InteractiveFly: GeneBrief

Hakai: Biological Overview | References

Gene name - Hakai

Synonyms -

Cytological map position - 37F2-37F2

Function - enzyme

Keywords - E3 ubiquitin ligase - required for stabilization of core components of the m6A mRNA methylation machinery - functions in the sex determination pathway - mediates splicing of Sex lethal

Symbol - Hakai

FlyBase ID: FBgn0032812

Genetic map position - chr2L:19,580,612-19,582,474

NCBI classification - RING finger, HC subclass, found in E3 ubiquitin-protein ligase Hakai, zinc finger protein 645 (ZNF645), and similar proteins

Cellular location - nuclear

NCBI links: EntrezGene, Nucleotide, Protein

Hakai orthologs: Biolitmine

N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNA, is installed by a multi-component writer complex; however, the exact roles of each component remain poorly understood. This study shows that a potential E3 ubiquitin ligase Hakai colocalizes and interacts with other m6A writer components, and Hakai mutants exhibit typical m6A pathway defects in Drosophila, such as lowered m6A levels in mRNA, aberrant Sxl alternative splicing, wing and behavior defects. Hakai, Vir, Fl(2)d and Flacc form a stable complex, and disruption of either Hakai, Vir or Fl(2)d led to the degradation of the other three components. Furthermore, MeRIP-seq indicates that the effective m6A modification is mostly distributed in 5' UTRs in Drosophila, in contrast to the mammalian system. Interestingly, it was demonstrated that m6A modification is deposited onto the Sxl mRNA in a sex-specific fashion, which depends on the m6A writer. Together, this work not only advances the understanding of mechanism and regulation of the m6A writer complex, but also provides insights into how Sxl cooperate with the m6A pathway to control its own splicing (Wang, 2021).

There are a variety of chemical modifications on biological macromolecules, such as proteins, nucleic acids, and glycolipids. Like DNA methylation and histone modification, RNA modification represents an extra layer of epigenetic regulatory mechanism. More than 150 chemical modifications in RNA have been discovered, and their biological functions are only starting to be revealed. Chemical modifications of RNA exist in all organisms and for all forms of RNA, including tRNA, rRNA, mRNA, and long noncoding RNA. Common RNA modifications include N6-methyladenosine (m6A), N6,2’-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), 5-methylcytidine (m5C), N4-acetylcytidine (ac4C), 7-methylguanosine (m7G), and pseudouridine (Ψ). Among them, m6A is the most abundant internal modification of mRNA in eukaryotes. Although m6A in mRNA was found more than 40 years ago, it was only recently that the field has made extensive progress owing to technological and experimental breakthroughs. By combining m6A-specific antibody and high-throughput sequencing, MeRIP-Seq or m6A-Seq allows the m6A mapping at the whole transcriptome level, thereby providing the possibility to correlate RNA modifications with their biological functions . These and subsequent studies revealed that m6A sites contain a consensus motif RRACH (R = G/A; H = U/A/C), and m6A peaks are enriched in the 3' untranslated region (UTR) and near the stop codon in yeast and mammals. In Arabidopsis, m6A is enriched not only in 3'UTRs and near the stop codon but also in 5'UTRs and around the start codon. In mammalian cells, m6A also accumulates in the 5'UTR region in response to stress conditions such as heat shock. The distribution of m6A is important since it implies the mechanism by which m6A modification regulates its mRNA (Wang, 2021).

Another major breakthrough is the gradual elucidation of the m6A modification pathway by biochemical and genetic studies. The m6A is deposited by a multicomponent methyltransferase complex ('writers'), mainly recognized by YTH domain-containing 'readers', and can be removed by FTO and ALKBH5 'erasers', although FTO was also indicated as an m6Am demethylase. The key catalytic component of the m6A writer complex, Mettl3, was purified and cloned in the 1990s. Since then, studies from yeast, Arabidopsis, Drosophila, and mammalian cells have identified several core components of the writer complex, including Mettl14, WTAP (Fl(2)d), VIRMA (Virilizer), RBM15/15B (Spenito), ZC3H13 (Flacc or Xio), and Hakai. Interestingly, Fl(2)d, Virilizer (Vir), Spenito (Nito), and Xio were first identified from Drosophila sex determination screens and later realized as part of the writer complex. They regulate Drosophila sex determination by controlling the alternative splicing of the master regulatory gene Sex-lethal (Sxl). Recently, Mettl3, Mettl14, as well as the reader Ythdc1, were also shown to be involved in this process. However, the detailed mechanism of how the m6A modification cooperates with Sxl protein to modulate its own splicing is still unclear. Thus, Drosophila can serve as a unique system to screen components in the m6A pathway and pinpoints a critical role for m6A in regulating splicing. Other than Sxl splicing, Drosophila m6A genes are highly expressed in the nervous system and exhibit similar wing and behavior defects when mutated. Mutants of several fly m6A factors are viable and thus provide an ideal model to study other processes, such as metabolism and immunity, in the future (Wang, 2021).

