing the Mical 5' UTR showed consistently stronger dependence on eIF4A and eIF3 than a regular UAS-GFP reporter. The important role of the Mical 5' UTR is also supported by the observation that Sox14 overexpression (which induces endogenous Mical mRNA) did not rescue the pruning defects induced by eIF4A RNAi, while overexpression of Mical from a UAS transgene (and thus lacking the endogenous 5' UTR) did. It is tempting to speculate that eIF3-eIF4A recognition signals may be abundant in 5' UTRs of ecdysone-induced genes (Rode, 2018).

    Several lines of evidence indicate that translation initiation of pupal pruning factors in c4 da neurons is still cap dependent: for one, overexpression of a cap-binding-deficient eIF3d mutant causes dominant dendrite-pruning defects, and in vitro translation of a 5' UTRMical reporter mRNA depends on a functional cap. While physical interactions were observed between eIF3 and a 5' UTR Mical reporter mRNA in S2 cells, cap binding by eIF3 could not be directly demonstrated in vivo. eIF3 does not bind to the isolated cap structure, and a biochemical cap-binding assay for eIF3 would require crosslinking eIF3 with a purified mRNA with a radioactively labeled cap. To further investigate developmental control of translation initiation in the future, it would be interesting to set up such an assay to address whether the Mical mRNA cap is also recognized via eIF3d or another eIF3 subunit (Rode, 2018).

    Sox14 expression seemed resistant to the inhibition of either eIF4E or eIF3, but this study found that these pathways can mediate Sox14 expression in a redundant fashion. Sox14 is upstream of the Cul-1 ubiquitin ligase that activates 4E-BP in c4da neurons. Its mRNA may be adapted to this position in the pruning pathway, as it could still use the regular eIF4F pathway early during the pupal phase and the eIF3 pathway later. Mical translation may only start when 4E-BP activity is already high, hence explaining its strong eIF3 dependence (Rode, 2018).

    Translation of long mRNAs is sensitive to the eIF4A cofactor eIF4B (Sen, 2016), and eIF4A dependence is also in part conferred by sequences in the coding region. eIF4B manipulation did not cause dendrite pruning defects, but the Mical construct used to rescue the pruning defects induced by eIF4A knockdown lacks an internal region non-essential for pruning. It is, therefore, possible that internal regions of the long Mical mRNA also contribute to its dependence on eIF4A (Rode, 2018).

    The strong similarities between the phenotypes caused by the manipulation of eIF4A and eIF3 suggested that these two factors cooperate functionally. eIF4A and eIF3 can be found in an eIF4A ATPase-dependent complex and that eIF4A clamping on the mRNA prevents eIF3 release from a 5' UTRMical reporter mRNA. Two recent in vitro studies found functional interactions between eIF4A and eIF3 in the context of canonical eIF4F-dependent translation initiation (Yourik, 2017, Sokabe, 2017): first, eIF3 stimulates eIF4A ATPase activity via its eIF3g subunit to promote PIC maturation (Yourik, 2017); and, second, eIF4A ATPase activity was required to reposition the eIF3j subunit within the PIC during maturation (Sokabe and Fraser, 2017). this study now demonstrate genetically that eIF4A has an eIF3-related function independently of eIF4F. These data showing that eIF3 and eIF4A interact in an ATPase-dependent manner and that eIF4A helicase activity is required for dendrite pruning are consistent with both the above proposals (Rode, 2018).

    Taken together, these data suggest that eIF3-eIF4A are part of a bypass mechanism that ensures translation of crucial ecdysone-induced mRNAs in the absence of an eIF4E-dependent translation initiation during developmental neuronal remodeling in the Drosophila PNS (Rode, 2018).

    Establishment of 5'-3' interactions in mRNA independent of a continuous ribose-phosphate backbone

    The functions of eukaryotic mRNAs are characterized by intramolecular interactions between their 5' and 3' ends. This study has addressed the question whether such 5'-3' interactions are established by diffusion-controlled encounter of the ends 'through solution' or by some type of scanning along the RNA backbone. For this purpose, in vitro translation system derived from Drosophila embryo extract was used that displays two types of 5'-3' interactions: cap-dependent translation initiation is stimulated by the poly(A) tail and inhibited by Smaug Recognition Elements (SREs) in the 3' UTR. Chimeric RNAs were constructed in which a luciferase open reading frame was separated from SREs and the poly(A) tail by a protein linker. Stimulation of translation by the poly(A) tail was fully functional with such RNAs even when disruption of the RNA backbone was combined with an inversion of the 5'-3' polarity between open reading frame and poly(A) segment. The stimulatory effect of the poly(A) tail also became weaker with increasing distance between the 5' end and the poly(A) segment. Both observations suggest that contacts between the poly(A) tail and the 5' end are established through solution, independently of the RNA backbone. In the same RNA constructs, SRE-dependent inhibition of translation was also insensitive to disruption of the RNA backbone. Thus, tracking of the RNA backbone is excluded as a mechanism for repression of cap-dependent initiation. However, SRE-dependent repression was insensitive to mRNA length, suggesting the possibility that the contact between the SREs in the 3' UTR and the 5' end of the RNA is established in a manner that differs from the contact between poly(A) tail and the cap (Kluge, 2020).

    Canonical nucleators are dispensable for stress granule assembly in intestinal progenitors

    Stressed cells downregulate translation initiation and assemble membrane-less foci termed stress granules (SGs). Extensively characterized in cultured cells, the existence of such structures in stressed adult stem cell pools remain poorly characterized. This study reports that Drosophila orthologs of mammalian SG components AGO1, ATX2, CAPRIN, eIF4E, FMRP, G3BP, LIN-28, PABP, and TIAR are enriched in adult intestinal progenitor cells where they accumulate in small cytoplasmic messenger ribonucleoprotein complexes (mRNPs). Treatment with sodium arsenite or rapamycin reorganized these mRNPs into large cytoplasmic granules. Formation of these intestinal progenitor stress granules (IPSGs) depended on polysome disassembly, led to translational downregulation, and was reversible. While canonical SG nucleators ATX2 and G3BP were sufficient for IPSG formation in the absence of stress, neither of them, nor TIAR, either individually or collectively, were required for stress-induced IPSG formation. This work therefore finds that IPSGs do not assemble via a canonical mechanism, raising the possibility that other stem cell populations employ a similar stress-response mechanism (Buddika, 2020).

    Premature termination codon readthrough in Drosophila varies in a developmental and tissue-specific manner

    Despite their essential function in terminating translation, readthrough of stop codons occurs more frequently than previously supposed. However, little is known about the regulation of stop codon readthrough by anatomical site and over the life cycle of animals. This study developed a set of reporters to measure readthrough in Drosophila melanogaster. A focused RNAi screen in whole animals identified upf1 as a mediator of readthrough, suggesting that the stop codons in the reporters were recognized as premature termination codons (PTCs). Readthrough rates of PTCs varied significantly throughout the life cycle of flies, being highest in older adult flies. Furthermore, readthrough rates varied dramatically by tissue and, intriguingly, were highest in fly brains, specifically neurons and not glia. This was not due to differences in reporter abundance or nonsense-mediated mRNA decay (NMD) surveillance between these tissues. Readthrough rates also varied within neurons, with cholinergic neurons having highest readthrough compared with lowest readthrough rates in dopaminergic neurons. Overall, these data reveal temporal and spatial variation of PTC-mediated readthrough in animals, and suggest that readthrough may be a potential rescue mechanism for PTC-harboring transcripts when the NMD surveillance pathway is inhibited (Chen, 2020).

    mRNA decapping is an evolutionarily conserved modulator of neuroendocrine signaling that controls development and ageing

    Eukaryotic 5'-3' mRNA decay plays important roles during development and in response to stress, regulating gene expression post-transcriptionally. In Caenorhabditis elegans, deficiency of DCAP-1/DCP1, the essential co-factor of the major cytoplasmic mRNA decapping enzyme, impacts normal development, stress survival and ageing. This study shows that overexpression of dcap-1 in neurons of worms is sufficient to increase lifespan through the function of the insulin/IGF-like signaling and its effector DAF-16/FOXO transcription factor. Neuronal DCAP-1 affects basal levels of INS-7, an ageing-related insulin-like peptide, which acts in the intestine to determine lifespan. Short-lived dcap-1 mutants exhibit a neurosecretion-dependent upregulation of intestinal ins-7 transcription, and diminished nuclear localization of DAF-16/FOXO. Moreover, neuronal overexpression of DCP1 in Drosophila melanogaster confers longevity in adults, while neuronal DCP1 deficiency shortens lifespan and affects wing morphogenesis, cell non-autonomously. This genetic analysis in two model-organisms suggests a critical and conserved function of DCAP-1/DCP1 in developmental events and lifespan modulation (Borbolis, 2020).

    Loss of the RNA trimethylguanosine cap is compatible with nuclear accumulation of spliceosomal snRNAs but not pre-mRNA splicing or snRNA processing during animal development

    The 2,2,7-trimethylguanosine (TMG) cap is one of the first identified modifications on eukaryotic RNAs. TMG, synthesized by the conserved Tgs1 enzyme, is abundantly present on snRNAs essential for pre-mRNA splicing. Results from ex vivo experiments in vertebrate cells suggested that TMG ensures nuclear localization of snRNAs. Functional studies of TMG using tgs1 mutations in unicellular organisms yield results inconsistent with TMG being indispensable for either nuclear import or splicing. Utilizing a hypomorphic Tgs1 mutation in Drosophila, this study shows that TMG reduction impairs germline development by disrupting the processing, particularly of introns with smaller sizes and weaker splice sites. Unexpectedly, loss of TMG does not disrupt snRNAs localization to the nucleus, disputing an essential role of TMG in snRNA transport. Tgs1 loss also leads to defective 3' processing of snRNAs. Remarkably, stronger Tgs1 mutations cause lethality without severely disrupting splicing, likely due to the preponderance of TMG-capped snRNPs. Tgs1, a predominantly nucleolar protein in Drosophila, likely carries out splicing-independent functions indispensable for animal development. Taken together, these results suggest that nuclear import is not a conserved function of TMG. As a distinctive structure on RNA, particularly non-coding RNA, it is suggested that TMG prevents spurious interactions detrimental to the function of RNAs that it modifies (Cheng, 2020).

