InteractiveFly: GeneBrief

Methyltransferase like 3: Biological Overview | References

BIOLOGICAL OVERVIEW

N⁶-methyladenosine (m⁶A) is the most common internal modification of eukaryotic messenger RNA (mRNA) and is decoded by YTH domain proteins. The mammalian mRNA m⁶A methylosome is a complex of nuclear proteins that includes a stable heterodimer [METTL3 (methyltransferase-like 3) and METTL14], WTAP (Wilms tumour 1-associated protein) and KIAA1429. Drosophila has corresponding homologues named Ime4 (Inducer of meiosis 4) , Mettl14 (Methyltransferase-like 14 ), the Wilms tumour 1-associated protein Female-lethal (2)d (Fl(2)d) and Virilizer (Vir). In Drosophila, fl(2)d and vir are required for sex-dependent regulation of alternative splicing of the sex determination factor Sex lethal (Sxl). However, the functions of m⁶A in introns in the regulation of alternative splicing remain uncertain. This study shows that m⁶A is absent in the mRNA of Drosophila lacking Ime4. In contrast to mouse and plant knockout model, Drosophila Ime4-null mutants remain viable, though flightless, and show a sex bias towards maleness. This is because m⁶A is required for female-specific alternative splicing of Sxl, which determines female physiognomy, but also translationally represses male-specific lethal 2 (msl-2) to prevent dosage compensation in females. The m⁶A reader protein YT521-B decodes m⁶A in the sex-specifically spliced intron of Sxl, as its absence phenocopies Ime4 mutants. Loss of m⁶A also affects alternative splicing of additional genes, predominantly in the 5' untranslated region, and has global effects on the expression of metabolic genes. The requirement of m⁶A and its reader YT521-B for female-specific Sxl alternative splicing reveals that this hitherto enigmatic mRNA modification constitutes an ancient and specific mechanism to adjust levels of gene expression (Haussmann, 2016).

In mature mRNA the m⁶A modification is most prevalently found around the stop codon as well as in 5' untranslated regions (UTRs) and in long exons in mammals, plants and yeast. Since methylosome components predominantly localize to the nucleus, it has been speculated that m⁶A localized in pre-mRNA introns could have a role in alternative splicing regulation in addition to such a role when present in long exons. This prompted an investigation of whether m⁶A is required for Sxl alternative splicing, which determines female sex and prevents dosage compensation in females. A null allele of the Drosophila METTL3 methyltransferase homologue Ime4 was induced by imprecise excision of a P element inserted in the promoter region. The excision allele Δ22-3 deletes most of the protein-coding region, including the catalytic domain, and is thus referred to as Ime4^null. These flies are viable and fertile, but both flightless and this phenotype can be rescued by a genomic construct restoring Ime4. Ime4 shows increased expression in the brain and, as in mammals and plants (Hongay, 2011), localizes to the nucleus (Haussmann, 2016).

Following RNase T1 digestion and ³²P end-labelling of RNA fragments, m⁶A was detected after guanosine (G) in poly(A) mRNA of adult flies at relatively low levels compared to other eukaryotes, but at higher levels in unfertilized eggs. After enrichment with an anti-m⁶A antibody, m⁶A is readily detected in poly(A) mRNA, but absent from Ime4^null flies (Haussmann, 2016).

As found in other systems, and consistent with a potential role in translational regulation, m⁶A was detected in polysomal mRNA, but not in the poly(A)-depleted rRNA fraction. This also confirmed that any m⁶A modification in rRNA is not after G in Drosophila (Haussmann, 2016).

Consistent with the hypothesis that m⁶A plays a role in sex determination and dosage compensation, the number of Ime4^null females was reduced to 60% compared to the number of males, whereas in the control strain female viability was 89%. The key regulator of sex determination in Drosophila is the RNA-binding protein Sxl, which is specifically expressed in females. Sxl positively auto-regulates expression of itself and its target transformer (tra) through alternative splicing to direct female differentiation. In addition, Sxl suppresses translation of msl-2 to prevent upregulation of transcription on the X chromosome for dosage compensation; full suppression also requires maternal factors. Accordingly, female viability was reduced to 13% by removal of maternal m⁶A together with zygotic heterozygosity for Sxl and Ime4 (Ime4^Δ22-3 females crossed with Sxl^7B0 males, a Sxl null allele). Female viability of this genotype is completely rescued by a genomic construct or by preventing ectopic activation of dosage compensation by removal of msl-2. Hence, females are non-viable owing to insufficient suppression of msl-2 expression, resulting in upregulation of gene expression on the X chromosome from reduced Sxl levels. In the absence of msl-2, disruption of Sxl alternative splicing resulted in females with sexual transformations displaying male-specific features such as sex combs, which were mosaic to various degrees, indicating that Sxl threshold levels are affected early during establishment of sexual identities of cells and/or their lineages. In the presence of maternal Ime4, Sxl and Ime4 do not genetically interact (Sxl^7B0/FM7 females crossed with Ime4^null males, 103% female viability. In addition, Sxl is required for germline differentiation in females and its absence results in tumorous ovaries. Consistent with this, tumorous ovaries in Sxl^7B0/+;Ime4^null/+ daughters from Ime4^null females or heterozygous Sxl^7B0 females (Haussmann, 2016).

Furthermore, levels of the Sxl female-specific splice form were reduced to approximately 50%, consistent with a role for m⁶A in Sxl alternative splicing . As a result, female-specific splice forms of tra and msl-2 were also significantly reduced in adult females (Haussmann, 2016).

To obtain more comprehensive insights into Sxl alternative splicing defects in Ime4^null females, splice junction reads were examined from RNA-seq. Besides the significant increase in inclusion of the male-specific Sxl exon in Ime4^null females, cryptic splice sites and increased numbers of intronic reads were detected in the regulated intron. Consistent with reverse transcription polymerase chain reaction (RT–PCR) analysis of tra, the reduction of female splicing in the RNA sequencing is modest, and as a consequence, alternative splicing differences of Tra targets dsx and fru were not detected in whole flies, suggesting that cell-type-specific fine-tuning is required to generate splicing robustness rather than being an obligatory regulator. In agreement with dosage-compensation defects as a main consequence of Sxl dysregulation in Ime4^null mutants, X-linked, but not autosomal, genes are significantly upregulated in Ime4^null females compared to controls (Haussmann, 2016).

Furthermore, Sxl mRNA is enriched in pull-downs with an m⁶A antibody compared to m⁶A-deficient yeast mRNA added for quantification. This enrichment is comparable to what was observed for m⁶A-pull-down from yeast mRNA (Haussmann, 2016).

To map m⁶A sites in the intron of Sxl, an in vitro m⁶A methylation assay was employed using Drosophila nuclear extracts and labelled substrate RNA. m⁶A methylation activity was detected in the vicinity of alternatively spliced exons. Further fine-mapping localized m⁶A in RNAs C and E to the proximity of Sxl-binding sites. Likewise, the female-lethal single amino acid substitution alleles fl(2)d¹ and vir^2F interfere with Sxl recruitment, resulting in impaired Sxl auto-regulation and inclusion of the male-specific exon. Female lethality of these alleles can be rescued by Ime4^null heterozygosity, further demonstrating the involvement of the m⁶A methylosome in Sxl alternative splicing (Haussmann, 2016).