Hakai, also known as CBLL1, was found as an interacting protein with several m6A writer components in proteomic studies. It encodes a RING finger-type E3 ubiquitin ligase and was originally identified as an E-cadherin-binding protein in human cell lines. It was proposed that Hakai ubiquitinates E-cadherin at the plasma membrane and induces its endocytosis, thus playing a negative role post-translationally. Due to the key role of E-cadherin in tumor metastasis, especially epithelial-mesenchymal transition, Hakai has been extensively studied mainly using cell culture and overexpression system, but a previous study using the Drosophila model did not observe an increase of E-cadherin level in Hakai mutants. In Arabidopsis, Hakai mutants show partially reduced m6A levels and the mutant phenotypes are weaker than other writer components. Importantly, the in vivo role of Hakai as a core m6A writer component has not been studied in any animal species. This study analyzed the role of Hakai in the Drosophila m6A modification pathway. The results demonstrated that Hakai is a bona fide member of the m6A writer complex, with its mutants showing reduced global m6A levels, typical m6A mutant phenotypes, and commonly-regulated gene sets. A high-quality fly m6A methylome was obtained using stringent MeRIP-seq, discovered a female-specific m6A methylation pattern for Sxl mRNA, characterized the role of Hakai in the m6A writer complex, and finally revisited the function of Hakai in E-cadherin regulation (Wang, 2021).

m6A modification has been known for more than 40 years but has recently gained great attention due to the emergence of technologies to map m6A methylome, as well as the identification of the writers, readers, and erasers in this pathway. Since the initial purification of the key methyltransferase Mettl3, other components of the writer complex were gradually identified through biochemical experiments and genetic screens. It is now known that m6A writer complex is comprised of multiple components including Mettl3, Mettl14, WTAP, VIRMA, RBM15/15B, ZC3H13. Hakai was first indicated as a WTAP interaction protein and was shown later to be required for full m6A methylation in Arabidopsis; however, its role in the m6A pathway in animals has not been studied. This study shows that Hakai interacts with other m6A writer subunits, and Hakai mutants exhibit characteristic m6A pathway phenotypes, such as lowered m6A levels in mRNA, aberrant alternative splicing of Sxl and other genes, held-out wings, and flightless flies, as well as reduced m6A peaks shared with Mettl3 and Mettl14 mutants in MeRIP-seq. Altogether, these data unambiguously argue that Hakai is the seventh, and likely last core component of the conserved m6A writer complex (Wang, 2021).

Each component in the m6A writer complex plays a role in mRNA methylation but their exact roles are not well understood. This systematic analysis of several m6A writer subunits has provided insights into the mechanism of this important complex. l(2)d, Vir, Hakai, and Flacc were found to form a stable complex, and knocking down either of Fl(2)d, Vir, or Hakai led to the degradation of the other three components. Mettl3, Mettl14, and Nito were not affected by the disruption of Fl(2)d, Vir or Hakai, suggesting that they have separate functions. Knocking down Flacc resulted in less nuclear staining of Fl(2)d, consistent with a role in nuclear localization of the writer complex. Based on these results, a model is proposed for the m6A methyltransferase complex. Mettl3 and Mettl14 form a stable heterodimer to catalyze the addition of the methyl group to mRNA. Nito/RBM15 contains three RRM domains and binds to positions adjacent to m6A sites, thus may provide target specificity for the m6A writer complex. Fl(2)-Vir-Hakai-Flacc form a platform to connect different components and may integrate environmental and cellular signals to regulate m6A methylation (Wang, 2021).