    Developmental regulation of canonical and small ORF translation from mRNAs

    Ribosomal profiling has revealed the translation of thousands of sequences outside annotated protein-coding genes, including small open reading frames of less than 100 codons, and the translational regulation of many genes. This study presents an improved version of Poly-Ribo-Seq and applied it to Drosophila melanogaster embryos. Highly correlated samples were obtained across five embryonic stages, with nearly 500 million putative ribosomal footprints mapped to mRNAs, and they were compared to existing Ribo-Seq and proteomic data. This analysis reveals, for the first time in Drosophila, footprints mapping to codons in a phased pattern, the hallmark of productive translation. A simple binomial probability metric is proposed to ascertain translation probability. The results also reveal reproducible ribosomal binding apparently not resulting in productive translation. This non-productive ribosomal binding seems to be especially prevalent amongst upstream short ORFs located in the 5' mRNA leaders, and amongst canonical ORFs during the activation of the zygotic translatome at the maternal-to zygotic transition. It is suggested that this non-productive ribosomal binding might be due to cis-regulatory ribosomal binding and to defective ribosomal scanning of ORFs outside periods of productive translation. The results are compatible with the main function of upstream short ORFs being to buffer the translation of canonical canonical ORFs and show that, in general, small ORFs in mRNAs display markers compatible with an evolutionary transitory state towards full coding function (Patraquim, 2020).

    Variants in SCAF4 Cause a Neurodevelopmental Disorder and Are Associated with Impaired mRNA Processing

    RNA polymerase II interacts with various other complexes and factors to ensure correct initiation, elongation, and termination of mRNA transcription. One of these proteins is SR-related CTD-associated factor 4 (SCAF4), which is important for correct usage of polyA sites for mRNA termination. Using exome sequencing and international matchmaking, nine likely pathogenic germline variants were identified in SCAF4 including two splice-site and seven truncating variants, all residing in the N-terminal two thirds of the protein. Eight of these variants occurred de novo, and one was inherited. Affected individuals demonstrated a variable neurodevelopmental disorder characterized by mild intellectual disability, seizures, behavioral abnormalities, and various skeletal and structural anomalies. Paired-end RNA sequencing on blood lymphocytes of SCAF4-deficient individuals revealed a broad deregulation of more than 9,000 genes and significant differential splicing of more than 2,900 genes, indicating an important role of SCAF4 in mRNA processing. Knockdown of the SCAF4 ortholog CG4266 in the model organism Drosophila melanogaster resulted in impaired locomotor function, learning, and short-term memory. Furthermore, an increased number of active zones was observed in larval neuromuscular junctions, representing large glutamatergic synapses. These observations indicate a role of CG4266 in nervous system development and function and support the implication of SCAF4 in neurodevelopmental phenotypes. In summary, these data show that heterozygous, likely gene-disrupting variants in SCAF4 are causative for a variable neurodevelopmental disorder associated with impaired mRNA processing (Fliedner, 2020).

    Nucleation by rRNA dictates the precision of nucleolus assembly

    Membrane-less organelles are intracellular compartments specialized to carry out specific cellular functions. There is growing evidence supporting the possibility that such organelles form as a new phase, separating from cytoplasm or nucleoplasm. However, a main challenge to such phase separation models is that the initial assembly, or nucleation, of the new phase is typically a highly stochastic process and does not allow for the spatiotemporal precision observed in biological systems. This study investigated the initial assembly of the nucleolus, a membrane-less organelle involved in different cellular functions including ribosomal biogenesis. The nucleolus formation is precisely timed in D. melanogaster embryos and follows the transcription of rRNA. This study provides evidence that transcription of rRNA is necessary for overcoming the highly stochastic nucleation step in the formation of the nucleolus, through a seeding mechanism. In the absence of rDNA, the nucleolar proteins studied are able to form high-concentration assemblies. However, unlike the nucleolus, these assemblies are highly variable in number, location, and time at which they form. In addition, quantitative study of the changes in the nucleoplasmic concentration and distribution of these nucleolar proteins in the wild-type embryos is consistent with the role of rRNA in seeding the nucleolus formation (Falahati, 2016).

    Formation of membrane-less organelles requires coordinated recruitment of tens to hundreds of different macromolecules. Several features of the formation of such membrane-less organelles including P granules, centrosomes, and nucleolus are consistent with a phase transition model. However, phase separation processes are often limited by the initial nucleation event that is highly stochastic. This study provides evidence that nucleolus formation in the wild-type embryos is not a highly variable nucleation-limited process. Rather, the transcription of rDNA renders the assembly process a well-controlled, growth-limited process. This accounts for the spatiotemporal precision of nucleolus formation observed in Drosophila embryos. In the absence of rDNA, the emergence of high-concentration assemblies of the nucleolar components reduces to a nucleation-limited process with high spatiotemporal variability. Also, apparent supersaturated levels of fibrillarin and RNA pol I prior to nucleolus formation at nuclear cycle (n.c.) 13 are consistent with the presence of a nucleation-limited process (Falahati, 2016).

    Several observations strongly suggest that pre-rRNA is the key player in seeding nucleolus formation. In all cases where nucleolus formation has been studied in metazoa, recruitment of nucleolar proteins occurs in the presence of unprocessed pre-rRNA. The current results show that the transcription of rDNA precedes the large-scale recruitment of the nucleolar proteins in Drosophila embryos. A similar chronological order has been observed for mouse embryos. X. laevis embryos drive nucleolus formation prior to any detectable transcription by RNA polymerase I by loading eggs with maternally provided pre-rRNA. Likewise when nucleoli re-assemble after mitosis in mammalian tissue culture cells, pre-rRNA transcribed in the previous interphase is present. Interestingly, the assembly of a synthetic nucleolus on an exogenous rDNA gene also requires transcription, and reducing transcription by RNA pol I can abolish nucleolus assembly in C. elegans, or delay the formation of the nucleolus in Drosophila embryos. Therefore, pre-rRNA seems to play a pivotal role in seeding the assembly of the nucleolar proteins (Falahati, 2016).

    The presence of local inhomogeneities throughout the nucleoplasm during early n.c. 14 and their absence at n.c. 13 is consistent with the role of pre-rRNA in nucleating the assemblies. The amount of pre-rRNA at n.c. 13 is ~30 times higher than n.c. 12, and a fraction of these transcripts may be carried over in the nucleus until the next cycle. Therefore, far more rRNA transcripts are present at the beginning of n.c. 14 compared to n.c. 13. These 'leftover' transcripts located throughout the nucleoplasm at n.c. 14 can seed the formation of assemblies ubiquitously in the nucleoplasm. Upon progression of interphase though, these local inhomogeneities are replaced by only two prominent assemblies located at nucleolus organizer regions (NORs). A subset of such small inhomogeneities disassemble during this time which could be due to the turnover of the seeding rRNA transcripts into ribosomes. Alternatively, in a process known as Ostwald ripening, reactivation of transcription can result in accumulation of further rRNA transcripts at NORs, which can in turn recruit more nucleolar components and destabilize smaller assemblies (Falahati, 2016).

    These data on fibrillarin and RNA pol I identify intriguing differences in their localization patterns during early cleavage cycles. While fibrillarin localizes to the nucleus during interphase and is excluded from the nuclei undergoing mitosis, RNA pol I has a peak of concentration with the onset of mitosis in these early nuclear cycles. Starting from n.c. 13 when the nucleolus forms, the dynamics of these two proteins overlap, and RNA pol I becomes enriched in the nuclei during interphase. However, although both fibrillarin and RNA pol I localize to the nucleolus, their involvement in the phase separation process appears to be different. After the nucleolus formation at n.c.13, the nucleoplasmic concentration of fibrillarin reaches a constant level, critical concentration (Ccr), which is indicative of a phase separation occurring and reaching equilibrium. In contrast, RNA pol I does not reach an equilibrium concentration, and its nucleoplasmic levels increase toward the end of n.c. 13. In addition, the localization of RNA pol I to high-concentration assemblies of nucleolar proteins (HANPs) relative to the nucleolus is much weaker than fibrillarin. These suggest that RNA pol I may not be a structural component of nucleolar phase separation but instead may be recruited by other factors (Falahati, 2016).

    Although the changing concentration of certain critical components and posttranscriptional modifications like phosphorylation may play broad roles in timing the formation of the nucleolus, the data suggest that seeding by rRNA may be the main mechanism regulating its temporal precision. A detectable nucleolus is not necessary for the initial transcription of rDNA. In mutants that lack rDNA, however, although fibrillarin and RNA pol I can still form high-concentration assemblies, the assembly process becomes variable and delayed. A similar loss in precision is observed when RNA pol-I-dependent transcription is reduced in embryos possessing normal rDNA, suggesting that it is the accumulation of rRNA transcripts, rather than the presence of the DNA itself, that drives nucleolar formation. By seeding the phase transition, this rRNA appears to dictate the spatiotemporal precision in the assembly, thereby turning an otherwise stochastic nucleation-limited process into a growth-limited event whose occurrence can be coordinated with other aspects of development (Falahati, 2016).

    tRNA Fragments Populations Analysis in Mutants Affecting tRNAs Processing and tRNA Methylation

    tRNA fragments (tRFs) are a class of small non-coding RNAs (sncRNAs) derived from tRNAs. tRFs are highly abundant in many cell types including stem cells and cancer cells, and are found in all domains of life. Beyond translation control, tRFs have several functions ranging from transposon silencing to cell proliferation control. However, the analysis of tRFs presents specific challenges and their biogenesis is not well understood. They are very heterogeneous and highly modified by numerous post-transcriptional modifications. This study describes a bioinformatic pipeline (tRFs-Galaxy) to study tRFs populations and shed light onto tRNA fragments biogenesis in Drosophila melanogaster. Indeed, small RNAs Illumina sequencing datasets were used that were extracted from wild type and mutant ovaries affecting two different highly conserved steps of tRNA biogenesis: 5'pre-tRNA processing (RNase-P subunit Rpp30) and tRNA 2'-O-methylation (dTrm7_34 and dTrm7_32). Using this pipeline, it was shown how defects in tRNA biogenesis affect nuclear and mitochondrial tRFs populations and other small non-coding RNAs biogenesis, such as small nucleolar RNAs (snoRNAs). This tRF analysis workflow will advance the current understanding of tRFs biogenesis, which is crucial to better comprehend tRFs roles and their implication in human pathology (Molla-Herman, 2020).