Next, alternative splicing changes was globally analyzed in Ime4^null females compared to the wild-type control strain. A statistically significant reduction in female-specific alternative splicing of Sxl was observed. In addition, 243 alternative splicing events in 163 genes were significantly different in Ime4^null females, equivalent to around 2% of alternatively spliced genes in Drosophila. Six genes for which the alternative splicing products could be distinguished on agarose gels were confirmed by RT-PCR. Notably, lack of Ime4 did not affect global alternative splicing and no specific type of alternative splicing event was preferentially affected. However, alternative first exon (18% versus 33%) and mutually exclusive exon (2% versus 15%) events were reduced in Ime4^null compared to a global breakdown of alternative splicing in wild-type Drosophila, mostly to the extent of retained introns (16% versus 6%), alternative donor (16% versus 9%) and unclassified events (14% versus 6%). Notably, the majority of affected alternative splicing events in Ime4^null were located to the 5' UTR, and these genes had a significantly higher number of AUG start codons in their 5' UTR compared to the 5' UTRs of all genes. Such a feature has been shown to be relevant to translational control under stress conditions. (Starck, 2016; Haussmann, 2016 and references therein).

The majority of the 163 differentially alternatively spliced genes in Ime4 females are broadly expressed (59%), while most of the remainder are expressed in the nervous system (33%), consistent with higher expression of Ime4 in this tissue. Accordingly, Gene Ontology analysis revealed a highly significant enrichment for genes involved synaptic transmission (Haussmann, 2016).

Since the absence of m⁶A affects alternative splicing, m⁶A marks are probably deposited co-transcriptionally before splicing. Co-staining of polytene chromosomes with antibodies against haemagglutinin (HA)-tagged Ime4 and RNA Pol II revealed broad co-localization of Ime4 with sites of transcription, but not with condensed chromatin-visualized with antibodies against histone H4. Furthermore, localization of Ime4 to sites of transcription is RNA-dependent, as staining for Ime4, but not for RNA Pol II, was reduced in an RNase-dependent manner (Haussmann, 2016).

Although m⁶A levels after G are low in Drosophila compared to other eukaryotes, broad co-localization of Ime4 to sites of transcription suggests profound effects on the gene expression landscape. Indeed, differential gene expression analysis revealed 408 differentially expressed genes where 234 genes were significantly upregulated and 174 significantly downregulated in neuron-enriched head/thorax of adult Ime4^null females. Cataloguing these genes according to function reveals prominent effects on gene networks involved in metabolism, including reduced expression of 17 genes involved in oxidative phosphorylation. Notably, overexpression of the m⁶A mRNA demethylase FTO in mice leads to an imbalance in energy metabolism resulting in obesity (Haussmann, 2016).

Next, tests were performed to see whether either of the two substantially divergent YTH proteins, YT521-B and CG6422, decodes m⁶A marks in Sxl mRNA. When transiently transfected into male S2 cells, YT521-B localizes to the nucleus, whereas CG6422 is cytoplasmic. Nuclear YT521-B can switch Sxl alternative splicing to the female mode and also binds to the Sxl intron in S2 cells. In vitro binding assays with the YTH domain of YT521-B demonstrate increased binding of m⁶A-containing RNA. In vivo, YT521-B also localizes to the sites of transcription (Haussmann, 2016).

To further examine the role of YT521-B in decoding m⁶A Drosophila strain YT521-B^MI02006 was analyzed, where a transposon in the first intron disrupts YT521-B. This allele is also viable, and phenocopies the flightless phenotype and the female Sxl splicing defect of Ime4^null flies. Likewise, removal of maternal YT521-B together with zygotic heterozygosity for Sxl and YT521-B reduces female viability and results in sexual transformations such as male abdominal pigmentation. In addition, overexpression of YT521-B results in male lethality, which can be rescued by removal of Ime4, further reiterating the role of m⁶A in Sxl alternative splicing. Since YT521-B phenocopies Ime4 for Sxl splicing regulation, it is the main nuclear factor for decoding m⁶A present in the proximity of the Sxl-binding sites. YT521-B bound to m⁶A assists Sxl in repressing inclusion of the male-specific exon, thus providing robustness to this vital gene regulatory switch (Haussmann, 2016).

Nuclear localization of m⁶A methylosome components suggested a role for this 'fifth' nucleotide in alternative splicing regulation. The discovery of the requirement of m6A and its reader YT521-B for female-specific Sxl alternative splicing has important implications for understanding the fundamental biological function of this enigmatic mRNA modification. Its key role in providing robustness to Sxl alternative splicing to prevent ectopic dosage compensation and female lethality, together with localization of the core methylosome component Ime4 to sites of transcription, indicates that the m⁶A modification is part of an ancient, yet unexplored mechanism to adjust gene expression. Hence, the recently reported role of m⁶A methylosome components in human dosage compensation (Moindrot, 2015; Patil, 2016) further support such a role and suggests that m⁶A-mediated adjustment of gene expression might be a key step to allow for the development of the diverse sex determination mechanisms found in nature (Haussmann, 2016).

m6A modulates neuronal functions and sex determination in Drosophila

N⁶-methyladenosine RNA (m⁶A) is a prevalent messenger RNA modification in vertebrates. Although its functions in the regulation of post-transcriptional gene expression are beginning to be unveiled, the precise roles of m⁶A during development of complex organisms remain unclear. This study carried out a comprehensive molecular and physiological characterization of the individual components of the methyltransferase complex, as well as of the YTH domain-containing nuclear reader protein in Drosophila melanogaster. A member of the split ends protein family, Spenito (Nito), was identified as a novel bona fide subunit of the methyltransferase complex. Important roles of this complex were identified in neuronal functions and sex determination, and the nuclear RNA binding protein YT521-B was identified as a main m⁶A effector in these processes. Altogether, this work substantially extends knowledge of m⁶A biology, demonstrating the crucial functions of this modification in fundamental processes within the context of the whole animal (Lence, 2016).

RNA modifications represent a critical layer of epigenetic regulation of gene expression. m⁶A is among the most abundant modifications in the mammalian system. m⁶A distribution has been determined in several organisms and cell types, including human, mouse, rice and yeast. The modification is found in a subset of the RRACH consensus sites (R, purine; H, non-guanine base) and is enriched around stop codons, in the 3'-untranslated regions (3'UTRs) and within long internal exons. m⁶A was shown to control several post-transcriptional processes, including pre-mRNA splicing, mRNA decay and translation, which are mediated in part via conserved members of the YTH protein family. The methyltransferase complex catalysing m⁶A formation in mammals consists of methyltransferase-like 3 (METTL3 - Drosophila ortholog: Ime4), methyltransferase-like 14 (METTL14) and a stabilizing factor called Wilms' tumour 1-associated protein (WTAP). In mammals, m⁶A can be reverted into adenosine via two identified demethylases: fat mass and obesity associated factor (FTO) and AlkB homologue 5 (ALKBH5) (Lence, 2016).