Hakai is a potential E3 ubiquitin ligase with an intact C3HC4 RING domain and a C2H2 domain. Its absence led to the degradation, rather than the accumulation of other m6A writer subunits, indicating that it may not act as an E3 ubiquitin ligase in this complex. Hakai was initially identified as an E-cadherin binding protein to downgrade its levels CR50, and the role of Hakai in cell proliferation and tumor progression was extensively studied in cell culture. However, the current in vivo analysis using various genetic tools did not find a role of Hakai in E-cadherin regulation. In addition, Hakai appeared as a ubiquitous nuclear protein showing little co-localization with E-cadherin in the membrane. Consistently, Hakai was shown to interact with PTB-associated splicing factor (PSF), a nuclear protein, and to affect its RNA-binding ability. Thus, the role of Hakai in E-cadherin regulation needs to be further investigated using the knockout mouse model and whether Hakai has other substrates for its E3 ligase activity also needs to be determined (Wang, 2021).

Recent emerging studies suggest that m6A is involved in numerous developmental processes and human diseases, mainly by regulating mRNA stability, translation, or splicing. Pioneer work has established the framework for the m6A pathway in Drosophila. However, only published Drosophila m6A methylome was performed in S2R + cells or embryos and was not done against writer mutants. Other than Sxl, few m6A target loci have been firmly mapped. By performing MeRIP-seq in wild-type adult flies as well as Mettl3, Mettl14, and Hakai mutants, this study demonstrated that although most m6A peaks are distributed in 3'UTRs, the functional peaks responding to the loss of m6A writers are mainly located in 5'UTRs. This finding indicates a major difference between Drosophila and mammalian m6A methylome, that mainly occurs in 3'UTRs, and is in agreement with a recently published manuscript using miCLIP. Interestingly, LC-MS data show that the overall level of m6A modification in Drosophila only accounted for 10-20% of that in mammalian cells. Mettl3 or Mettl14 mutants are embryonic lethal in mice while they develop into adults in flies. It is possible that the m6A pathway acquires additional functions during evolution (Wang, 2021).

m6A modification in 3'UTRs usually causes mRNA instability and m6A in 5'UTRs is linked to translation enhancement. In agreement with the view that functional m6A peaks are located in 5'UTRs in Drosophila, this study did not observe an increase in mRNA half-life of m6A targets in Mettl3 mutants compared to wild-type. These results imply that the major role of m6A modification in Drosophila is not on mRNA degradation, but possibly on translation upregulation, which can be tested by combining ribosome profiling and functional analysis of a single transcript in the future. The current data by combining MeRIP-seq and splicing analysis shed light on how the m6A modification contributes to splicing regulation. In all five cases analyzed, four (Dsp1, CG8929, fl(2)d, Aldh-III) in 5'UTRs and one (Sxl) in exon/intron, reduction of m6A modification was correlated with enhanced splicing, arguing that the normal role of these modifications might be to repress splicing events nearby (Wang, 2021).

Last but probably the most interesting finding from this work is to demonstrate the female-specific m6A modification around Sxl exon3. Sxl is a textbook paradigm to study alternative splicing and has been intensively investigated for more than thirty years. Sxl protein binds to its own mRNA to control the alternative splicing, but its binding sites are located ~200 nucleotides downstream or upstream of the male exon, meaning other regulators should be involved. Recently, the m6A modification pathway was shown to modulate Sxl alternative splicing, but the detailed mechanism has not been resolved. The MeRIP-seq data revealed that several m6A peaks were deposited only in females on and around Sxl exon3, and they were in the vicinity of Sxl-binding sites. This finding was further validated by independent m6A-IP-qPCR and showed that these modifications were reduced in Mettl3 mutant females. This unexpected finding suggests a model that one main function of Sxl may be to recruit the m6A writer complex that methylates nearby m6A sites. The m6A reader Ythdc1 in turn binds to these sites and might interact with the splicing machinery to repress splicing. Future experiments, such as interactions between Sxl and Mettl3/Mettl14, interactions between Ythdc1 and general splicing factors, mapping of the exact m6A methylation site in Sxl at the single nucleotide level, comparison of transcriptome-wide binding sites of Sxl with m6A modification sites, will be required to firmly prove the model (Wang, 2021).

