A unified model for the function of YTHDF proteins in the regulation of m6A-modified mRNA
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PROVIDER: PRJNA554903 | ENA |
REPOSITORIES: ENA
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Project description:N6-methyladenosine (m6A) is the most abundant mRNA nucleotide modification and regulates critical aspects of cellular physiology and differentiation. m6A is thought to mediate its effects through a complex network of interactions between different m6A sites and three functionally distinct cytoplasmic YTHDF m6A-binding proteins (DF1, DF2, and DF3). In contrast to the prevailing model, we show that DF proteins bind the same m6A-modified mRNAs, rather than different mRNAs. Furthermore, we find that DF proteins do not induce translation in HeLa cells. Instead, the DF paralogs act redundantly to mediate mRNA degradation and cellular differentiation. The ability of DF proteins to regulate stability and differentiation becomes evident only when all three DF paralogs are simultaneously depleted. Our studies reveal a unified model of m6A function in which all m6A-modified mRNAs are subjected to the combined action of the YTHDF proteins in proportion to the number of m6A sites.
2020-06-02 | GSE134380 | GEO
Project description:N6-methyladenosine (m6A) is the most abundant internal mRNA nucleotide modification in mammals, regulating critical aspects of cell physiology and differentiation. The YTHDF proteins are the primary readers of m6A modifications and exert physiological functions of m6A in the cytosol. Elucidating the regulatory mechanisms of YTHDF proteins is critical to understanding m6A biology. Here, we report a mechanism that protein post-translational modifications control the biological functions of the YTHDF proteins. We find that YTHDF1 and YTHDF3, but not YTHDF2, carry high levels of nutrient-sensing O-GlcNAc modifications. O-GlcNAc modification attenuates the translation promoting function of YTHDF1 and YTHDF3 by blocking their interactions with proteins associated with mRNA translation. We further demonstrate that O-GlcNAc modifications on YTHDF1 and YTHDF2 regulate the assembly, stability, and disassembly of stress granule, facilitating rapid exchange of m6A-modified mRNAs in stress granules for recovery from stress. Therefore, our results discover an important regulatory pathway of YTHDF functions, adding an additional layer of complexity to the post-transcriptional regulation function of mRNA m6A.
2023-08-31 | GSE216570 | GEO
Project description:N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNAs (mRNAs), and plays important roles in cell differentiation and organism development. It regulates multiple steps throughout the RNA life cycle including RNA processing, translation, and metabolism, via the recognition by selective binding proteins. In cytoplasm, m6A binding protein YTHDF1 facilitates translation of m6A-modified mRNAs, and YTHDF2 accelerates the decay of m6A-modified transcripts. The biological function of YTHDF3, another cytoplasmic m6A binder of the YTH domain family, remains unknown. Here, we report that YTHDF3 promotes protein synthesis in synergy with YTHDF1, and affects methylated mRNA decay mediated by YTHDF2. Cells deficient in all of YTHDF proteins experience the most dramatic accumulation of the m6A-methylated transcripts. These results indicate that in cytoplasm, YTHDF proteins act in an integrated and cooperative network to accelerate metabolism of m6A-modified mRNAs. The combinative and dynamic nature of YTHDF proteins may collectively impact fundamental biological processes and diseases related to m6A RNA methylation.
2016-09-15 | GSE86214 | GEO
Project description:The roles of epitranscriptomic modifications in mRNA regulation have recently received substantial attention, with appreciation growing for their phenotypically selective impacts within the animal. We adopted Drosophila melanogaster as a model system to study m6A, the most abundant internal modification of mRNA. Here, we report proteomic and functional analyses of fly m6A-binding proteins, confirming nuclear (YTHDC) and cytoplasmic (YTHDF) YTH domain proteins as the major m6A binders. Since all core m6A pathway mutants are viable, we assessed in vivo requirements of the m6A pathway in cognitive processes. Assays of short term memory revealed an age-dependent requirement of m6A writers working via YTHDF, but not YTHDC, comprising the first phenotypes assigned to Drosophila mutants of the cytoplasmic m6A reader. These factors promote memory via neural-autonomous activities, and are required in the mushroom body, the center for associative learning. To inform their basis, we mapped m6A from wild-type and mettl3 null mutant heads, allowing robust discrimination of Mettl3-dependent m6A sites. In contrast to mammalian m6A, which is predominant in 3' UTRs, Drosophila m6A is highly enriched in 5' UTRs and occurs in an adenosine-rich context. Genomic analyses demonstrate that Drosophila m6A does not directionally affect RNA stability, but is preferentially deposited on genes with low translational efficiency. However, functional tests indicate a role for m6A in translational activation, since we observe reduced nascent protein synthesis in mettl3-KO cells. Finally, we show that ectopic YTHDF can increase m6A target reporter output in an m6A-binding dependent manner, and that this activity is required for in vivo neural function of YTHDF in memory. Altogether, we provide the first tissue-specific m6A maps in this model organism and reveal selective behavioral and translational defects for m6A/YTHDF mutants.