Several studies have uncovered crucial roles for METTL3 during development and cell differentiation. Knockout of Mettl3 in murine naive embryonic stem cells blocks differentiation, while its deletion in mice causes early embryonic lethality. Similarly, in Drosophila, loss of the METTL3 orthologue Ime4 is reported to be semi-lethal during development, with adult escapers having reduced fertility owing to impaired Notch signalling (Hongay, 2011). Depletion of the METTL3 orthologue MTA in Arabidopsis thaliana also affects embryonic development, while in yeast ime4 has an essential role during meiosis. All of these observations indicate the importance of m⁶A in the gonads and during early embryogenesis. Recent crystal structure studies investigated the molecular activities of the two predicted catalytic proteins; however, their respective roles in vivo remain unclear. This study has characterized members of the methyltransferase complex in Drosophila and identifies the split ends (SPEN) family protein, Spenito (Nito), as a novel bona fide subunit. Expression of complex components is substantially enriched in the nervous system, and flies with mutations in Ime4 and Mettl14 suffer from impaired neuronal functions. Methyltransferase complex components also influence the female-specific splicing of Sex-lethal (Sxl ), revealing a role in fine-tuning sex determination and dosage compensation. Notably, knockout of the nuclear m⁶A reader YT521-B resembles the loss of the catalytic subunits, implicating this protein as a main effector of m⁶A in vivo (Lence, 2016).

To investigate potential functions of m⁶A in Drosophila, its levels were monitored on mRNA samples isolated at different developmental stages of wild-type flies using mass spectrometry. m⁶A was found to be remarkably enriched in early embryogenesis but drops dramatically 2 h after fertilization and remains low throughout the rest of embryogenesis and early larval stages. During the third larval instar, m⁶A rises again to reach a peak at pupal phases. While the overall level of m⁶A decreases in adults, it remains substantially elevated in heads and ovaries (Lence, 2016).

A phylogenetic analysis of the Drosophila METTL3 orthologue Ime4 identifies two closely related factors, CG7818 and CG14906. Depletion of Ime4 and CG7818 in embryonic-derived Schneider (S2R+) cells decreases m⁶A levels by about 70%, whereas depletion of CG14906 had no effect. These results indicate that Ime4 and CG7818 are required to promote m⁶A activity in Drosophila. Because of its sequence and functional conservation with human METTL14, CG7818 was renamed dMettl14. Fl(2)d and Virilizer (Vir) are the Drosophila homologues of WTAP and KIAA1429, respectively, which are integral components of the complex in mammals. Both transcripts follow the same developmental distribution as other methyltransferase complex components and their depletion also affects m⁶A levels. Ime4 and Fl(2)d co-immunoprecipitate with dMettl14 in an RNA-independent manner. Likewise, Vir, Fl(2)d and Ime4 are found in the same complex. Notably, Fl(2)d depletion reduces the interaction between Ime4 and dMettl14, confirming its proposed role as a stabilizing factor. All components localize in the nucleus and are ubiquitously expressed in early embryonic stages but show substantial enrichment in the neuroectoderm at later stages. Altogether, the results demonstrate the existence of a conserved functional methyltransferase complex in Drosophila and reveal its particular abundance in the nervous system (Lence, 2016).

To obtain insight into the transcriptome-wide m⁶A distribution in S2R+ cells, methylated RNA immunoprecipitation was performed followed by sequencing (MeRIP-seq). In total, 1,120 peaks representing transcripts of 812 genes were identified. The consensus sequence RRACH is present in most m⁶A peaks. Additional sequences are also enriched, suggesting their potential involvement in providing specificity to the methyltransferase complex. As shown in other species, enrichment near start and stop codons was observed (Lence, 2016).

Transcriptome analyses was performed in S2R+ cells lacking m⁶A components. Knockdown of Fl(2)d leads to strong changes in gene expression (n = 2,129 differentially expressed genes; adjusted P value < 0.05), while knockdowns of Ime4 and dMettl14 have milder effects. Gene ontology analyses revealed that genes involved in diverse metabolic processes, anion transport and cell adhesion are significantly overrepresented. Despite the fact that S2R+ cells are of non-neuronal origin, the affected genes are also enriched for neuronal functions, including roles in axon guidance and synapse activity. Consistent with the larger average size of neuronal genes, affected genes are significantly larger than the non-affected ones. The genes affected upon Ime4/dMettl14 double knockdown were compared with the m⁶A profile. Overall, about 15% of the affected genes contain at least one m⁶A peak. A slight but significant positive influence of m⁶A on mRNA levels was found and this effect seems independent of the location of the m⁶A peak along the transcript. Several splicing changes upon knockdown of individual complex components were also observed. fl(2)d itself is among the affected transcripts in any of the knockdowns tested. Generally, each knockdown results in alternative 5' splice site usage and intron retention, which was also observed in human cells (Lence, 2016).

YTH proteins are critical readers of m⁶A in mammals. While vertebrates contain five proteins of this family, only two members exist in flies, CG6422 and YT521-B. CG6422 was found to be localized in the cytoplasm and strongly enriched during the first 2 h after fertilization but then declines and remains at low levels during development and adulthood. By contrast, YT521-B is strictly nuclear and shows strong enrichment in the embryonic nervous system and adult brains. Using dot-blot assays and pull-down experiments it was confirmed that YT521-B binds m⁶A in Drosophila. RNA-sequencing (RNA-seq) experiments show that depletion of CG6422 only marginally affects splicing while YT521-B knockdown significantly impairs this process (103 differentially regulated splicing events). The overlap of mis-spliced events between YT521-B knockdown and knockdown of methyltransferase complex subunits is about 70%, revealing that YT521-B might be the main mediator of m⁶A function in pre-mRNA splicing (Lence, 2016).

To investigate potential roles of m⁶A during Drosophila development, Ime4- and dMettl14-knockout flies were generated. Two deletions in Ime4 were created, removing the entire coding sequence (Ime4null) or only the C-terminal part containing the catalytic domain (Ime4Δcat). Flies homozygous for either mutant allele as well as transheterozygous flies survive until adulthood. No encapsulation defects were observed in ovaries as previously shown using different alleles (Hongay, 2011). However, the mutant flies have a reduced lifespan and exhibit multiple behavioural defects: flight and locomotion are severely affected and they spend more time grooming. They also display a mild held-out wing appearance resulting from failure to fold their wings together over the dorsal surface of the thorax and abdomen. dMettl14 mutant flies have normal wings but their locomotion is also deficient . To test whether Ime4 and dMettl14 can compensate for each other in vivo, double-mutant animals were generated. Removing one copy of Ime4 in the dMettl14 mutant background mimics the held-out wing phenotype observed upon loss of Ime4. Double-homozygous mutants give similar phenotypes as the Ime4 single knockout, albeit with increased severity. Altogether, these phenotypic analyses strongly suggest that Ime4 and dMettl14 control similar physiological processes in vivo, indicating that they probably regulate common targets. Furthermore, the function of Ime4 appears to be slightly predominant over dMettl14 and most activities require its catalytic domain (Lence, 2016).

To quantify the locomotion phenotype better, the so-called Buridan's paradigm was applied. The activity and walking speed of Ime4 mutant flies is reduced by twofold compared with control flies. In addition, orientation defects were observed. All phenotypes were rescued by ubiquitous (Tub-GAL4) and neuronal (elav-GAL4), but not mesodermal (24B10-GAL4) expression of Ime4 complementary DNA. These findings demonstrate that m⁶A controls Drosophila behaviour by specifically influencing neuronal functions. To investigate potential neurological defects underlying the behavioural phenotype, the neuromuscular junction (NMJ) of Ime4-mutant larvae was examined. Notably, NMJ synapses grow exuberantly in the Ime4 mutant, displaying a 1.5-fold increase in the number of boutons and a 1.3-fold increase of active zones per bouton\, indicating that Ime4 may regulate locomotion via control of synaptic growth at the NMJ. To identify target genes involved in locomotion, adult heads of 1-2 day-old female flies were dissected and subjected to RNA-seq. In total, 1,681 genes display significant changes in expression and splicing upon Ime4 loss of function. Notably, many of the affected genes control fly locomotion. Next the list of affected genes with the MeRIP data from S2R+ cells anda dozen locomotion-related genes were detected as potential direct targets of m⁶A. Hence, it is likely that more than a single gene accounts for the locomotion phenotype observed in the absence of a functional methyltransferase complex (Lence, 2016).