    Mutations in Drosophila tRNA processing factors cause phenotypes similar to Pontocerebellar Hypoplasia

    Mature transfer (t)RNAs are generated by multiple RNA processing events, which can include the excision of intervening sequences. The tRNA splicing endonuclease (TSEN) complex is responsible for cleaving these intron-containing pre-tRNA transcripts. In humans, TSEN copurifies with CLP1, an RNA kinase. Despite extensive work on CLP1, its in vivo connection to tRNA splicing remains unclear. Interestingly, mutations in CLP1 or TSEN genes cause neurological diseases in humans that are collectively termed Pontocerebellar Hypoplasia (PCH). In mice, loss of Clp1 kinase activity results in premature death, microcephaly and progressive loss of motor function. To determine if similar phenotypes are observed in Drosophila, this study characterized mutations in crowded-by-cid (cbc), the CLP1 ortholog, as well as in the fly ortholog of human TSEN54. Analyses of organismal viability, larval locomotion and brain size revealed that mutations in both cbc and Tsen54 phenocopy those in mammals in several details. In addition to an overall reduction in brain lobe size, increased cell death was found in mutant larval brains. Ubiquitous or tissue-specific knockdown of cbc in neurons and muscles reduced viability and locomotor function. These findings indicate that PCH can be successfully modeled in a genetically-tractable invertebrate (Schmidt, 2022).

    Structural and Genetic Determinants of Convergence in the Drosophila tRNA Structure-Function Map

    The evolution of tRNA multigene families remains poorly understood, exhibiting unusual phenomena such as functional conversions of tRNA genes through anticodon shift substitutions. This study has improved FlyBase tRNA gene annotations from twelve Drosophila species, incorporating previously identified ortholog sets to compare substitution rates across tRNA bodies at single-site and base-pair resolution. All rapidly evolving sites fell within the same metal ion-binding pocket that lies at the interface of the two major stacked helical domains. A tRNA Structure-Function Mapper (tSFM) method was applied independently to each Drosophila species and one outgroup species Musca domestica; although predicted tRNA structure-function maps are generally highly conserved in flies, one tRNA Class-Informative Feature (CIF) within the rapidly evolving ion-binding pocket-Cytosine 17 (C17), ancestrally informative for lysylation identity, independently gained asparaginylation identity and substituted in parallel across tRNA(Asn) paralogs at least once, possibly multiple times, during evolution of the genus. In D. melanogaster, most tRNA(Lys) and tRNA(Asn) genes are co-arrayed in one large heterologous gene cluster, suggesting that heterologous gene conversion as well as structural similarities of tRNA-binding interfaces in the closely related asparaginyl-tRNA synthetase (AsnRS) and lysyl-tRNA synthetase (LysRS) proteins may have played a role in these changes. A previously identified Asn-to-Lys anticodon shift substitution in D. ananassae may have arisen to compensate for the convergent and parallel gains of C17 in tRNA(Asn) paralogs in that lineage. These results underscore the functional and evolutionary relevance of the tRNA structure-function map predictions and illuminate multiple genomic and structural factors contributing to rapid, parallel and compensatory evolution of tRNA multigene families (Phillips, 2021).

    Effects of codon usage on gene expression are promoter context dependent

    Codon usage bias is a universal feature of all genomes. Although codon usage has been shown to regulate mRNA and protein levels by influencing mRNA decay and transcription in eukaryotes, little or no genome-wide correlations between codon usage and mRNA levels are detected in mammalian cells, raising doubt on the significance of codon usage effect on gene expression. This study shows that gene-specific regulation reduces the genome-wide codon usage and mRNA correlations: Constitutively expressed genes exhibit much higher genome-wide correlations than differentially expressed genes from fungi to human cells. Using Drosophila S2 cells as a model system, this study showed that the effect of codon usage on mRNA expression level is promoter-dependent. Regions downstream of the core promoters of differentially expressed genes can repress the codon usage effects on mRNA expression. An element in the Hsp70 promoter was identified to be necessary and sufficient for this inhibitory effect. The promoter-dependent codon usage effects on mRNA levels are regulated at the transcriptional level through modulation of histone modifications, nucleosome densities and premature termination. Together, these results demonstrate that promoters play a major role in determining whether codon usage influences gene expression and further establish the transcription-dependent codon usage effects on gene expression (Yang, 2021).

    Distinct responses to rare codons in select Drosophila tissues

    Codon usage bias has long been appreciated to influence protein production. Yet, relatively few studies have analyzed the impacts of codon usage on tissue-specific mRNA and protein expression. This study used codon-modified reporters to perform an organism-wide screen in Drosophila melanogaster for distinct tissue responses to codon usage bias. These reporters reveal a cliff-like decline of protein expression near the limit of rare codon usage in endogenously expressed Drosophila genes. Near the edge of this limit, however,the testis and brain are uniquely capable of expressing rare codon-enriched reporters. A new metric of tissue-specific codon usage, the tissue-apparent Codon Adaptation Index (taCAI), to reveal a conserved enrichment for rare codon usage in the endogenously expressed genes of both Drosophila and human testis. A role was further demonstrate for rare codons in an evolutionarily young testis-specific gene, RpL10Aa. Optimizing RpL10Aa codons disrupts female fertility. This work highlights distinct responses to rarely used codons in select tissues, revealing a critical role for codon bias in tissue biology (Allen, 2022).

    Tissue-Specific Ribosome Profiling in Drosophila

    Robust mechanisms exist that serve to dynamically regulate the translation of mRNA into proteins across heterogeneous tissues. These processes ensure timely generation of proteins in quantities that scale with the demands of specific cell types. Importantly, this translational regulation occurs with spatiotemporal precision and is capable of recalibration as conditions change. Aberrant regulation of translation contributes to and exacerbates a wide range of diseases. Although dynamic control of translation is an essential and fundamental process shared by organisms, specific tissues and cell types can be differentially impacted by circumstances that challenge and impair basal translation, highlighting the heterogeneous nature of translational regulation. To understand how translation is differentially regulated during changing environments and across specific cells and tissues, methods capable of profiling translation in specific tissues and cells are crucial. This paper describes a method for profiling genome-wide translation in specific tissues or cell types in Drosophila melanogaster, in which ribosome affinity purification is combined with ribosome profiling to enable a simplified protocol for robust analysis of translation in specific tissues (Chen, 2021).

    Cell competition removes segmental aneuploid cells from Drosophila imaginal disc-derived tissues based on ribosomal protein gene dose

    Aneuploidy causes birth defects and miscarriages, occurs in nearly all cancers and is a hallmark of aging. Individual aneuploid cells can be eliminated from developing tissues by unknown mechanisms. Cells with ribosomal protein (Rp) gene mutations are also eliminated, by cell competition with normal cells. Because Rp genes are spread across the genome, their copy number is a potential marker for aneuploidy. Elimination of imaginal disc cells with irradiation-induced genome damage often required cell competition genes. Segmentally aneuploid cells derived from targeted chromosome excisions were eliminated by the RpS12-Xrp1 cell competition pathway if they differed from neighboring cells in Rp gene dose, whereas cells with normal doses of the Rp and eIF2γ genes survived and differentiated adult tissues. Thus, cell competition, triggered by differences in Rp gene dose between cells, is a significant mechanism for the elimination of aneuploid somatic cells, likely to contribute to preventing cancer (Ji, 2021).

    A neural m(6)A/Ythdf pathway is required for learning and memory in Drosophila

    Epitranscriptomic modifications can impact behavior. This study used Drosophila melanogaster to study N(6)-methyladenosine (m6A), the most abundant modification of mRNA. Proteomic and functional analyses confirm its nuclear (Ythdc1) and cytoplasmic (Ythdf) YTH domain proteins as major m(6)A binders. Assays of short term memory in m6A mutants reveal neural-autonomous requirements of m6A writers working via Ythdf, but not Ythdc1. Furthermore, m6A/Ythdf operate specifically via the mushroom body, the center for associative learning. m6A from wild-type and Mettl3 mutant heads was mapped, allowing robust discrimination of Mettl3-dependent m6A sites that are highly enriched in 5' UTRs. Genomic analyses indicate that Drosophila m6A is preferentially deposited on genes with low translational efficiency and that m6A does not affect RNA stability. Nevertheless, functional tests indicate a role for m6A/Ythdf in translational activation. Altogether, this molecular genetic analyses and tissue-specific m(6)A maps reveal selective behavioral and regulatory defects for the Drosophila Mettl3/Ythdf pathway (Kan, 2021).

    In an effort to identify factors that regulate memory, the 'epitranscriptome', the multitude of modified bases that exist beyond the standard RNA nucleotides was of primary interest. The most abundant and most well-studied internal modification of mRNA is N6-methyladenosine (m6A). While m6A has been recognized to exist in mRNA since the 1970s, its functional significance has been elusive until recently. Key advances included (1) techniques to determine individual methylated transcripts, and in particular specific methylated sites, and (2) mechanistic knowledge of factors that install m6A ('writers') and mediate their regulatory consequences ('readers'). The core m6A methytransferase complex acting on mRNA consists of the Mettl3 catalytic subunit and its heterodimeric partner Mettl14. These associate with other proteins that play broader roles in splicing, mRNA processing and gene regulation, but that are collectively required for normal accumulation of m6A (Kan, 2021).

    Downstream of the writers, various readers are sensitive to the presence or absence of m6A, and thereby mediate differential regulation by this mRNA modification. The most well-characterized readers contain YTH domains, for which atomic insights reveal how a tryptophan-lined pocket selectively binds methylated adenosine and discriminates against unmodified adenosine. In addition, some other proteins were proposed as m6A readers, based primarily on preferential in vitro binding to methylated vs. unmethylated RNA probes. In mammals, m6A readers confer diverse regulatory fates onto modified transcripts, including splicing and nuclear export via the nuclear reader YTHDC1, and RNA decay via cytoplasmic readers Ythdf1-3. Certain YTHDF and YTHDC2 were also reported to regulate translation via m6A under specific contexts (Kan, 2021).

    Despite intense efforts into m6A mechanisms and genomics using cell systems, genetic analyses of the m6A pathway have only begun in earnest in the past few years, mostly in vertebrates. Notably, many studies have revealed sensitivity of the mammalian nervous system to manipulation of m6A factors. Mutants in writer (Mettl3 and Mettl14), reader (primarily ythdf1), and eraser (FTO) factors have collectively been shown to exhibit aberrant neurogenesis and/or differentiation. Moreover, these mutants impact neural function and behavior, including during learning and memory paradigms. Overall, these observations may reflect some heightened requirements for m6A in neurons, perhaps owing to their unique architectures and/or regulatory needs (Kan, 2021).