Hakai is required for stabilization of core components of the m6A mRNA methylation machinery

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. This study shows that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. Its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, this study demonstrates that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. This work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex (Bawankar, 2021)

N6-methyladenosine (m6A) is one of the most abundant and well-studied mRNA modifications in eukaryotes. This modification plays a central role in almost every aspect of mRNA metabolism, and is essential for several biological processes such as cell differentiation, DNA repair, circadian rhythm, neurogenesis and sex determination, among others. Its dysregulation in humans is associated with numerous diseases, including metabolic alteration, neuronal disorders and various types of cancers. The downstream effects of m6A are generally mediated by YTH domain RNA-binding proteins, known as 'm6A readers' that preferentially bind m6A modified RNAs and affect their fate (Bawankar, 2021)

m6A on mRNA is deposited co-transcriptionally by a conserved multiprotein complex that can be divided into two stable sub-complexes: the heterodimer METTL3/METTL14 also known as m6A-METTL Complex (MAC) that contains the catalytic activity, and the m6A-METTL Associated Complex (MACOM) that is required for full MAC activity and includes WTAP (Fl(2)d), VIRMA (Virilizer), RBM15/RMB15B (Spenito) and ZC3H13 (Flacc). More recently, another factor named HAKAI was found associated with components of MACOM in plants and human cells and required to maintain m6A level (Ruzicka, 2017; Yue, 2018; Bawankar, 2021 and references therein)

The precise function of MAC and MACOM components has been the subject of intense research over the past years. Structural studies revealed that METTL3 and METTL14 form a stable heterodimer and that METTL3 is the only factor that contains the catalytic activity since METTL14 is unable to bind the methyl group donor S-Adenosylmethionine. Nevertheless, METTL14 is essential to support the interaction of the complex with its RNA targets and to enhance METTL3 activity. WTAP was shown to stabilize the interaction between METTL3 and METTL14 and to recruit the METTL3/METTL14 heterodimer into the nuclear speckles. RBM15/RBM15B is an RNA-binding protein that recognizes U-rich sequences on the mRNA and is suggested to recruit the m6A machinery in close proximity to these sites. Furthermore, VIRMA was proposed to facilitate selective m6A installation near the stop codon and in the 3' UTR of mRNAs through its interaction with polyadenylation cleavage factors CPSF5 and CPSF627. Lastly, four recent studies identified ZC3H13 as part of MACOM. ZC3H13 was found to stabilize the interaction between WTAP and RBM15 in mouse embryonic stem cells as well as in flies and to contribute to the localization of the writer complex to the nucleus. The only m6A writer component whose function is still poorly explored is HAKAI (Bawankar, 2021)

HAKAI, also known as CBLL1, is a RING-finger type E3 ubiquitin ligase that mediates ubiquitination and subsequent endocytosis of the E-cadherin complex, leading to cell-cell adhesion loss and increased cell motility. HAKAI can also regulate cell proliferation in an E-cadherin-independent manner by affecting the ability of the PTB-associated splicing factor to bind some of its RNA targets. During the last decades, HAKAI has been mostly studied in the context of epithelial-mesenchymal transitions and cancer progression. However, as aforementioned, it became increasingly clear that HAKAI is also a component of the m6A biogenesis machinery in vertebrates as well as in plants. HAKAI was identified as one of the strongest WTAP interactors in mammalian cells and as part of an evolutionary conserved protein complex including WTAP, VIRMA and ZC3H1339. Furthermore, Hakai mutant in Arabidopsis thaliana displayed mild developmental defects together with reduced m6A levels. Recently, Hakai was identified among the top enriched proteins in th Spenito (Nito) interactome in Drosophila S2R+ cells, suggesting its evolutionary conserved role within the m6A pathway (Bawankar, 2021)

This study reports that Hakai is a conserved member of MACOM and is essential for m6A deposition in flies. In line with its role in the m6A pathway, Hakai functions in the sex determination pathway and mediates splicing of Sex lethal. Moreover, its depletion results in altered gene expression and splicing changes that resemble the loss of other MACOM components. This study found that Hakai in flies encodes short and long protein isoforms that display distinct subcellular localization. Its ubiquitin ligase domain is required for homodimerization and interaction with other MACOM components. Finally, it was shown that Hakai removal leads to a severe reduction of Virilizer (Vir), Fl(2)d and Flacc protein levels, indicating that Hakai is essential for maintaining the stability of MACOM components (Bawankar, 2021)