Among the top hits showing changes in alternative splicing upon Ime4 knockout was Sxl , encoding a master regulator of sex determination and dosage compensation37. Sxl is expressed in both females and males, but the transcript in males contains an additional internal exon introducing a premature stop codon. To confirm the role of Ime4 and potentially dMettl14 in Sxl splicing, RNA extracts from the heads of both sexes were examined by polymerase chain reaction with reverse transcription (RT-PCR). While splicing is unaffected in males, mutant females of both genotypes show inclusion of the male-specific exon and decrease of the female-specific isoform. This decrease is less pronounced when analysing isoform levels from whole flies, possibly reflecting the specific enrichment of m⁶A in the brain. Consistent with these findings, splicing of two Sxl target transcripts, transformer (tra) and msl-2, is also altered. These results indicate that the methyltransferase complex facilitates splicing of Sxl pre-mRNA, suggesting a role in sex determination and dosage compensation. To validate this hypothesis, whether Ime4 genetically interacts with Sxl was examined. Transheterozygous Ime4 females were crossed with males carrying a deficiency in the Sxl locus and the survival rate of the progeny was quantified for both sexes. Females lacking one wild-type copy of both Ime4 and Sxl had severely reduced survival, while males were unaffected. This effect probably arises from impairment of the dosage compensation pathway. Thus, these findings indicate that Ime4 interacts with Sxl to control female survival (Lence, 2016).

Given that YT521-B specifically recognizes m⁶A and influences most m⁶A-dependent splicing events in S2R+ cells, whether its deletion in vivo mimics the knockout of members of the methyltransferase complex was investigated. A deletion in the YT521-B locus that disrupts expression of both YT521-B isoforms was generated. Similar to Ime4 and dMettl14 mutants, YT521-B mutant flies survive until adulthood but exhibit flight defects and poor locomotion. Comparison of the transcriptome of Ime4-knockout with YT521-B-knockout female flies identified 397 splicing events regulated by Ime4 and, among those, 243 (61% of Ime4-affected events) are also regulated by YT521-B, indicating a similar overlap as from S2R+ cells. While alternative 5' splice site usage is not specifically enriched in vivo, intron retention is still overrepresented. Notably, loss of YT521-B also leads to the male-specific splicing of Sxl , tra and msl-2 and to the decrease of the female-specific Sxl isoform in females. Collectively, these experiments strongly suggest that the m⁶A methyltransferase complex regulates adult locomotion and sex determination primarily via YT521-B binding to m⁶A (Lence, 2016).

To investigate the mechanisms of YT521-B-mediated splicing control, specific interacting partners were sought using stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics upon immunoprecipitation of a Myc-tagged YT521-B protein from S2R+ cells. 73 factors were identified that show more than twofold enrichment in the YT521-B-Myc sample. Almost half (n = 30) are predicted mRNA-binding proteins. To investigate whether some of these mRNA-binding proteins regulate m⁶A-dependent splicing, they were depleted in S2R+ cells, and the effects on fl(2)d splicing were assessed. Notably, three proteins, Hrb27C, Qkr58E-1 and Nito, were found to similarly control fl(2)d splicing. Expanding this analysis to six additional m⁶A-regulated splicing events reveals that Hrb27C and Qkr58E-1 regulate only a subset, while loss of Nito consistently leads to similar splicing defects as observed upon depletion of YT521-B and members of the methyltransferase complex. To get further insights into the interplay between YT521-B and the three mRNA-binding proteins, co-immunoprecipitation experiments were performed. While Qkr58E-1 interacts with YT521-B in an RNA-independent fashion, interactions with Hrb27C and Nito could not be reproduced. However, this study found that Nito interacts with both Fl(2)d and Ime4 independently of the presence of RNA. These findings indicate that Nito might be a component of the methyltransferase complex. Accordingly, nito mRNA expression correlates with m⁶A levels during development and Nito knockdown leads to a severe m⁶A decrease. This decrease is not an indirect consequence of reduced levels of methyltransferase complex components upon Nito knockdown. Finally, YT521-B binding to mRNA depends on the presence of Nito. Collectively, these results demonstrate that Nito is a bona fide member of the m⁶A methyltransferase complex (Lence, 2016).

This analysis argues against a vital role for Ime4 in Drosophila as both deletion alleles give rise to homozygous adults without prominent lethality during development. This cannot be explained by compensation via dMettl14, as its knockout produces similar effects as the Ime4 knockout. Furthermore, depleting both genes only slightly intensifies the locomotion phenotype without affecting fly survival, supporting the idea that Ime4 and dMettl14 act together to regulate the same target genes. Accordingly, loss of either component in vivo dramatically affects stability of the other (Lence, 2016).

Loss of function of either of the methyltransferases produces severe behavioural defects. All of them can be rescued by specific expression of Ime4 cDNA in the nervous system of Ime4 mutants, indicating neuronal functions. This is consistent with the substantial enrichment of m⁶A and its writer proteins in the embryonic neuroectoderm, as well as with the affected genes upon depletion in S2R+ cells. These analyses further reveal notable changes in the architecture of NMJs, potentially explaining the locomotion phenotype. In the mouse, m⁶A is enriched in the adult brain, whereas in zebrafish, METTL3 and WTAP show high expression in the brain region of the developing embryo. Furthermore, a crucial role for the mouse m⁶A demethylase FTO in the regulation of the dopaminergic pathway was clearly demonstrated. Thus, together with previous studies, this work reveals that m⁶A RNA methylation is a conserved mechanism of neuronal mRNA regulation contributing to brain function (Lence, 2016).

Ime4 and dMettl14 also control the splicing of the Sxl transcript, encoding for the master regulator of sex determination in Drosophila. This is in agreement with the previously demonstrated roles of Fl(2)d and Vir in this process. However, in contrast to these mutants, mutants for Ime4, dMettl14 and YT521-B are mostly viable, ruling out an essential role in sex determination and dosage compensation. Only when one copy of Sxl is removed, Ime4 mutant females start to die. Notably, m⁶A effect on Sxl appears more important in the brain compared to the rest of the organism, possibly allowing fly survival in the absence of this modification (Lence, 2016).

The targeted screen identifies Nito as a bona fide methlytransferase complex subunit. The vertebrate homologue of Nito, RBM15, was recently shown to affect XIST gene silencing via recruitment of the methyltransferase complex to XIST RNA, indicating that its role in m⁶A function and dosage compensation is conserved. In summary, this study provides a comprehensive in vivo characterization of m⁶A biogenesis and function in Drosophila, demonstrating the crucial importance of the methyltransferase complex in controlling neuronal functions and fine-tuning sex determination via its nuclear reader YT521-B (Lence, 2016).

m⁶A RNA methylation regulates promoter- proximal pausing of RNA polymerase II

RNA polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. This study shows that the m⁶A RNA modification regulates promoter-proximal RNAP II pausing in Drosophila cells. The m⁶A methyltransferase complex (MTC) and the nuclear reader Ythdc1 are recruited to gene promoters. Depleting the m⁶A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m⁶A catalytic domain. Collectively, these data reveal an important link between RNAP II pausing and the m⁶A RNA modification, thus adding another layer to m⁶A-mediated gene regulation (Akhtar, 2021).

