InteractiveFly: GeneBrief

Methyltransferase like 3: Biological Overview | References


Gene name - Methyltransferase like 3

Synonyms - Ime4

Cytological map position - 95D8-95D8

Function - enzyme

Keywords - methyltransferase, internal modification of mRNA, alternative splicing, Notch signaling, sex determination and dosage compensation, modulation of neural functions

Symbol - Mettl3

FlyBase ID: FBgn0039139

Genetic map position - chr3R:24,032,157-24,034,257

NCBI classification - MT-A70 is the S-adenosylmethionine-binding subunit of human mRNA:m6A methyl-transferase (MTase)

Cellular location - nucleus



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Guo, J., Tang, H. W., Li, J., Perrimon, N. and Yan, D. (2018). Xio is a component of the Drosophila sex determination pathway and RNA N(6)-methyladenosine methyltransferase complex. Proc Natl Acad Sci U S A. PubMed ID: 29555755
Summary:
N(6)-methyladenosine (m(6)A), the most abundant chemical modification in eukaryotic mRNA, has been implicated in Drosophila sex determination by modifying Sex-lethal (Sxl) pre-mRNA and facilitating its alternative splicing. This study identified a sex determination gene, CG7358, and renamed it xio according to its loss-of-function female-to-male transformation phenotype. xio encodes a conserved ubiquitous nuclear protein of unknown function. Xio was shown to colocalize and interacts with all previously known m(6)A writer complex subunits (METTL3, METTL14, Fl(2)d/WTAP, Vir/KIAA1429, and Nito/Rbm15) and that loss of xio is associated with phenotypes that resemble other m(6)A factors, such as sexual transformations, Sxl splicing defect, held-out wings, flightless flies, and reduction of m(6)A levels. Thus, Xio encodes a member of the m(6)A methyltransferase complex involved in mRNA modification. Since its ortholog ZC3H13 (or KIAA0853) also associates with several m(6)A writer factors, the function of Xio in the m(6)A pathway is likely evolutionarily conserved.
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 Ime4null. 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 Ime4null 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 Ime4null 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 (Ime4Δ22-3 females crossed with Sxl7B0 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 (Sxl7B0/FM7 females crossed with Ime4null 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 Sxl7B0/+;Ime4null/+ daughters from Ime4null females or heterozygous Sxl7B0 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 Ime4null females, splice junction reads were examined from RNA-seq. Besides the significant increase in inclusion of the male-specific Sxl exon in Ime4null 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 Ime4null mutants, X-linked, but not autosomal, genes are significantly upregulated in Ime4null 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 vir2F 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 Ime4null heterozygosity, further demonstrating the involvement of the m6A methylosome in Sxl alternative splicing (Haussmann, 2016).

Next, alternative splicing changes was globally analyzed in Ime4null 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 Ime4null 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 Ime4null 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 Ime4null 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 Ime4null 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-BMI02006 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 Ime4null 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).

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).


REFERENCES

Search PubMed for articles about Drosophila Ime4

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

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


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