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
  • 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

    Lee, C. H., Kiparaki, M., Blanco, J., Folgado, V., Ji, Z., Kumar, A., Rimesso, G. and Baker, N. E. (2018). A regulatory response to ribosomal protein mutations controls translation, growth, and cell competition. Dev Cell 46(4): 456-469. PubMed ID: 30078730

    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).



    Factors involved in translation initiation and repression

    belle
    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

    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 4a (eIF-4a)
    ATP-dependent RNA helicase which is a subunit of the eIF4F complex involved in cap recognition and is required for mRNA binding to 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 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

    GCN2
    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).

    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).


    References

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    Lee, C. H., Kiparaki, M., Blanco, J., Folgado, V., Ji, Z., Kumar, A., Rimesso, G. and Baker, N. E. (2018). A regulatory response to ribosomal protein mutations controls translation, growth, and cell competition. Dev Cell 46(4): 456-469. PubMed ID: 30078730

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    Shan, L., Wu, C., Chen, D., Hou, L., Li, X., Wang, L., Chu, X., Hou, Y. and Wang, Z. (2017). Regulators of alternative polyadenylation operate at the transition from mitosis to meiosis. J Genet Genomics [Epub ahead of print]. PubMed ID: 28190776

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    Zygotically transcribed genes

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