It was recently shown that two conserved sub-complexes, MAC and MACOM interact to deposit m6A on mRNA in flies and mice (Knuckles, 2018). While the structure of the catalytic MAC, which consists of the heterodimer METTL3 and METTL14, has been thoroughly characterized, knowledge of MACOM is limited. In particular, the full composition, assembly and exact function of each subunit have remained unclear. This study identifies Hakai as an integral component of MACOM in Drosophila and human cells. In line with this function, it was shown that Hakai interacts with Vir and other MACOM components, and its depletion reduced m6A levels and led to altered gene expression, resembling loss of other MACOM subunits. Furthermore, flies lacking Hakai are lethal and display aberrant splicing of Sex lethal, consistent with the role of MACOM in sex determination and dosage compensation pathways. The few individuals that escape lethality are flightless, as shown earlier in other mutants of the m6A pathway. Mechanistically, it was found that Hakai is required to stabilize several MACOM components, likely explaining its requirement for m6A deposition (Bawankar, 2021)

The question that arises is whether all MACOM components have now been identified. Five factors, which include Fl(2)d, Vir, Flacc, Nito and Hakai have been validated. Earlier biochemical studies estimated a molecular weight of 875 kDa for the large form of the human methyltransferase complex. The calculated total molecular weight of the combined five factors corresponds to 600 kDa, which suggests that the complex contains additional factors or multiple copies of the known factors. The data show that Hakai, Fl(2)d and Nito have the ability to self-interact. If it is assumed that these three factors are present as dimers, the total weight reaches up to 868 kDa, which would be consistent with the predicted mass of MACOM. Nevertheless, additional biochemical and structural characterization will be required to confirm the exact identity and stoichiometry of the different complex components (Bawankar, 2021)

While Hakai is undoubtedly a core component of the complex, it is surprising that its gene inactivation results in milder phenotypes in comparison to the inactivation of other MACOM components. Indeed, the few females that escaped developmental lethality in the Hakai loss-of-function mutant did not display male sex combs and this phenotype was also not observed in a sensitized background with reduced Hakai dosage. However, the females did show male pigmentation on their abdomen, indicating tissue-specific alteration of sex determination. These milder defects are consistent with quantification of the m6A level in the Hakai mutant, which appeared reduced but not completely absent, as observed in the Mettl3 mutant. Also, Sxl splicing showed tissue-specific alterations, indicative of local requirement for this factor. These results are consistent with a previous study in Arabidopsis showing less pronounced impact of Hakai on the m6A levels as well as on organismal development. This apparent discrepancy may be explained by the function uncovered in this work. The data show that upon depletion of Hakai the level of some of the other MACOM components is reduced but not completely lost, which suggests that the remaining MACOM could still support methylation. In this case, tissue-specific requirement may be mediated through differential expression of factors that may impact on this residual interaction and could determine tissue-specific levels of m6A. Additional work would be required to test this hypothesis (Bawankar, 2021)

In vertebrates, Hakai was shown to ubiquitinate E-cadherin and promote its degradation. In contrast, the current data from Drosophila cells provide no evidence for ubiquitination activity towards MACOM components or any other proteins. Instead, this work strongly suggests that Hakai is required for the stability of three MACOM components, Vir, Fl(2)d and Flacc, independently of its enzymatic activity. The question that remains is how does Hakai exert this function. It was previously shown that the so-called 'orphan proteins' are unstable and get degraded if their protein partners that constitute a common complex are absent. Destabilization can be triggered due to aberrant protein folding, altered localization or because of exposure of normally protected protein binding interfaces. It is therefore possible that Hakai stabilizes other MACOM components via one of these mechanisms. If this model is true, other components of the complex are also expected to stabilize each other. Indeed, it was found that protein levels of Vir, Fl(2)d and Flacc were also strongly reduced upon loss of Vir or Fl(2)d. Interestingly, in mES cells a strong reduction of Wtap levels was previously reported upon loss of Virma and a strong destablization of Zc3h13 was observed upon depletion of Virma, Wtap or Hakai34, suggesting a conserved mechanism for Hakai-Vir-Fl(2)d-Flacc stabilization between flies and mice. It was further demonstrated that protein levels of Hakai and Nito are unperturbed upon depletion of other MACOM components, suggesting that Hakai and Nito might function in additional processes not linked to MACOM or m6A-deposition. This is in agreement with observations that Hakai also localizes in the cytoplasm, and with a study that found Nito (RBM15) in an evolutionary conserved protein complex with proteins unrelated to remaining MACOM components (Wen, 2018; Bawankar, 2021 and references therein).