    Among invertebrates, Caenorhabditis elegans lacks the core m6A machinery, but the presence of a Drosophila ortholog of Mettl3 (originally referred to as IME4) opened this model system. While mammals contain multiple members of both nuclear and cytoplasmic YTH domain families, the fly system is simplified in containing only one of each, referred to as Ythdc1 (YT-521B or CG12076) and Ythdf (CG6422), respectively. Recently, several labs established biochemical, genetic, and genomic foundations for studying the m6A pathway in Drosophila. Surprisingly, these studies jointly reported that knockout of all core m6A writer factors in Drosophila is compatible with viability and largely normal exterior patterning. Nevertheless, mutants of Mettl3, Mettl14, and Ythdc1 exhibit a common suite of molecular and phenotypic defects. These include several behavioral abnormalities as well as aberrant splicing of the master female sex determination factor Sex lethal (Sxl). The suite of locomotor and postural defects in Drosophila m6A mutants was again consistent with the notion that the nervous system might be especially sensitive (Kan, 2021).

    However, a major open question from these studies concerns the regulatory and biological roles of the sole Drosophila cytoplasmic YTH factor, Ythdf. In contrast to other core m6A factors, overt defects were not previously observe in the Ythdf mutants, nor did it seem to exhibit robust m6A-specific binding activity. This study used proteomic analyses to reveal Ythdc1 and Ythdf as the major m6A-specific binders in Drosophila, and focused biochemical tests show that Ythdf prefers a distinct sequence context than tested previously. Hypothesizing that the nervous system might exhibit particular needs for the m6A pathway, a paradigm of aversive olfactory conditioning was used to reveal an m6A/Ythdf pathway that is important for STM in older animals. These phenotypic data were complemented with high-stringency maps of methylated transcript sites from fly heads, and it was shown that m6A does not impact transcript levels but is preferentially deposited on genes with lower translational efficiency. Nevertheless, functional tests reveal that Mettl3/Ythdf can enhance protein output. Finally, this study showed that physiological Mettl3/Ythdf function is explicitly required within mushroom body neurons to mediate normal conditioned odor memory during aging. Overall, this study provides insights into the in vivo function of this mRNA modification pathway for normal behavior (Kan, 2021).

    Despite tremendous interests in the regulatory utilities and biological impacts of mRNA methylation, there has been relatively little study from invertebrate models. Given that the m6A pathway seems to have been lost from C. elegans, Drosophila is an ideal choice for this. Since the initial report that Mettl3 mutants affect germline development, it has been shown that Drosophila harbors an m6A pathway similar to that of mammals, but simplified in that it has a single nuclear and cytoplasmic YTH reader. Nevertheless, Drosophila has proven to be a useful system to discover and characterize novel m6A factors. Expanding the breadth of model systems can increase appreciation for the utilization and impact of this regulatory modification (Kan, 2021).

    It is widely presumed, based on mammalian profiling, that metazoan m6A is enriched at stop codons and 3' UTRs. However, high-resolution maps indicate that 5' UTRs are by far the dominant location of methylation in mature Drosophila mRNAs. Although further study is required, many of these m6A 5' UTR regions coincide with previous embryo miCLIP data, while other miCLIP CIMs calls located in other transcript regions proved usually not to be Mettl3-dependent. Thus, the current data indicate a fundamentally different distribution of m6A in Drosophila mRNAs compared to mammals (Kan, 2021).

    While mammalian m6A clearly elicits a diversity of regulatory consequences, depending on genic and cellular context and other factors, a dominant role is to induce target decay through one or more cytoplasmic YTH readers. This harkens back to classic observations that m6A is correlated with preferential transcript decay, and more recent data that loss of m6A writers or cytoplasmic YTH readers results in directional upregulation of m6A targets. However, several lines of study did not yield convincing evidence for a broad role for the Drosophila m6A pathway in target decay. Instead, the dominant localization of m6A in fly 5' UTRs is suggestive of a possible impact in translational regulation. Genomic and genetic evidence support the notion that m6A is preferentially deposited in transcripts with overall lower translational efficiency, but that m6A/Ythdf may potentiate translation. However, it is possible to rationalize a regulatory basis for these apparently opposite trends, if the greater modulatory window of poorly translated loci is utilized for preferred targeting by m6A/Ythdf (Kan, 2021).

    As is generally the case for mammalian m6A, the choice of how appropriate targets are selected for modification, and which gene regions are preferentially methylated, remains to be understood. The minimal context for m6A is insufficient to explain targeting, and as mentioned also seems to be different between Drosophila and vertebrates. A further challenge for the future will be to elucidate a mechanism for m6A/Ythdf-mediated translational regulation. This will reveal possible similarities or distinctions with the multiple strategies proposed for translational regulation by mammalian m6A, which include both cap-independent translation via 5' UTRs during the heat-shock response via eIF383 or YTHDF236; cap-dependent mRNA circularization via Mettl3-eIF3H84; and activity-dependent translational activation in neurons (Kan, 2021).

    Recent studies have highlighted neuronal functions of mammalian m6A pathway factors. There is a growing appreciation that mouse mutants of multiple components in the m6A RNA-modification machinery affect learning and memory. This study provides substantial evidence that, in Drosophila, neural m6A is critical for STM. This study specifically focused on STM as this paradigm has been extensively characterized in Drosophila. Mouse studies have almost exclusively examined effects on LTM, and these two memory phases are mechanistically distinct. One main distinction is that LTM requires protein synthesis after training, while STM does not. So, while direct comparisons between the two systems are not possible, it is nevertheless instructive to consider the parallels and distinctions of how m6A facilitates normal memory function in these species. This is especially relevant given that both mouse and fly central nervous systems require a cytoplasmic YTH factor for memory (Kan, 2021).

    In mice, the m6A writer Mettl3 enhance long-term memory consolidation, potentially by promoting the expression of genes such as Arc, c-Fos and others. Another study found that Mettl14 is required for LTM formation and neuronal excitability. Conversely, knockdown of the m6A demethylase FTO in the mouse prefrontal cortex resulted in enhanced memory consolidation. Amongst mammalian YTH m6A readers, YTHDF1 was shown to induce the translation of m6A-marked mRNA specifically in stimulated neurons. In cultured hippocampal neurons, levels of YTHDF1 in the PSD fraction were found to increase by ~30% following KCl treatment. This suggests that YTHDF1 concentration at the synapse could be critical for regulating the expression levels of proteins (such as CaMK2a) involved in synaptic plasticity. Taken together, these studies suggest the m6A pathway is a crucial mechanism of LTM consolidation in mammals that optimizes animal behavioral responses (Kan, 2021).

    Of note, the genetics and sample sizes possible in Drosophila permit comprehensive, stringent, and anatomically resolved analyses. Thus, this study systematically analyzed all writer and reader factors, and revealed a notable functional segregation, suggesting that the cytoplasmic reader Ythdf is a major effector of Mettl3/Mettl14 m6A in memory. Given that Ythdf mutants otherwise exhibit few overt developmental or behavioral defects in normal or sensitized backgrounds (while Ythdc1 mutants generally phenocopy Mettl3/Mettl14 mutants) its role in STM is a surprising insight into the contribution of Ythdf to a critical adaptive function. Moreover, the spatial requirements of m6A for STM can be pinpointed by showing that (1) neuronal-specific and MB-specific depletion of Mettl3/Ythdf can induce defective STM, and (2) neuronal and MB-specific restoration of Mettl3 or Ythdf to their respective whole-animal knockouts restores normal STM. Moreover, the fact that Ythdf gain-of-function in the MB can also disrupt STM, but does not generally alter other aspects of development or behavior, points to a homeostatic role of m6A regulation in Drosophila learning and memory (Kan, 2021).

    It was observed that STM defects in fly m6A mutants are age-dependent, which has not been reported in mammals. Although many physiological capacities decline with life history, the observed STM defects seem to be decoupled from other age-related phenotypes, since mutation of Ythdf or neural overexpression of Ythdf can interfere with STM but does not substantially impact lifespan or locomotion. In this regard, Mettl3 and Ythdf are different from classical memory genes such as rutabaga because STM impairment in m6A mutants was absent in young flies and only became apparent with progressing age (Kan, 2021).

    One interpretation is that there is a cumulative effect of deregulated m6A networks that has a progressive impact specific to mushroom-body neurons. To gain further mechanistic insights, future studies will need to examine age-related changes in gene expression and/or translation, in a cell-specific manner. It remains to be seen whether specific deregulated targets downstream of Ythdf have large individual effects, or whether the STM deficits arise from myriad small effects on translation. Ythdf-CLIP and ribosome profiling from the CNS may prove useful to decipher this. Assuming that loss of translational enhancement of m6A/Ythdf targets mediates STM defects, one possibility, to be explored in future studies, is that some targets may already be known from prior genetic studies of memory (Kan, 2021).

    Ribosome heterogeneity in Drosophila melanogaster gonads through paralog-switching

    Ribosomes have long been thought of as homogeneous macromolecular machines, but recent evidence suggests they are heterogeneous and could be specialised to regulate translation. This study characterised ribosomal protein heterogeneity across 4 tissues of Drosophila melanogaster. Testes and ovaries were found to contain the most heterogeneous ribosome populations, which occurs through a combination of paralog-enrichment and paralog-switching. Structures were solved of ribosomes purified from in vivo tissues by cryo-EM, revealing differences in precise ribosomal arrangement for testis and ovary 80S ribosomes. Differences in the amino acid composition of paralog pairs and their localisation on the ribosome exterior indicate paralog-switching could alter the ribosome surface, enabling different proteins to regulate translation. One testis-specific paralog-switching pair is also found in humans, suggesting this is a conserved site of ribosome heterogeneity. Overall, this work leads to a proposal that mRNA translation might be regulated in the gonads through ribosome heterogeneity, providing a potential means of ribosome specialisation (Hopes, 2021).

    Unconventional translation initiation factor EIF2A is required for Drosophila spermatogenesis

    EIF2A is an unconventional translation factor required for initiation of protein synthesis from non-AUG codons from a variety of transcripts, including oncogenes and stress related transcripts in mammalian cells. Its function in multicellular organisms has not been reported. This study identified and characterize mutant alleles of the CG7414 gene, which encodes the Drosophila EIF2A ortholog. CG7414 was shown to undergo sex-specific splicing that regulates its male-specific expression. A Mi{Mic} transposon insertion was identified that disrupts the coding regions of all predicted isoforms and is a likely null allele, and a PBac transposon insertion into an intron was identified that is a hypomorph. The Mi{Mic} allele is homozygous lethal, while the viable progeny from the hypomorphic PiggyBac allele are male sterile and female fertile. In dEIF2A mutant flies, sperm failed to individualize due to defects in F-actin cones and failure to form and maintain cystic bulges, ultimately leading to sterility. These results demonstrate that EIF2A is essential in a multicellular organism, both for normal development and spermatogenesis, and provide an entrée into the elucidation of the role of EIF2A and unconventional translation in vivo (Lowe, 2021).