N6-methyladenosine (m6A) has recently emerged as the most-prevalent mRNA modification in eukaryotes, controlling various developmental, cellular, and molecular processes, such as pre-mRNA splicing, alternative polyadenylation, mRNA decay, and translation. Genome-wide mapping of m6A in vertebrates revealed a preferred enrichment at specific subsets of sites centered mostly around stop codons, last exons, and 3' untranslated regions (UTRs) of many mRNAs, as well as, to a lesser extent, at 5' UTRs. m6A is deposited on mRNA by a multi-subunit complex, including methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms-tumor-1-associating protein (WTAP in vertebrates and Fl(2)d in Drosophila), and four additional factors (RBM15/RBM15B in vertebrates, Spenito in Drosophila; VIRMA in vertebrates, Virilizer in Drosophila; ZC3H13 in vertebrates, and Xio/Flacc and HAKAI in both vertebrates and Drosophila). This complex is commonly referred to as the m6A 'writer' or methyltransferase complex (MTC). The downstream function of m6A is mediated by the so-called 'reader' proteins; among which, YTH-domain-containing protein families are the best described. Most m6As are deposited co-transcriptionally, allowing the coupling of this modification with transcription. Indeed, recent studies have shown that m6A complex members can be recruited to the chromatin by chromatin-associated proteins, post-translational modifications of histones, or transcription factors. One interesting aspect, recently uncovered, is the crosstalk between the transcription machinery and translational regulation through the m6A RNA modification. Specifically, RNA polymerase II (RNAP II) kinetics was shown to affect m6A deposition, with slower transcriptional dynamics enhancing the m6A deposition on mRNAs and impeding translation. In addition, m6A was found at many regulatory RNAs, regulating their abundance and causing indirect downstream effects on transcription and chromatin accessibility (Akhtar, 2021).

Transcription is one of the most important cellular mechanism controlling eukaryotic gene expression and follows three different highly regulated stages: initiation, elongation, and termination. After initiation and promoter clearance, RNAP II undergoes a key rate-limiting step of pausing, whereby it stalls and accumulates 30-60 nt downstream of the transcription start site. This mechanism is particularly important for developmentally and stimulus-controlled genes and serves as a checkpoint that coordinates transcription elongation, chromatin modifications, and mRNA processing (Akhtar, 2021).

Pausing has been linked to the phosphorylation state in the heptapeptide repeat of the C-terminal domain (CTD) of the large subunit of RNAP II. The phosphorylation state of CTD correlates with various stages of transcription, for example: Ser5 phosphorylation of CTD is required for mRNA capping and Ser2 phosphorylation is necessary for the recruitment of splicing factors and polyadenylation and corresponds to the elongating form of RNAP II. The transition from pausing to early elongation is critical for gene expression in higher metazoans and requires several factors. In Drosophila, paused genes predominantly fall into two categories: one is associated with GAGA factor (GAF) binding at their promoter, whereas the other binds M1BP. Together with these pause-enhancing factors, eukaryotic cells have also evolved a number of specialized elongation factors, such as polymerase associated factor 1 (PAF1), FACT, and Spt6. Various studies have underlined the significance of this transition and demonstrated its function as a key checkpoint in mRNA processing. Despite the co-transcriptional deposition of m6A, its role in regulation of RNAP II transcription is understudied (Akhtar, 2021).

This study found that the m6A MTC directly regulates the release of RNAP II from its paused state. In Drosophila cells, the complex is predominantly recruited to gene promoters, in an RNA- and transcription-dependent manner but independent of splicing. This study found predictive enrichment of motifs and binding of pause-regulating factors, such as GAF and M1BP, in regions occupied by the m6A MTC and validated their involvement in recruiting m6A MTC at the promoter. Furthermore, evidence is provided that the m6A complex components interact with the histone chaperone Spt6, previously shown to enhance elongation in Drosophila. Moreover, tethering Mettl3 to a heterologous gene promoter is sufficient to enhance the release of RNAP II toward the gene body. Lastly, it was shown that the catalytic domain of Mettl3 is essential for its recruitment to the chromatin and for promoting transcriptional effect. Collectively, these results support a model in which m6A, through its MTC, directly feeds back on the transcription machinery via the control of promoter proximal pausing of RNAP II (Akhtar, 2021).

These data uncovered a novel mechanism in which transcription-dependent recruitment of the m6A complex components positively feedbacks on the transcription machinery to promote release of RNAP II from the paused state. Although this study provides the first evidence for a role of m6A in RNAP II pausing, that link is not unexpected. First, m6A complex components and pause factors are both found at gene promoters, and the most-prevalent paused genes (i.e., developmentally regulated and stress-associated genes) tend to also be bound by the m6A MTC at their promoter. Second, inhibiting elongation by DRB, a well-known elongation inhibitor, had no effect on the recruitment of m6A MTC recruitment, suggesting that this recruitment is independent of, and happens before, transition into elongation. Third, the m6A RNA modification has a critical role during cell fate decision in early mouse development and in stem cell differentiation. These same processes are highly subjected to RNAP II pausing and might involve m6A RNA for their precise control. Interestingly, the current results suggest that m6A RNA modification affects pause regulation on many genes, demonstrating its involvement as a generic contributor in fine-tuning the transcriptional activity of paused genes. Furthermore, the effect of m6A MTC on regulation of pause escape is more pronounced on most paused genes in which m6A MTC recruitment is also most prevalent. Surprisingly, we could also observe binding of the m6A MTC complex on well-characterized enhancers, suggesting an additional uncharacterized function of m6A MTC on gene expression (Akhtar, 2021).

Another interesting aspect of the m6A in pause release comes from studies on DNA:RNA hybrids or R-loops, where m6A was shown to regulate R-loops removal (Abakir, 2020). Many of the pause sites have higher propensities of forming R-loops, and factors favorable for R-loops formation and RNAP II pausing are interconnected. These findings suggest that m6A could potentially mediate R-loop-dependent RNAP II pausing (Akhtar, 2021).

An unsupervised approach was implemented to investigate one of the key questions in the field regarding the specificity of the m6A deposition. Indeed, the degenerate consensus motif RRCAH (in which A = m6A) has been identified as a key motif for m6A deposition in several genome-wide mapping studies. However, the motif itself is more prevalent than the experimental validated frequency of m6A on RNA. This suggests that other factors have a crucial role in targeting m6A on RNA. Coherently, chromatin-associated proteins drive the deposition of m6A on RNA through m6A MTC recruitment on chromatin. One study, using acute myeloid leukemia (AML) cells identified CEBPZ-dependent recruitment of Mettl3 to promoters of a selected set of genes. Another intriguing study demonstrated H3K36me3 as a key determinant of the deposition of m6A on RNA. However, the lack of H3K36me3 at promoters and the occurrence of m6A in the 5' UTR of the genes, especially in Drosophila, suggest that additional features determine the deposition of this mark. Indeed, the distribution of m6A on RNA is different between vertebrates and Drosophila, suggesting that the deposition and ensuing regulation can be species dependent. However, the deposition of m6A in vertebrates has also been reported to be developmental-stage- and cell-type-dependent, with distinct deposition patterns, including on 5' UTR. The approach used in this study identified RNAP II, CTD-state, and pause factors (GAF and M1BP) as the most important features predictive of m6A complex binding at promoters. That is also consistent with its observed effect on RNAP II pause regulation and the transition to elongation. Consistently, the KD of kinases responsible for the phosphorylation of the CTD resulted in the decrease of Mettl3 binding at promoters. Although, that effect could potentially be mediated through other targets of those kinases. Lastly, transcriptional kinetics has also been shown to influence the deposition of this mark on RNA. All the evidence points to the existence of context-dependent features and of extensive crosstalk in guiding m6A and m6A MTC on the RNA and chromatin, respectively. Further investigations are required to identify all the factors responsible for guiding the deposition and removal of this mark in a dynamic biological process (Akhtar, 2021).