Given the insights from the current study, the following model of MACOM assembly is proposed: (1) Fl(2)d-Vir-Hakai form a minimal protein unit that is required for the assembly and functionality of the remaining methyltransferase complex. (2) Stability of Fl(2)d and Vir depends on each other and Hakai, but is largely independent of Flacc and Nito. (3) Fl(2)d-Vir-Hakai can interact with MAC, however, this is not sufficient for m6A deposition. (4) Joining of Flacc and Nito is essential for the formation of a complete MACOM complex that can bind and methylate its targets together with MAC (Bawankar, 2021)

In conclusion, this work revealed the requirement of Hakai for m6A deposition in Drosophila. The apparent dependency of Fl(2)d, Vir and Flacc for each other’s stability might be an important mechanism that maintains an equilibrium of protein stoichiometry for a complete complex assembly in order to prevent aberrant interactions of orphan subunits and unwanted m6A installation. Given the critical role of m6A in multiple physiological processes, it will be important to address whether perturbation of this equilibrium, for instance, by single nucleotide polymorphism, may impact the development or severity of pathological conditions (Bawankar, 2021)

Essential requirement for RING finger E3 ubiquitin ligase Hakai in early embryonic development of Drosophila

Hakai is a RING finger type E3 ubiquitin ligase that is highly conserved in metazoans. Mammalian Hakai was shown to bind and ubiquitinate the intracellular domain of E-cadherin, and this activity is implicated in down-regulation of E-cadherin during v-Src-induced cellular transformation. To evaluate this model in vivo, the function of the Drosophila homologue of Hakai was studied. In cultured S2 cells, Drosophila Hakai and E-cadherin (Shotgun) formed a complex in a way distinct from the interaction described for mammalian counterparts. Hakai null mutants died during larval stages but this lethality could be offset by a HA-tagged Hakai construct. While zygotic Hakai function was dispensable for cell proliferation and differentiation in the wing disc epithelium, maternal Hakai mutants showed a variety of defects in epithelial integrity, including stochastic loss of E-cadherin expression and reduction of aPKC; defects in cell specification and cell migration were also observed. No increase of E-cadherin, however, was observed. Regulation of multiple target proteins under control of Hakai is, therefore, essential for early embryonic morphogenesis in Drosophila (Kaido, 2009).

Functions of Hakai orthologs in other species

Zc3h13 Regulates Nuclear RNA m6A Methylation and Mouse Embryonic Stem Cell Self-Renewal

N(6)-methyladenosine (m6A) is an abundant modification in eukaryotic mRNA, regulating mRNA dynamics by influencing mRNA stability, splicing, export, and translation. However, the precise m6A regulating machinery still remains incompletely understood. This study demonstrates that ZC3H13, a zinc-finger protein, plays an important role in modulating RNA m6A methylation in the nucleus. Knockdown of Zc3h13 in mouse embryonic stem cell significantly decreases global m6A level on mRNA. Upon Zc3h13 knockdown, a great majority of WTAP, Virilizer, and Hakai translocate to the cytoplasm, suggesting that Zc3h13 is required for nuclear localization of the Zc3h13-WTAP-Virilizer-Hakai complex, which is important for RNA m6A methylation. Finally, Zc3h13 depletion, as does WTAP, Virilizer, or Hakai, impairs self-renewal and triggers mESC differentiation. Taken together, these findings demonstrate that Zc3h13 plays a critical role in anchoring WTAP, Virilizer, and Hakai in the nucleus to facilitate m6A methylation and to regulate mESC self-renewal (Wen, 2018).