    Somatic genetic rescue of a germline ribosome assembly defect

    Indirect somatic genetic rescue (SGR) of a germline mutation is thought to be rare in inherited Mendelian disorders. This study established that acquired mutations in the EIF6 gene are a frequent mechanism of SGR in Shwachman-Diamond syndrome (SDS), a leukemia predisposition disorder caused by a germline defect in ribosome assembly. Biallelic mutations in the SBDS or EFL1 genes in SDS impair release of the anti-association factor eIF6 (see Drosophila eIF6) from the 60S ribosomal subunit, a key step in the translational activation of ribosomes. This study identified diverse mosaic somatic genetic events (point mutations, interstitial deletion, reciprocal chromosomal translocation) in SDS hematopoietic cells that reduce eIF6 expression or disrupt its interaction with the 60S subunit, thereby conferring a selective advantage over non-modified cells. SDS-related somatic EIF6 missense mutations that reduce eIF6 dosage or eIF6 binding to the 60S subunit suppress the defects in ribosome assembly and protein synthesis across multiple SBDS-deficient species including yeast, Dictyostelium and Drosophila. These data suggest that SGR is a universal phenomenon that may influence the clinical evolution of diverse Mendelian disorders and support eIF6 suppressor mimics as a therapeutic strategy in SDS (Tan, 2021).

    The DEAD-Box Protein Rok1 Coordinates Ribosomal RNA Processing in Association with Rrp5 in Drosophila

    Ribosome biogenesis and processing involve the coordinated action of many components. The DEAD-box RNA helicase (Rok1) is essential for cell viability, and the depletion of Rok1 inhibits pre-rRNA processing. Previous research on Rok1 and its cofactor Rrp5 has been performed primarily in yeast. Few functional studies have been performed in complex multicellular eukaryotes. This study used a combination of genetics and developmental experiments to show that Rok1 and Rrp5, which localize to the nucleolus, play key roles in the pre-rRNA processing and ribosome assembly in D. melanogaster. The accumulation of pre-rRNAs caused by Rok1 depletion can result in developmental defects. The loss of Rok1 enlarged the nucleolus and led to stalled ribosome assembly and pre-rRNA processing in the nucleolus, thereby blocking rRNA maturation and exacerbating the inhibition of mitosis in the brain. This study also discovered that rrp5(4-2/4-2) displayed significantly increased ITS1 signaling by fluorescence in situ hybridization, and a reduction in ITS2. Rrp5 signal was highly enriched in the core of the nucleolus in the rok1(167/167) mutant, suggesting that Rok1 is required for the accurate cellular localization of Rrp5 in the nucleolus. This study has thus uncovered functions of Rok1 that reveal important implications for ribosome processing in eukaryotes (Chen, 2022).

    The mRNA decapping protein 2 (DCP2) is a major regulator of developmental events in Drosophila-insights from expression paradigms

    The Drosophila genome codes for two decapping proteins, DCP1 and DCP2, out of which DCP2 is the active decapping enzyme. The present endeavour explores the endogenous promoter firing, transcript and protein expression of DCP2 in Drosophila wherein, besides a ubiquitous expression across development, an active expression paradigm during dorsal closure and a plausible moonlighting expression in the Corazonin neurons of the larval brain were identified. It was also demonstrated that the ablation of DCP2 leads to embryonic lethality and defects in vital morphogenetic processes whereas a knockdown of DCP2 in the Corazonin neurons reduces the sensitivity to ethanol in adults, thereby ascribing novel regulatory roles to DCP2. These findings unravel novel putative roles for DCP2 and identify it as a candidate for studies on the regulated interplay of essential molecules during early development in Drosophila, nay the living world (Kunar, 2021).

    Ribbon boosts ribosomal protein gene expression to coordinate organ form and function

    Cell growth is well defined for late (postembryonic) stages of development, but evidence for early (embryonic) cell growth during postmitotic morphogenesis is limited. This study reports early cell growth as a key characteristic of tubulogenesis in the Drosophila embryonic salivary gland (SG) and trachea. A BTB/POZ domain nuclear factor, Ribbon (Rib), mediates this early cell growth. Rib binds the transcription start site of nearly every SG-expressed ribosomal protein gene (RPG) and is required for full expression of all RPGs tested. Rib binding to RPG promoters in vitro is weak and not sequence specific, suggesting that specificity is achieved through cofactor interactions. Accordingly, this study demonstrates Rib's ability to physically interact with each of the three known regulators of RPG transcription. Surprisingly, Rib-dependent early cell growth in another tubular organ, the embryonic trachea, is not mediated by direct RPG transcription. These findings support a model of early cell growth customized by transcriptional regulatory networks to coordinate organ form and function (Loganathan, 2022).

    Puromycin Labeling Coupled with Proximity Ligation Assays to Define Sites of mRNA Translation in Drosophila Embryos and Human Cells

    Genetic mutations, whether they occur within protein-coding or noncoding regions of the genome, can affect various aspects of gene expression by influencing the complex network of intra- and intermolecular interactions that occur between cellular nucleic acids and proteins. One aspect of gene expression control that can be impacted is the intracellular trafficking and translation of mRNA molecules. To study the occurrence and dynamics of translational regulation, researchers have developed approaches such as genome-wide ribosome profiling and artificial reporters that enable single molecule imaging. In this study, A complementary and optimized approach that combines puromycin labeling with a proximity ligation assay (Puro-PLA) to define sites of translation of specific mRNAs in tissues or cells. This method can be used to study the mechanisms driving the translation of select mRNAs and to access the impact of genetic mutations on local protein synthesis. This approach involves the treatment of cell or tissue specimens with puromycin to label nascently translated peptides, rapid fixation, followed by immunolabeling with appropriate primary and secondary antibodies coupled to PLA oligonucleotide probes, ligation, amplification, and signal detection via fluorescence microscopy. Puro-PLA can be performed at small scale in individual tubes or in chambered slides, or in a high-throughput setup with 96-well plate, for both in situ and in vitro experimentation (Chin, 2021).

    The Drosophila ribosome protein S5 paralog RpS5b promotes germ cell and follicle cell differentiation during oogenesis

    Emerging evidence suggests that ribosome heterogeneity may have important functional consequences in the translation of specific mRNAs within different cell types and under various conditions. Ribosome heterogeneity comes in many forms, including post-translational modification of ribosome proteins (RPs), absence of specific RPs and inclusion of different RP paralogs. The Drosophila genome encodes two RpS5 paralogs: RpS5a and RpS5b. While RpS5a is ubiquitously expressed, RpS5b exhibits enriched expression in the reproductive system. Deletion of RpS5b results in female sterility marked by developmental arrest of egg chambers at stages 7-8, disruption of vitellogenesis and posterior follicle cell (PFC) hyperplasia. While transgenic rescue experiments suggest functional redundancy between RpS5a and RpS5b, molecular, biochemical and ribo-seq experiments indicate that RpS5b mutants display increased rRNA transcription and RP production, accompanied by increased protein synthesis. Loss of RpS5b results in microtubule-based defects and in mislocalization of Delta and Mindbomb1, leading to failure of Notch pathway activation in PFCs. The results indicate that germ cell-specific expression of RpS5b promotes proper egg chamber development by ensuring the homeostasis of functional ribosomes (Jang, 2021).

    The Disease-Associated Proteins Drosophila Nab2 and Ataxin-2 Interact with Shared RNAs and Coregulate Neuronal Morphology

    Nab2 encodes the Drosophila melanogaster member of a conserved family of zinc finger polyadenosine RNA-binding proteins (RBPs) linked to multiple steps in post-transcriptional regulation. Mutation of the Nab2 human ortholog ZC3H14 gives rise to an autosomal recessive intellectual disability but understanding of Nab2/ZC3H14 function in metazoan nervous systems is limited, in part because no comprehensive identification of metazoan Nab2/ZC3H14-associated RNA transcripts has yet been conducted. Moreover, many Nab2/ZC3H14 functional protein partnerships remain unidentified. This study presents evidence that Nab2 genetically interacts with Ataxin-2 (Atx2), which encodes a neuronal translational regulator, and that these factors coordinately regulate neuronal morphology, circadian behavior, and adult viability. The first high-throughput identifications are presented of Nab2- and Atx2-associated RNAs in Drosophila brain neurons using RNA immunoprecipitation-sequencing (RIP-Seq). Critically, the RNA interactomes of each RBP overlap, and Nab2 exhibits high specificity in its RNA associations in neurons in vivo, associating with a small fraction of all polyadenylated RNAs. The identities of shared associated transcripts (e.g., drk, me31B, stai) and of transcripts specific to Nab2 or Atx2 (e.g., Arpc2 and tea) promise insight into neuronal functions of, and genetic interactions between, each RBP. Consistent with prior biochemical studies, Nab2-associated neuronal RNAs are overrepresented for internal A-rich motifs, suggesting these sequences may partially mediate Nab2 target selection. These data support a model where Nab2 functionally opposes Atx2 in neurons, demonstrate Nab2 shares associated neuronal RNAs with Atx2, and reveal Drosophila Nab2 associates with a more specific subset of polyadenylated mRNAs than its polyadenosine affinity alone may suggest (Rounds, 2021).

    The Transcriptional Response to Oxidative Stress is Independent of Stress-Granule Formation

    Cells respond to stress with translational arrest, robust transcriptional changes, and transcription-independent formation of mRNP assemblies termed stress granules (SGs). Despite considerable interest in the role of SGs in oxidative, unfolded-protein and viral stress responses, whether and how SGs contribute to stress-induced transcription has not been rigorously examined. To address this, this study characterized transcriptional changes in Drosophila S2 cells induced by acute oxidative-stress and assessed how these were altered under conditions that disrupted SG assembly. Oxidative stress for 3-hours predominantly resulted in induction or upregulation of stress-responsive mRNAs whose levels peaked during recovery after stress cessation. The stress-transcriptome is enriched in mRNAs coding for chaperones, including HSP70s, small heat shock proteins, glutathione transferases, and several non-coding RNAs. Oxidative stress also induced cytoplasmic SGs that disassembled 3-hours after stress cessation. As expected, RNAi-mediated knockdown of the conserved G3BP1/Rasputin protein inhibited SG assembly. However, this disruption had no significant effect on the stress-induced transcriptional response or stress-induced translational arrest. Thus, SG assembly and stress-induced gene expression alterations appear to be driven by distinctive signaling processes. It is suggested that while SG assembly represents a fast, transient mechanism, the transcriptional response enables a slower, longer-lasting mechanism for adaptation to and recovery from cell stress (Singh, 2022).