This study also identified Spt6, previously reported as elongation factor, interacting with the m6A complex. Recent studies in yeast and plant suggest an additional role of Spt6 in precisely selecting the TSS and preventing cryptic transcription starts. These results indicating a role for Spt6 in the recruitment of MTC at TSS could also implicate m6A in the fidelity of TSS selection. Furthermore, Spt6 participates in mRNA homeostasis by inducing mRNA decay through interactions with RNAP II and the Ccr4-Not deadenylation complex. m6A RNA modification has been also described to control mRNA decay, partly through Ccr4-Not. Those studies, combined with the. current findings, show another potential crosstalk between Spt6 and m6A components and support the existence of a feedback loop between mRNA synthesis and decay, allowing for rapid changes of global mRNA levels of developmental and stress regulated genes (Akhtar, 2021).

Finally, m6A was recently shown to indirectly inhibit transcription by regulating the stability of chromatin-associated RNAs (Liu et al., 2020). Although this result might seem to contradict the current findings (which, rather, point toward a positive action of m6A on transcription regulation), this potential multifaceted and context-dependent regulation is reminiscent of a similar action of the polymerase-associated factor 1 (PAF1). Indeed, depending on the cell type, PAF1 is required for either stabilizing RNAP II pausing or promoting its pause release (Akhtar, 2021).

In conclusion, the m6A-mediated control of RNAP II pausing adds a novel layer to an already highly regulated process. The feedback mechanism reported here establishes m6A as a critical regulator of the transcriptional checkpoint involving RNA processing (Akhtar, 2021).

It remains to be determined whether this function of m6A MTC is conserved across higher metazoans. Moreover, it would be interesting to demonstrate this function in a developmental context for e.g., during embryogenesis. This study, using a machine learning approach, attempted to determine the features associated with the recruitment of m6A MTC to chromatin; however, the resulting binding matrix is certainly not exhaustive. Several additional characteristics could determine the specificity of m6A deposition across the genome. In addition, it would be extremely beneficial to understand whether the chromatin recruitment of m6A MTC is a prerequisite for RNA modification or vice versa (Akhtar, 2021).

mTORC1-chaperonin CCT signaling regulates m(6)A RNA methylation to suppress autophagy

Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism that senses and integrates nutritional and environmental cues with cellular responses. Recent studies have revealed critical roles of mTORC1 in RNA biogenesis and processing. This study finds that the m(6)A methyltransferase complex (MTC) is a downstream effector of mTORC1 during autophagy in Drosophila and human cells. Furthermore, the Chaperonin Containing Tailless complex polypeptide 1 (CCT) complex, which facilitates protein folding, acts as a link between mTORC1 and MTC. The mTORC1 activates the chaperonin CCT complex to stabilize MTC, thereby increasing m(6)A levels on the messenger RNAs encoding autophagy-related genes, leading to their degradation and suppression of autophagy. Altogether, this study reveals an evolutionarily conserved mechanism linking mTORC1 signaling with m(6)A RNA methylation and demonstrates their roles in suppressing autophagy (Tang, 2021).

mTORC1, an evolutionarily conserved serine/threonine kinase, is a master regulator of cell growth, metabolism, and proliferation coupling different nutritional and environmental cues, including growth factors, energy levels, cellular stress, and amino acids, with metabolic programs. For example, insulin activates PI3K/AKT and inhibits the Tuberous Sclerosis Complex (TSC) 1/2, a negative regulator of mTORC1, thus promoting mTORC1 activation. Activated mTORC1 then phosphorylates multiple downstream effectors that control a wide range of anabolic and catabolic processes. Phosphorylation of the ribosomal S6 kinase 1 (S6K1) and eIF4E-binding protein 1 (4E-BP1) by mTORC1 promotes protein translation and enhances cell growth and proliferation. Moreover, autophagy, an intracellular degradation system that delivers cytoplasmic components to lysosomes, is inhibited by mTORC1 through phosphorylation of Atg13 that, in turn, inhibits ULK1 kinase activity (Tang, 2021).

Recent studies have highlighted a role for mTORC1 in regulating RNA metabolism. Through the phosphorylation of RNA metabolic proteins, mTORC1 modulates various RNA biogenesis and processing events. Phosphorylation of the SR protein kinase SRPK2 by S6K1 promotes its transport into the nucleus where it activates SR proteins and induces splicing of lipogenic pre-messenger RNAs (pre-mRNAs) for de novo synthesis of fatty acids and cholesterol, suggesting that SRPK2 is a critical mediator of mTORC1-dependent lipogenesis. In addition, mTORC1 regulates alternative splicing and polyadenylation of autophagic and metabolic genes to control autophagy, lipid, protein, and energy metabolism through the cleavage and polyadenylation complex. Furthermore, mTORC1 mediates phosphorylation of the decapping enzyme Dcp2. Phosphorylated Dcp2 associates with RNA helicase RCK family members and binds to transcripts of Autophagy-related genes (Atg) to degrade them, thereby suppressing autophagy. Altogether, these studies suggest an essential role for mTORC1 in controlling RNA biogenesis and processing, revealing a major function for mTORC1 in the regulation of protein diversity and in reshaping cellular metabolism and autophagy (Tang, 2021).

N6-methyl-adenosine (m6A) is one of the most abundant chemical modifications in eukaryotic mRNA, which is preferentially enriched in 3' UTRs and around stop codons. m6A modification affects almost all aspects of mRNA metabolism, such as splicing, translation, and stability, and plays essential roles in a wide range of cellular processes, including Drosophila sex determination and metabolism. The m6A methyltransferase complex (MTC) catalyzes m6A formation and is composed of the methyltransferase-like protein 3 (METTL3), the methyltransferase-like protein 14 (METTL14), WTAP (the ortholog of Drosophila Fl(2)d), and RBM15/RBM15B (the ortholog of Drosophila Nito). Although METTL3 is the only catalytic component of the MTC, its interaction with METTL14 is necessary for RNA substrate recognition and efficient m6A deposition. WTAP stabilizes the interaction between the two METTL proteins, and RBM15/RBM15B have been proposed to recruit the MTC to its target transcripts (Tang, 2021).

Using autophagy as a readout of mTORC1 signaling in Drosophila, the MTC was identified as a downstream effector of mTORC1 signaling. From the analysis of high-confidence Drosophila and human MTC proteomic data, the Chaperonin Containing Tailless complex polypeptide 1 (CCT) complex was identified as an MTC interactor that mediates the effects of mTORC1 on m6A modification and autophagy. In mammalian cells, it was also found that the CCT complex plays critical roles in the regulation of MTC protein stability and m6A RNA modification, suggesting that the mTORC1-CCT-MTC axis is conserved from Drosophila to mammals. These studies thus unveil a mechanism linking mTORC1 signaling and the chaperonin CCT complex to RNA methylation and also uncover a layer of mTORC1 regulation of autophagy (Tang, 2021).