VIRMA mediates preferential m6A mRNA methylation in 3'UTR and near stop codon and associates with alternative polyadenylation

N(6)-methyladenosine (m6A) is enriched in 3'untranslated region (3'UTR) and near stop codon of mature polyadenylated mRNAs in mammalian systems and has regulatory roles in eukaryotic mRNA transcriptome switch. Significantly, the mechanism for this modification preference remains unknown, however. This study reports a characterization of the full m6A methyltransferase complex in HeLa cells identifying METTL3/METTL14/WTAP/VIRMA/HAKAI/ZC3H13 as the key components, and it was shown that VIRMA mediates preferential mRNA methylation in 3'UTR and near stop codon. Biochemical studies reveal that VIRMA recruits the catalytic core components METTL3/METTL14/WTAP to guide region-selective methylations. Around 60% of VIRMA mRNA immunoprecipitation targets manifest strong m6A enrichment in 3'UTR. Depletions of VIRMA and METTL3 induce 3'UTR lengthening of several hundred mRNAs with over 50% targets in common. VIRMA associates with polyadenylation cleavage factors CPSF5 and CPSF6 in an RNA-dependent manner. Depletion of CPSF5 leads to significant shortening of 3'UTR of over 2800 mRNAs, 84% of which are modified with m6A and have increased m6A peak density in 3'UTR and near stop codon after CPSF5 knockdown. Together, these studies provide insights into m6A deposition specificity in 3'UTR and its correlation with alternative polyadenylation (Yue, 2018).

Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI

N6-adenosine methylation (m6A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, this study identified a core set of mRNA m6A writer proteins in Arabidopsis thaliana. The components required for m6A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m6A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m6A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m6A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences (Ruzicka, 2017).


Search PubMed for articles about Drosophila Hakai

Bawankar, P., Lence, T., Paolantoni, C., Haussmann, I. U., Kazlauskiene, M., Jacob, D., Heidelberger, J. B., Richter, F. M., Nallasivan, M. P., Morin, V., Kreim, N., Beli, P., Helm, M., Jinek, M., Soller, M. and Roignant, J. Y. (2021). Hakai is required for stabilization of core components of the m6A mRNA methylation machinery. Nat Commun 12(1): 3778. PubMed ID: 34145251

Kaido, M., Wada, H., Shindo, M. and Hayashi, S. (2009). Essential requirement for RING finger E3 ubiquitin ligase Hakai in early embryonic development of Drosophila. Genes Cells 14(9): 1067-1077. PubMed ID: 19682089

Knuckles, P., Lence, T., Haussmann, I. U., Jacob, D., Kreim, N., Carl, S. H., Masiello, I., Hares, T., Villasenor, R., Hess, D., Andrade-Navarro, M. A., Biggiogera, M., Helm, M., Soller, M., Buhler, M. and Roignant, J. Y. (2018). c3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m6A machinery component Wtap/Fl(2)d. Genes Dev. PubMed ID: 29535189

Ruzicka, K., Zhang, M., Campilho, A., Bodi, Z., Kashif, M., Saleh, M., Eeckhout, D., El-Showk, S., Li, H., Zhong, S., De Jaeger, G., Mongan, N. P., Hejatko, J., Helariutta, Y. and Fray, R. G. (2017). Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI. New Phytol 215(1): 157-172. PubMed ID: 28503769

Wang, Y., Zhang, L., Ren, H., Ma, L., Guo, J., Mao, D., Lu, Z., Lu, L. and Yan, D. (2021). Role of Hakai in m6A modification pathway in Drosophila. Nat Commun 12(1): 2159. PubMed ID: 33846330

Wen, J., Lv, R., Ma, H., Shen, H., He, C., Wang, J., Jiao, F., Liu, H., Yang, P., Tan, L., Lan, F., Shi, Y. G., He, C., Shi, Y. and Diao, J. (2018). Zc3h13 regulates nuclear RNA m6A methylation and mouse embryonic stem cell self-renewal. Mol Cell 69(6): 1028-1038 e1026. PubMed ID: 29547716

Yue, Y., Liu, J., Cui, X., Cao, J., Luo, G., Zhang, Z., Cheng, T., Gao, M., Shu, X., Ma, H., Wang, F., Wang, X., Shen, B., Wang, Y., Feng, X., He, C. and Liu, J. (2018). VIRMA mediates preferential m6A mRNA methylation in 3'UTR and near stop codon and associates with alternative polyadenylation. Cell Discov 4: 10. PubMed ID: 29507755

Biological Overview

date revised: 20 October 2021

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