    Modulation of RNA stability regulates gene expression in two opposite ways: through buffering of RNA levels upon global perturbations and by supporting adapted differential expression

    The steady state levels of RNAs, often referred to as expression levels, result from a well-balanced combination of RNA transcription and decay. Alterations in RNA levels will therefore result from tight regulation of transcription rates, decay rates or both. This study explored the role of RNA stability in achieving balanced gene expression and present genome-wide RNA stabilities in Drosophila melanogaster male and female cells as well as male cells depleted of proteins essential for dosage compensation. Two distinct RNA-stability mediated responses were found to be involved in regulation of gene expression. The first of these responds to acute and global changes in transcription and thus counteracts potentially harmful gene mis-expression by shifting the RNA stability in the direction opposite to the transcriptional change. The second response enhances inter-individual differential gene expression by adjusting the RNA stability in the same direction as a transcriptional change. Both mechanisms are global, act on housekeeping as well as non-housekeeping genes and were observed in both flies and mammals. Additionally, this study showed that, in contrast to mammals, modulation of RNA stability does not detectably contribute to dosage compensation of the sex-chromosomes in D. melanogaster (Faucillion, 2022).

    Non-canonical initiation factors modulate repeat-associated non-AUG translation

    Repeat associated non-AUG (RAN) translation of expanded repeat mutation mRNA produces toxic peptides in neurons of patients suffering from neurodegenerative diseases. Recent findings indicate that RAN translation in diverse model systems is not inhibited by cellular stressors that impair global translation through phosphorylation of the alpha subunit of eIF2, the essential eukaryotic translation initiation factor that brings the initiator tRNA to the 40S ribosome. Using in vitro, cell-based, and Drosophila models, this study examined the role of alternative ternary complex factors that may function in place of eIF2, including eIF2A, eIF2D, DENR, and MCTS1. Among these factors, DENR knockdown had the greatest inhibitory effect on RAN translation of expanded GGGGCC and CGG repeat reporters, and its reduction improved survival of Drosophila expressing expanded GGGGCC repeats. Taken together, these data support a role for alternative initiation factors in RAN translation and suggest these may serve as novel therapeutic targets in neurodegenerative disease (Green, 2022).

    A Novel Mistranslating tRNA Model in Drosophila melanogaster has Diverse, Sexually Dimorphic Effects

    Transfer RNAs (tRNAs) are the adaptor molecules required for reading the genetic code and producing proteins. tRNA variants can lead to genome-wide mistranslation, the misincorporation of amino acids not specified by the standard genetic code into nascent proteins. While genome sequencing has identified putative mistranslating tRNA variants in human populations, little is known regarding how mistranslation affects multicellular organisms. This study created a multicellular model of mistranslation by integrating a serine tRNA variant that mistranslates serine for proline (tRNASerUGG, G26A) into the Drosophila melanogaster genome. Mistranslation was confirmed via mass spectrometry, and it was found that tRNASerUGG, G26A misincorporates serine for proline at a frequency of ~ 0.6% per codon. tRNASerUGG, G26A extends development time and decreases the number of flies that reach adulthood. While both sexes of adult flies containing tRNASerUGG, G26A present with morphological deformities and poor climbing performance, these effects are more pronounced in female flies and the impact on climbing performance is exacerbated by age. This model will enable studies into the synergistic effects of mistranslating tRNA variants and disease-causing alleles (Isaacson, 2022).

    CMTr mediated 2'-O-ribose methylation status of cap-adjacent nucleotides across animals

    Cap methyltransferases (CMTrs) O methylate the 2' position of the ribose (cOMe) of cap-adjacent nucleotides of animal, protist, and viral mRNAs. Animals generally have two CMTrs, whereas trypanosomes have three, and many viruses encode one in their genome. In the splice leader of mRNAs in trypanosomes, the first four nucleotides contain cOMe, but little is known about the status of cOMe in animals. This study showed that cOMe is prominently present on the first two cap-adjacent nucleotides with species- and tissue-specific variations in Caenorhabditis elegans, honeybees, zebrafish, mouse, and human cell lines. In contrast, Drosophila contains cOMe primarily on the first cap-adjacent nucleotide. De novo RoseTTA modeling of CMTrs reveals close similarities of the overall structure and near identity for the catalytic tetrad, and for cap and cofactor binding for human, Drosophila and C. elegans CMTrs. Although viral CMTrs maintain the overall structure and catalytic tetrad, they have diverged in cap and cofactor binding. Consistent with the structural similarity, both CMTrs from Drosophila and humans methylate the first cap-adjacent nucleotide of an AGU consensus start. Because the second nucleotide is also methylated upon heat stress in Drosophila, these findings argue for regulated cOMe important for gene expression regulation (Dix, 2022).

    eIF3j inhibits translation of a subset of circular RNAs in eukaryotic cells

    Increasing studies have revealed that a subset of circular RNAs (circRNAs) harbor an open reading frame and can act as protein-coding templates to generate functional proteins that are closely associated with multiple physiological and disease-relevant processes, and thus proper regulation of synthesis of these circRNA-derived proteins is a fundamental cellular process required for homeostasis maintenance. However, how circRNA translation initiation is coordinated by different trans-acting factors remains poorly understood. In particular, the impact of different eukaryotic translation initiation factors (eIFs) on circRNA translation and the physiological relevance of this distinct regulation have not yet been characterized. In this study, all 43 Drosophila eIFs were screened and it was revealed the conflicting functions of eIF3 subunits in the translational control of the translatable circRNA circSfl: eIF3 is indispensable for circSfl translation, while the eIF3-associated factor eIF3j is the most potent inhibitor. Mechanistically, the binding of eIF3j to circSfl promotes the disassociation of eIF3. The C-terminus of eIF3j and an RNA regulon within the circSfl untranslated region (UTR) are essential for the inhibitory effect of eIF3j. Moreover, the physiological relevance of eIF3j-mediated circSfl translation repression in response to heat shock was revealed. Finally, additional translatable circRNAs were identified to be similarly regulated in an eIF3j-dependent manner. Altogether, this study provides a significant insight into the field of cap-independent translational regulation and undiscovered functions of eIF3 (Song, 2022).

    Serotonergic neuron ribosomal proteins regulate the neuroendocrine control of Drosophila development

    The regulation of ribosome function is a conserved mechanism of growth control. While studies in single cell systems have defined how ribosomes contribute to cell growth, the mechanisms that link ribosome function to organismal growth are less clear. This study explored this issue using Drosophila Minutes, a class of heterozygous mutants for ribosomal proteins. These animals exhibit a delay in larval development caused by decreased production of the steroid hormone ecdysone, the main regulator of larval maturation. This developmental delay is not caused by decreases in either global ribosome numbers or translation rates. Instead, this study showed that they are due in part to loss of Rp function specifically in a subset of serotonin (5-HT) neurons that innervate the prothoracic gland to control ecdysone production. These effects do not occur due to altered protein synthesis or proteostasis, but that Minute animals have reduced expression of synaptotagmin, a synaptic vesicle protein, and that the Minute developmental delay can be partially reversed by overexpression of synaptic vesicle proteins in 5-HTergic cells. These results identify a 5-HT cell-specific role for ribosomal function in the neuroendocrine control of animal growth and development (Deliu, 2022).

    The Drosophila hnRNP F/H homolog Glorund recruits dFMRP to inhibit nanos translation elongation

    Translational control of maternal mRNAs generates spatial and temporal patterns of protein expression necessary to begin animal development. Translational repression of unlocalized nanos (nos) mRNA in late-stage Drosophila oocytes by the hnRNP F/H homolog, Glorund (Glo), is important for embryonic body patterning. While previous work has suggested that repression occurs at both the translation initiation and elongation phases, the molecular mechanism by which Glo regulates nos translation remains elusive. This study identified the Drosophila fragile X mental retardation protein, dFMRP, as a Glo interaction partner with links to the translational machinery. Using an oocyte-based in vitro translation system, it was confirmed that Glo regulates both initiation and elongation of a nos translational reporter and showed that dFMRP specifically represses translation elongation and promotes ribosome stalling. Furthermore, mutational analysis and in vivo and in vitro binding assays were combined to show that Glo's qRRM2 domain specifically and directly interacts with dFMRP. These findings suggest that Glo regulates nos translation elongation by recruiting dFMRP and that Glo's RNA-binding domains can also function as protein-protein interaction interfaces critical for its regulatory functions. Additionally, they reveal a mechanism for targeting dFMRP to specific transcripts (Peng, 2022).

    Extended disordered regions of ribosome-associated NAC proteins paralogs belong only to the germline in Drosophila melanogaster

    The nascent polypeptide-associated complex (NAC) consisting of α- and β-subunits is an essential ribosome-associated protein conserved in eukaryotes. NAC is a ubiquitously expressed co-translational regulator of nascent protein folding and sorting providing for homeostasis of cellular proteins. This study reports on discovering the germline-specific NACαβ paralogs (gNACs), whose β-subunits, non-distinguishable by ordinary immunodetection, are encoded by five highly homologous gene copies, while the α-subunit is encoded by a single αNAC gene. The gNAC expression is detected in the primordial embryonic and adult gonads via immunostaining. The germline-specific α and β subunits differ from the ubiquitously expressed paralogs by the extended intrinsically disordered regions (IDRs) acquired at the N- and C-termini of the coding regions, predicted to be phosphorylated. The presence of distinct phosphorylated isoforms of gNAC-β subunits is confirmed by comparing of their profiles by 2D-isoeletrofocusing resolution before and after phosphatase treatment of testis ribosomes. It was revealed that the predicted S/T sites of phosphorylation in the individual orthologous IDRs of gNAC-β sequences of Drosophila species are positionally conserved despite these disordered regions are drastically different. The IDR-dependent molecular crowding and specific coordination of NAC and other proteostasis regulatory factors at the ribosomes of germinal cells is proposed. These findings imply that there may be a functional crosstalk between the germinal and ubiquitous α- and β-subunits based on assessing their depletion effects on the fly viability and gonad development (Kogan, 2022).