This study has demonstrate that the MTC acts as a downstream effector of mTORC1 to regulate m6A RNA methylation of Atg transcripts, inducing their degradation and thus suppressing autophagy. Furthermore, the CCT complex was identified as a link between mTORC1 and MTC. CCT downstream of mTORC1 signaling can stabilize METTL3 and METTL14 to up-regulate m6A levels and inhibit autophagy. Accordingly, depletion of either mTORC1, CCT, METTL3, or METTL14 compromises m6A RNA methylation and promotes autophagy. Importantly, the role of mTORC1-CCT-MTC signaling in regulating autophagy is conserved from Drosophila to mammals. Thus, this study discovered a function of mTORC1 in regulating m6A RNA methylation during autophagy (Tang, 2021).

mTORC1 inhibition suppresses protein translation but also affects gene expression at different levels. This study identified an epitranscriptomic mechanism by which mTORC1 activates m6A RNA methylation to promote Atg mRNA turnover and inhibits autophagy. This m6A-mediated mRNA degradation represents a layer of gene regulation by mTORC1. Moreover, as mTORC1 activity regulates global m6A levels, it is likely that the MTC also mediates additional physiological functions of mTORC1. It is noted that depletion of METTL3, METTL14, or CCT8 cannot fully rescue TSC1-induced effects. Although these results could be caused by partial RNAi knockdown, they may also indicate that other pathways contribute to mTORC1 regulation of autophagy. Indeed, studies have reported that mTORC1 suppresses autophagy through modulation of transcription factors, RNA-processing complexes, and mRNA degradation machinery, further highlighting that mTORC1 utilizes multiple RNA biogenesis processes to control autophagy (Tang, 2021).

The catalytic core components of the MTC, METTL3/METTL14, have a substrate sequence specificity for a DRA*CH motif (D = G/A/U, R = G/A, A* = methylated adenosine, H = A/U/C). However, only a subset of consensus sites across the mRNA transcriptome are methylated. Thus, it has been speculated that other factors in the MTC specify METTL3/METTL14 methylation patterns. Proteomic results combined with biochemical validation in both Drosophila and mammalian cells identified multiple splicing factors that interact with known MTC components. Future work will be needed to confirm whether these factors are directly involved in the regulation of m6A methylation and how they coordinate with the m6A machinery to affect RNA processing. It will also be interesting to investigate whether mTORC1 controls other regulators of RNA m6A methylation, in addition to METTL3 and METTL14. Moreover, the proteomics data revealed that multiple components of E3 ubiquitin ligase complex interact with MTC, suggesting that they may be involved in ubiquitination of MTC. Ubiquitination of METTL3 has also been observed, but its function and related E3 ubiquitin ligases remain unclear (Tang, 2021).

Previous genetic analyses showed that the CCT complex functions downstream of mTORC1 and that mTORC1 positively regulates the transcriptional levels of the CCT complex (Kim, 2019). Another study identified CCT2 as a substrate of S6 kinase, a downstream effector of mTOR, in mammalian cells, suggesting that both transcriptional and posttranslational regulations contribute to CCT complex activation by mTORC1. However, the phosphorylation site (Ser-260) of mammalian CCT2 is not conserved in Drosophila and how this phosphorylation modulates CCT function is not clear. Multiple phosphorylation sites have been detected in CCT components. Interestingly, a previous study showed that CCT8 was phosphorylated following insulin stimulation (Vinayagam, 2016), suggesting that other phosphorylation sites are involved in mTORC1-regulated CCT activation. Future studies are needed to comprehensively map the phosphorylation sites on CCT components and investigate their physiological roles (Tang, 2021).

The CCT complex is a highly conserved complex that assists the folding of about 10% of the eukaryotic proteome. The interactions of the CCT complex with METTL3 and METTL14 were observed in a previous study using AP/MS in human cells. Consistently, genetic and biochemical data from this study further confirmed their interactions and characterized the functions of CCT in stabilizing METTL3 and METTL14 and controlling m6A RNA methylation. These findings thus further expand the impact of the CCT complex on RNA metabolism (Tang, 2021).

Multiple studies have reported that CCT complex protein levels dramatically increase in autophagy mutants, proposing that CCT is one of the substrates of autophagy. Future studies will be needed to test whether autophagy is able to degrade the CCT complex and whether autophagy feedback inhibits CCT (Tang, 2021).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Drosophila Inducer of MEiosis 4 (IME4) is required for Notch signaling during oogenesis

N⁶-methyladenosine is a nonediting RNA modification found in mRNA of all eukaryotes, from yeast to humans. Although the functional significance of N⁶-methyladenosine is unknown, the Inducer of MEiosis 4 (IME4) gene of Saccharomyces cerevisiae, which encodes the enzyme that catalyzes this modification, is required for gametogenesis. This study found that the Drosophila IME4 homolog, Dm ime4, is expressed in ovaries and testes, indicating an evolutionarily conserved function for this enzyme in gametogenesis. In contrast to yeast, but as in Arabidopsis, ime4 is essential for viability. Lethality is rescued fully by a wild-type transgenic copy of ime4 but not by introducing mutations shown to abrogate the catalytic activity of yeast Ime4, indicating functional conservation of the catalytic domain. The phenotypes of hypomorphic alleles of ime4 that allow recovery of viable adults reveal critical functions for this gene in oogenesis. Ovarioles from ime4 mutants have fused egg chambers with follicle-cell defects similar to those observed when Notch signaling is defective. Indeed, using a reporter for Notch activation, this study found markedly reduced levels of Notch signaling in follicle cells of ime4 mutants. This phenotype of ime4 mutants is rescued by inducing expression of a constitutively activated form of Notch. This study reveals the function of IME4 in a metazoan. In yeast, this enzyme is responsible for a crucial developmental decision, whereas in Drosophila it appears to target the conserved Notch signaling pathway, which regulates many vital aspects of metazoan development (Hongay, 2011).

This study describes the role of the IME4 mRNA N⁶-adenosine methyltransferase in the development of Drosophila. In contrast to the homologous gene in the unicellular eukaryote S. cerevisiae, Drosophila ime4 is an essential gene. In adults, IME4 is required for male and female fertility. In females, IME4 is essential for oogenesis, and loss of function shows defects consistent with failure in soma–germ line interactions. Notch signaling is reduced in ime4 mutants, suggesting a function for IME4 in the Notch signaling pathway. Furthermore, IME4 probably functions upstream of this signaling pathway, because the expression of a constitutively activated form of Notch rescues the compound egg chamber phenotype of ime4 homozygous females (Hongay, 2011).

The essential function of ime4 probably is a common feature in multicellular organisms. In A. thaliana, MTA, the ime4 homolog, is essential for embryogenesis, because loss-of-function homozygous mutants are unable to proceed through embryogenesis past the globular stage and thus are unable to form differentiated tissues. Although the focus of this report is the function of IME4 in oogenesis, homozygous mutant males also have reduced fertility; thus it will be interesting to determine the function of ime4 in spermatogenesis and uncover similarities or differences in the roles of IME4 in the ovary and testis. Further investigation of IME4 function before its role in adult gametogenesis will reveal whether the protein controls cell differentiation in a variety of developmental contexts. Interestingly, the rare ime4 mutant adults that are obtained have a high incidence of Notched wings, raising the possibility that IME4 is required for Notch signaling in other developmental stages and tissues (Hongay, 2011).

The defects in oogenesis that were observe when ime4 function is compromised can be explained by failure of Notch signaling in follicle cells starting early in the germaria. ime4 mutants and ime4 ablation via RNAi show defects in germ line–soma interactions leading to failure of follicle-cell differentiation, as shown by absence of stalks and polar cells and aberrant germ-line cyst encapsulation, similar to defects previously reported for Notch signaling mutants. Defects in soma–germ-line communication, like those described for Notch signaling mutants, lead to the formation of aberrant egg chambers, which are eliminated via apoptosis. In addition to the phenotypic similarities between ime4 and Notch signaling mutants, significantly lower Notch reporter activity was seen in ime4 mutants than in sibling controls, indicating that Notch signaling is compromised by low levels of IME4. Because the oogenesis phenotype of ime4 homozygous mutants can be rescued fully by expressing an activated form of Notch, this study shows that Notch signaling is the pathway primarily affected in oogenesis in ime4 mutants. Taken together, these data indicate that Dm IME4 is a key player in Notch signaling, probably functioning upstream of Notch activation. It will be interesting to determine how the enzymatic function of IME4 affects this signaling pathway and whether transcripts harboring N⁶-mA–modified mRNA are involved in Notch signaling during soma–germ line interactions (Hongay, 2011).

The function of yeast IME4 is to allow entry into meiosis; thus a defect was expected in meiotic entry in Drosophila ime4 mutants. Because a complete deletion of ime4 is lethal, it was not possible to investigate the phenotypic consequences of total absence of Dm IME4 protein in oogenesis. With this caveat, ime4 mutants that cause reduced fertility and ovary degeneration do not affect the onset of meiosis, as synaptonemal complex assembly was detected in the oocytes of mutant egg chambers (Hongay, 2011).

The protein expression of Dm IME4 and the phenotypes indicate a requirement in both the soma and the germ line. The mutant phenotype of compound egg chambers is caused by the inability of follicle cells to encapsulate a single 16-cell germ-line cyst, suggesting that the major role of IME4 in oogenesis is in the somatic follicle cells. It is possible, however, that soma and germ line have different threshold requirements for IME4 protein levels, and the reduction of IME4 in the hypomorphic alleles may have affected the follicle cells primarily. This study observed, albeit at low frequency, an extra round of mitotic germ-line cyst divisions. This phenotype is a consequence of IME4 acting in the germ line, because it can be reproduced by RNAi knockdowns using germ-line drivers. Taken together, these results suggest that IME4 acts in both germ line and soma and plays a role in signaling between these two lineages during gametogenesis. In follicle cells this signaling appears to be accomplished via the Notch pathway (Hongay, 2011).

In yeast, IME4 controls a crucial developmental decision in this unicellular eukaryote's life cycle: to continue mitosis or to enter the gametogenesis program. The present demonstration of developmental functions of Drosophila IME4 shows how a conserved function can be expanded in evolution, in this case for use in multiple developmental decisions and to target a signal transduction pathway that does not exist in yeast (Hongay, 2011).

Search PubMed for articles about Drosophila Ime4

Abakir, A., Giles, T. C., Cristini, A., Foster, J. M., Dai, N., Starczak, M., Rubio-Roldan, A., Li, M., Eleftheriou, M., Crutchley, J., Flatt, L., Young, L., Gaffney, D. J., Denning, C., Dalhus, B., Emes, R. D., Gackowski, D., Correa, I. R., Jr., Garcia-Perez, J. L., Klungland, A., Gromak, N. and Ruzov, A. (2020). N(6)-methyladenosine regulates the stability of RNA:DNA hybrids in human cells. Nat Genet 52(1): 48-55. PubMed ID: 31844323

Akhtar, J., Renaud, Y., Albrecht, S., Ghavi-Helm, Y., Roignant, J. Y., Silies, M. and Junion, G. (2021). m⁶A RNA methylation regulates promoter- proximal pausing of RNA polymerase II. Mol Cell 81(16): 3356-3367. PubMed ID: 34297910

Haussmann, I. U., Bodi, Z., Sanchez-Moran, E., Mongan, N. P., Archer, N., Fray, R. G. and Soller, M. (2016). m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination. Nature 540(7632): 301-304. PubMed ID: 27919081

Hongay, C. F. and Orr-Weaver, T. L. (2011). Drosophila Inducer of MEiosis 4 (IME4) is required for Notch signaling during oogenesis. Proc Natl Acad Sci U S A 108(36): 14855-14860. PubMed ID: 21873203

Kan, L., Ott, S., Joseph, B., Park, E. S., Dai, W., Kleiner, R. E., Claridge-Chang, A. and Lai, E. C. (2021). A neural m(6)A/Ythdf pathway is required for learning and memory in Drosophila. Nat Commun 12(1): 1458. PubMed ID: 33674589

Kim, A. R. and Choi, K. W. (2019). TRiC/CCT chaperonins are essential for organ growth by interacting with insulin/TOR signaling in Drosophila. Oncogene 38(24): 4739-4754. PubMed ID: 30792539

Lence, T., Akhtar, J., Bayer, M., Schmid, K., Spindler, L., Ho, C. H., Kreim, N., Andrade-Navarro, M. A., Poeck, B., Helm, M. and Roignant, J. Y. (2016). m6A modulates neuronal functions and sex determination in Drosophila. Nature 540(7632): 242-247. PubMed ID: 27919077

Moindrot, B., Cerase, A., Coker, H., Masui, O., Grijzenhout, A., Pintacuda, G., Schermelleh, L., Nesterova, T. B. and Brockdorff, N. (2015). A Pooled shRNA Screen Identifies Rbm15, Spen, and Wtap as Factors Required for Xist RNA-Mediated Silencing. Cell Rep 12(4): 562-572. PubMed ID: 26190105

Patil, D. P., Chen, C. K., Pickering, B. F., Chow, A., Jackson, C., Guttman, M. and Jaffrey, S. R. (2016). m(6)A RNA methylation promotes XIST-mediated transcriptional repression. Nature 537(7620): 369-373. PubMed ID: 27602518

Starck, S. R., Tsai, J. C., Chen, K., Shodiya, M., Wang, L., Yahiro, K., Martins-Green, M., Shastri, N. and Walter, P. (2016). Translation from the 5' untranslated region shapes the integrated stress response. Science 351(6272): aad3867. PubMed ID: 26823435

Tang, H. W., Weng, J. H., Lee, W. X., Hu, Y., Gu, L., Cho, S., Lee, G., Binari, R., Li, C., Cheng, M. E., Kim, A. R., Xu, J., Shen, Z., Xu, C., Asara, J. M., Blenis, J. and Perrimon, N. (2021). mTORC1-chaperonin CCT signaling regulates m(6)A RNA methylation to suppress autophagy. Proc Natl Acad Sci U S A 118(10). PubMed ID: 33649236

Vinayagam, A., Kulkarni, M. M., Sopko, R., Sun, X., Hu, Y., Nand, A., Villalta, C., Moghimi, A., Yang, X., Mohr, S. E., Hong, P., Asara, J. M. and Perrimon, N. (2016). An integrative analysis of the InR/PI3K/Akt network identifies the dynamic response to insulin signaling. Cell Rep 16(11): 3062-3074. PubMed ID: 27626673

date revised: 23 June 2023

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