    A translation control module coordinates germline stem cell differentiation with ribosome biogenesis during Drosophila oogenesis

    Ribosomal defects perturb stem cell differentiation, and this is the cause of ribosomopathies. How ribosome levels control stem cell differentiation is not fully known. This study discovered that three DExD/H-box proteins govern ribosome biogenesis (RiBi) and Drosophila oogenesis. Loss of these DExD/H-box proteins, which were named Aramis, Athos, and Porthos, aberrantly stabilizes p53, arrests the cell cycle, and stalls germline stem cell (GSC) differentiation. Aramis controls cell-cycle progression by regulating translation of mRNAs that contain a terminal oligo pyrimidine (TOP) motif in their 5' UTRs. TOP motifs confer sensitivity to ribosome levels that are mediated by La-related protein (Larp). One such TOP-containing mRNA codes for novel nucleolar protein 1 (Non1), a conserved p53 destabilizing protein. Upon a sufficient ribosome concentration, Non1 is expressed, and it promotes GSC cell-cycle progression via p53 degradation. Thus, a previously unappreciated TOP motif in Drosophila responds to reduced RiBi to co-regulate the translation of ribosomal proteins and a p53 repressor, coupling RiBi to GSC differentiation (Martin, 2022).

    All life depends on the ability of ribosomes to translate mRNAs into proteins. Despite this universal requirement, perturbations in ribosome biogenesis (RiBi) affect some cell types more than others. Stem cells, a cell type that underlies the generation and expansion of tissues, have an increased ribosomal requirement. Ribosome production is dynamically regulated to maintain higher amounts in stem cells. Reduction of ribosome levels in several stem cell systems can cause differentiation defects. In Drosophila, perturbations that reduce ribosome levels in the germline stem cells (GSCs) result in differentiation defects, causing infertility. Similarly, humans with impaired RiBi are afflicted with clinically distinct diseases known as ribosomopathies, such as Diamond-Blackfan anemia, that often result from loss of proper differentiation of tissue-specific progenitor cells. However, the mechanisms by which RiBi is coupled to proper stem cell differentiation remain incompletely understood (Martin, 2022).

    RiBi requires the transcription of ribosomal RNAs (rRNAs) and of mRNAs encoding ribosomal proteins (RPs) . Hundreds of factors, including DExD/H-box proteins, transiently associate with maturing rRNAs to facilitate rRNA processing, modification, and folding. RPs are imported into the nucleus, where they assemble with rRNAs in the nucleolus to form precursors to the 40S and 60S ribosomal subunits, which are then exported to the cytoplasm (Martin, 2022).

    In mammals, mRNAs that encode the RPs contain a terminal oligo pyrimidine (TOP) motif within their 5' untranslated region (UTR), which regulates their translation in response to nutrient levels. Under growth-limiting conditions, La-related protein 1 (Larp1) binds to the TOP sequences and to mRNA caps to inhibit translation of RPs. When growth conditions are suitable, Larp1 is phosphorylated by the mammalian target of rapamycin complex 1 (mTORC1) and does not efficiently bind the TOP sequence, allowing for translation of RPs. Whether TOP motifs exist in Drosophila to coordinate RP synthesis is unclear. The Drosophila ortholog of Larp1, Larp is required for proper cytokinesis and meiosis in Drosophila testis, as well as for female fertility, but its targets remain undetermined (Martin, 2022).

    Germline depletion of RiBi factors results in a stereotypical GSC differentiation defect during Drosophila oogenesis. Female Drosophila maintain 2-3 GSCs in the germarium. Asymmetric cell division of GSCs produces a self-renewing daughter GSC and a differentiating daughter, called the cystoblast (CB). This asymmetric division is unusual: following mitosis, the abscission of the GSC and CB is not completed until the following G2 phase. The GSC is marked by a round structure called the spectrosome, which elongates and eventually bridges the GSC and CB, similar to the fusomes that connect differentiated cysts. During abscission, the extended spectrosome structure is severed and a round spectrosome is established in the GSC and the CB. RiBi defects result in failed GSC-CB abscission, causing cells to accumulate as interconnected cysts called the 'stem cysts' that are marked by a fusome-like structure. In contrast with differentiated cysts, these stem cysts do not express the differentiation factor bag of marbles (Bam), do not differentiate, and typically die, resulting in sterility. How proper RiBi promotes GSC abscission and differentiation is not known (Martin, 2022).

    During Drosophila oogenesis, efficient RiBi is required in the germline for proper GSC cytokinesis and differentiation. The outstanding questions that needed to be addressed were: (1) Why does disrupted RiBi impair GSC abscission? And (2) How does the GSC monitor and couple RiBi to differentiation? The results suggest that a germline RiBi defect stalls the cell cycle, resulting a loss of differentiation and the formation of stem cysts. Proper RiBi was found to be monitored through a translation control module that allows for co-regulation of RPs and a p53 repressor. Ais, Ath, and Pths support RiBi and allowing for translation of a p53 repressor, preventing p53 stabilization, cell-cycle arrest, and loss of stem cell differentiation (Martin, 2022).

    The developmental upregulation of p53 during GSC differentiation concomitant with reduced RiBi parallels observations in disease states, such as ribosomopathies. This study found that p53 levels in GSCs are regulated by the conserved p53 regulator Non1. Although Non1 has been shown to directly interact with p53, how it regulates p53 levels in both humans and Drosophila is not known (Martin, 2022).

    TOP-containing mRNAs are known to be coregulated to coordinate ribosome production in response to environmental cues. Surprisingly, the observation that loss of ais reduces translation, albeit indirectly via regulation of RiBi, of a cohort of TOP-containing mRNAs, including Non1, suggests that the TOP motif also sensitizes their translation to lowered levels of RiBi. This notion is supported by TOP reporter assays demonstrating that reduced translation upon loss of ais requires the TOP motif. It is hypothesized that limiting TOP mRNA translation lowers RP production to maintain a balance with reduced rRNA production. This feedback mechanism would prevent the production of excess RPs that cannot be integrated into ribosomes and the ensuing harmful aggregates (Martin, 2022).

    The translation and stability of TOP-containing mRNAs are mediated by Larp1 and its phosphorylation. This study found that perturbing rRNA production and thus RiBi, without directly targeting RPs, also results in dysregulation of TOP mRNAs. The current data show that Drosophila Larp binds the RpL30 and Non1 5' UTR in a TOP-dependent manner in vitro and to 97% of the translation targets identified in vivo. Together, these data suggest that rRNA production regulates TOP mRNAs via Larp albeit indirectly. Furthermore, the cytokinesis defect caused by OE of Larp-DM15 in the germline suggests that Larp regulation could maintain the homeostasis of RiBi by balancing the expression of RP production with the rate of other aspects of RiBi, such as rRNA processing, during development (Martin, 2022).

    Ribosomopathies arise from RiBi defects. The underlying mechanisms of tissue specificity remain unresolved. This study demonstrates that loss of proteins involved in rRNA processing lead to cell-cycle arrest. Given that Drosophila GSCs undergo an atypical cell cycle as a normal part of their development it may be that this underlying cellular program in the germline leads to the tissue-specific phenotype of stem-cyst formation (Sanchez et al., 2016). This model implies that other tissues would likewise exhibit tissue-specific manifestations of ribosomopathies due to their underlying cell state. These data suggest two other sources of potential tissue specificity: (1) tissues express different cohorts of mRNAs, such as Non1, which are sensitive to ribosome levels (2). p53 activation, as previously described, is differentially tolerated in different tissues . Together, these mechanisms could begin to explain the tissue-specific nature of ribosomopathies and their link to differentiation (Martin, 2022).

    The exact processing steps that Ais, Ath, and Pths promote in Drosophila RiBi remain unknown; it is hypothesized that the processing step they act on the rRNA would be similar to what has been reported in yeast and mammals. Lack of a full rescue from ais, ath, and pths GKD in p53 mutants suggest that multiple genes likely influence the cell-cycle arrest. Finally, it is possible that the roles of Ais, Ath, and Pths in indirectly promoting Non1 translation does not represent a general effect of RiBi defects and is specific to these three proteins. However, this is thout unlikely as nearly all genes involved in RiBi outside of RPs share the same phenotype when depleted during Drosophila oogenesis (Martin, 2022).

    The transcription factor Xrp1 orchestrates both reduced translation and cell competition upon defective ribosome assembly or function

    Ribosomal Protein (Rp) gene haploinsufficiency affects translation rate, can lead to protein aggregation, and causes cell elimination by competition with wild type cells in mosaic tissues. This study finds that the modest changes in ribosomal subunit levels observed were insufficient for these effects, which all depended on the AT-hook, bZip domain protein Xrp1. Xrp1 reduced global translation through PERK-dependent phosphorylation of eIF2α. eIF2α phosphorylation was itself suficient to enable cell competition of otherwise wild type cells, but through Xrp1 expression, not as the downstream effector of Xrp1. Unexpectedly, many other defects reducing ribosome biogenesis or function (depletion of TAF1B, eIF2, eIF4G, eIF6, eEF2, eEF1alpha1, or eIF5A), also increased eIF2α phosphorylation and enabled cell competition. This was also through the Xrp1 expression that was induced in these depletions. In the absence of Xrp1, translation differences between cells were not themselves sufficient to trigger cell competition. Xrp1 is shown here to be a sequence-specific transcription factor that regulates transposable elements as well as single-copy genes. Thus, Xrp1 is the master regulator that triggers multiple consequences of ribosomal stresses and is the key instigator of cell competition (Kiparaki, 2022).

    This study has explored the mechanisms by which Rp mutations affect Drosophila imaginal disc cells, causing reduced translation and elimination by competition with wild-type cells in mosaics. The findings reinforced the key role played by the AT-hook bZip protein Xrp1, which is a sequence-specific transcription factor responsible for multiple aspects of not only the Rp phenotype, but also other ribosomal stresses. It was Xrp1, rather than the reduced levels of ribosomal subunits, that affected overall translation rate, primarily through PERK-dependent phosphorylation of eIF2α. Phosphorylation of eIF2α, as well as other disruptions to ribosome biogenesis and function such as reduction in rRNA synthesis or depletion of translation factors, were all sufficient to cause cell competition with nearby wild type cells, but this occurred because all these perturbations activated Xrp1, not because differences in translation levels between cells were sufficient to cause cell competition directly. In fact, the data show that differences in translation are neither sufficient nor necessary to trigger cell competition, which therefore depends on other Xrp1-dependent processes. Protein aggregation and activation of 'oxidative stress response' genes were also downstream effects of Xrp1 activity. While this paper was in preparation, other groups have also reported relationships between eIF2α phosphorylation, cell competition, and Xrp1, but none have reached the same overall conclusions as this study (Kiparaki, 2022).

    Our findings lead to a picture of Xrp1 as the key instigator of cell competition in response to multiple genetic triggers. Failure to appreciate the role of Xrp1 may have led to questionable conclusions in some previous studies. The current findings confirm the central importance of the transcriptional response to Rp mutations, and to other disruptions of ribosome biogenesis and function. They suggest therapeutic approaches to ribosomopathies, and have implications for the surveillance of aneuploid cells (Kiparaki, 2022).

    Rp gene haploinsufficiency has been proposed to affect ribosome concentrations, and hence translation, lead to the accumulation of ribosome components and assembly intermediates, and cause proteotoxic stress. Any of these could have been responsible for activating Xrp1 in Rp+/- cells (Kiparaki, 2022).

    The current data show that in fact ribosome subunit concentration is only moderately affected by Rp haploinsufficiency. 15-20% reduction in LSU concentrations in several RpL mutants, and 20-25% reduction in SSU (small subunit) concentrations in several RpS mutants. RpL14+/- also reduced SSU ~ 25%. Ribosomal subunit levels were unaffected by Xrp1. Broadly similar results have been reported in yeast, and by mass spec quantification of ribosomal proteins in RpS3+/- and RpS23+/- Drosophila wing discs (Kiparaki, 2022).

    Multiple explanations for the modest effects on ribosome subunit number are possible. It is particularly pointed out that, even if expression of a particular Rp is reduced in proportion to a 50% reduction in mRNA level, the respective protein concentration (i.e. number of molecules/cell volume) is unlikely to fall to 50%, because ribosomes are required for cellular growth, so that an Rp mutation affects the denominator in the concentration equation, as well as the numerator. It is even possible that a 50% reduction in its rate of Rp synthesis could leave steady state ribosome subunit concentration unaffected, if cellular growth rate was slowed by the same amount (Kiparaki, 2022).

    Modest changes in SSU and LSU levels could still affect ribosome function, which may depend more on the concentrations of free subunits than on total subunits. The data suggests, however, that cellular and animal models of ribosomopathy Diamond Blackfan Anemia (DBA) that have generally sought to achieve a 50% reduction in Rp protein expression could be significantly more severe than occurs in DBA patients, and that actual ribosome subunit concentrations should be measured in DBA patient cells to guide future models (Kiparaki, 2022).

    This study confirmed that ribosome assembly intermediates accumulate in Drosophila wing discs following Rp haploinsufficiency. In yeast, aggregates of unused Rp rapidly trigger transcriptional changes. It has been suggested proteotoxic stress might lead to eIF2α phosphorylation in Drosophila, with Xrp1 amplifying this effect, but this study found that while Perk was responsible for eIf2α phosphorylation, it was not required for Xrp1 expression in Rp mutants, placing Perk and eIF2α phosphorylation downstream. Consistent with this, it was shown that the protein aggregates reported in Rp+/- cells were only seen in some Rp mutants, all affecting the SSU, and were also a downstream consequence of Xrp1 activity, as also now seen by others. It remains plausible that unused ribosomal components are the initial trigger for cellular responses in Drosophila as in yeast, but in Drosophila the species involved have not yet been identified. Because Xrp1 expression depends particularly on RpS12, an RpS12-containing signaling species is one possibility (Kiparaki, 2022).

    PERK-dependent phosphorylation of eIF2α was the mechanism by which Xrp1 suppresses global translation in Rp+/- mutants (Kiparaki, 2022).

    It is interesting that Xrp1 protein levels increase under conditions of reduced global translation. Perhaps Xrp1 is one of the few genes whose translation is enhanced when eIF2α is phosphorylated. Although PERK is known to be activated by ER stress, the IRE/Xbp1 branch of the UPR was not unequivocally detected in Rp+/- mutants. It is suspected that the UPR might be suppressed in Rp+/- mutants by Xrp1-dependent changes in transcription of Perk, BiP, and other UPR genes. Perhaps in proliferative tissues it is preferable to replace stressed cells than to repair them (Kiparaki, 2022).

    It will be interesting to determine whether eIF2α phosphorylation occurs in human ribosomopathies. Notably, knock-out of CReP, one of the two mouse PPP1R15 homologs, causes anemia, similar to DBA, and PERK-dependent eIF2α phosphorylation occurs in RpL22-deficient mouse αβ T-cells and activates p53 there. Thus, inhibitors of eIF2α phosphorylation could be explored as potential DBA drugs. TAF1B depletion, which also acted through Xrp1 and eIF2α phosphorylation in Drosophila, is a model of Treacher Collins Syndrome, and failure to release eIF6, leading to defective LSU maturation and 80 S ribosome formation, causes Schwachman Diamond syndrome, two other ribosomopathies where potential contributions of eIF2α phosphorylation are possible (Kiparaki, 2022).

    Because eIF2α phosphorylation alone was sufficient to target cells for competitive elimination, at first it seemed that eIF2α phosphorylation was the mechanism by which Xrp1 caused cell competition, which often correlates with differences in cellular translation levels. One group has suggested this. Another group concluded that eIF2α phosphorylation in Rp+/- cells did not lead to cell competition, but the opposite conclusion is corroborated by the independent finding that haploinsufficiency for the γ subunit of eIF2 also causes cell competition. It is concluded that eIF2α phosphorylation can cause cell competition but not directly. Instead, phosphorylation of eIF2α is itself sufficient to activate Xrp1 expression, as found by this and several other groups. Crucially, Perk inactivation restored eIF2α phosphorylation and global translation to normal in Rp+/- cells, without preventing cell competition, which must therefore depend on other Xrp1 targets. Elimination of eIF2γ haploinsufficient cells is also Xrp1-dependent, as expected if Xrp1 is downstream of eIF2 activity in cell competition (Kiparaki, 2022).

    Knock-down of factors directly involved in the translation mechanism further distinguished cell competition from differential translation levels. Different factors affected translation in diverse ways. In Rp+/- mutants, PERK-dependent phosphorylation of eIF2α suppressed global translation, which was normalized by Perk or Xrp1 depletion. PERK-dependent phosphorylation of eIF2α also contributed to the translation deficits of cells depleted for TAF1B, eIF6, and possibly eEF1α1, which were all partially restored by eIF2α dephosphorylation and fully by Xrp1 depletion, suggesting that Xrp1 can also affect translation by additional mechanisms. By contrast, translation deficits caused by eIF4G, eIF5A, or eEF2 depletion were restored little by eIF2α dephosphorylation or Xrp1 depletion, indicating Xrp1-independent effects of these factors on translation (Kiparaki, 2022).

    Several conclusions follow from studies of these factors. As noted above, reduced translation cannot be required for cell competition, because perk-/- Rp+/- mutant cells are eliminated by perk+/- Rp+/+ cells. Secondly, lower translation is not sufficient for competitive elimination, because no competitive cell death was observed in eIF4G Xrp1-depleted, eIF5A Xrp1-depleted, and eEF2 Xrp1-depleted cells, even though their translation was lower than the nearby wild type cells. Another group also concluded that lower translation alone was not sufficient for cell competition, based on different data (Kiparaki, 2022).

    The current findings focus attention on Xrp1 activity as the key factor marking cells for competition, distinct from its effects on global translation, which only trigger cell competition when Xrp1 is induced (Kiparaki, 2022).

    It was confirmed that Xrp1 is a sequence-specific transcriptional activator, and it is proposed that direct transcriptional targets of Xrp1 predispose Rp+/- cells, and other genotypes, to elimination by wild-type cells. Expression of several hundred single copy genes is regulated by Xrp1 in Rp mutant cells, and this study reports that expression of some transposable elements is affected in addition, whose potential contribution to cell competition might also be interesting. One or more of these transcriptional targets may lead to competitive interactions with wild-type cells (Kiparaki, 2022).

    These Xrp1 targets include genes that also contribute to oxidative stress responses, such as GstD genes, which has previously led to the suggestion that an oxidative stress response is responsible for cell competition. Because the oxidative stress reporter used in previous studies is probably activated in Rp+/- cells by direct Xrp1-binding, and not by the Nrf2-dependent ARE site, it is not now certain whether Rp+/- cells experience oxidative stress or Nrf2 activity. An alternative explanation of cell competition in response to Nrf2 over-expression could be induction of Xrp1 expression by Nrf2 (Kiparaki, 2022).

    These results reveal the central importance of Xrp1 as the driver of cell competition. Far from being expressed specifically in Rp mutants, this study now finds that Xrp1 is induced by multiple challenges, not only to ribosome biogenesis, such as by depletion of the polI cofactor TAF1B or LSU maturation factor eIF6, but also challenges to ribosome function, both at the levels of initiation or elongation, all leading to cell competition and to Xrp1-dependent eIF2α phosphorylation (Kiparaki, 2022).

    Had Xrp1 expression and function not been evaluated in PPP1R15-depleted cells, it would have been concluded that eIF2α phosphorylation was the likely downstream effector of competition in Rp mutant cells, rather than an example of another upstream stress that induces Xrp1. It is becoming apparent that other triggers of cell competition, including depletion for Helicase at 25E (Hel25E), a helicase that plays roles in mRNA splicing and in mRNA nuclear export, over-expression of Nrf2, the transcriptional master regulator of the oxidative stress response, and loss of mahjong, a ubiquitin ligase implicated in planar cell polarity, all lead to Xrp1 expression. Earlier models regarding these cell competition mechanisms, in which the role of Xrp1 was not recognized, may be questionable. It would be important now to check for possible activation of Xrp1 in cells with other defects affecting translation, including mutations of an eIF5A-modifying enzyme and mutations of a pre-rRNA processing enzyme. It would not be surprising if other conditions that lead to eIF2α phosphorylation, such as ER stress, nutrient deprivation, or viral infection, also activate Xrp1 and are thereby marked for elimination by more normal neighbors. It will be particularly interesting to determine whether any of these environmental perturbations could interfere with surveillance and removal of aneuploid cells, given the potential importance for tumor surveillance (Kiparaki, 2022).


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    Zygotically transcribed genes

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