2021-01-22 | GSE147230 | GEO
Project description:N6-methyladenosine (m6A) in mRNA is key to eukaryotic gene regulation. Many m6A functions involve RNA-binding proteins that recognize m6A via a YT521-B Homology (YTH) domain. YTH domain proteins contain long intrinsically disordered regions (IDRs) that may mediate phase separation and interaction with protein partners, but whose precise biochemical functions remain largely unknown. The Arabidopsis thaliana YTH domain proteins ECT2, ECT3 and ECT4 accelerate organogenesis through stimulation of cell division in organ primordia. Here, we use ECT2 to reveal molecular underpinnings of this function. We show that stimulation of leaf formation requires the long N-terminal IDR, and we identify two short IDR-elements required for ECT2-mediated organogenesis. Of these two, a 19-amino acid region containing a tyrosine-rich motif conserved in both plant and metazoan YTHDF proteins is necessary for binding to the major cytoplasmic poly(A)-binding proteins PAB2, PAB4 and PAB8. Remarkably, overexpression of PAB4 in leaf primordia partially rescues the delayed leaf formation in ect2 ect3 ect4 mutants, suggesting that the ECT2-PAB2/4/8 interaction on target mRNAs of organogenesis-related genes may overcome limiting PAB concentrations in primordial cells.
2023-11-28 | PXD045801 | Pride
Project description:N6-methyladenosine (m6A) plays critical roles in gene expression control by recruiting the cytoplasmic reader proteins YTHDF1,2 and 3. Recently, the function of YTHDF proteins and whether they bind to shared or distinct sets of methylated mRNAs in cells has been the subject of debate. Here, we developed TRIBE-STAMP, an approach for single-molecule detection of the target RNAs of two RNA binding proteins simultaneously in cells. Applying TRIBE-STAMP to the YTHDF proteins revealed that most target mRNAs are shared among the three YTHDF proteins. Surprisingly, we also found that individual mRNA molecules can be bound by more than one YTHDF protein throughout their lifetime. Furthermore, we show that YTHDF proteins bind sequentially to target methylated mRNAs, with YTHDF1 binding before YTHDF2 or YTHDF3. Our data reveal shared molecular interactions among the YTHDF proteins and support a model in which YTHDF1 and YTHDF3 are unlikely to promote rapid mRNA decay.
2022-10-28 | GSE201538 | GEO
Project description:N6-methyladenosine (m6A) plays critical roles in gene expression control by recruiting the cytoplasmic reader proteins YTHDF1,2 and 3. Recently, the function of YTHDF proteins and whether they bind to shared or distinct sets of methylated mRNAs in cells has been the subject of debate. Here, we developed TRIBE-STAMP, an approach for single-molecule detection of the target RNAs of two RNA binding proteins simultaneously in cells. Applying TRIBE-STAMP to the YTHDF proteins revealed that most target mRNAs are shared among the three YTHDF proteins. Surprisingly, we also found that individual mRNA molecules can be bound by more than one YTHDF protein throughout their lifetime. Furthermore, we show that YTHDF proteins bind sequentially to target methylated mRNAs, with YTHDF1 binding before YTHDF2 or YTHDF3. Our data reveal shared molecular interactions among the YTHDF proteins and support a model in which YTHDF1 and YTHDF3 are unlikely to promote rapid mRNA decay.
2022-10-28 | GSE200712 | GEO
Project description:m6A and YTHDF proteins control the subcellular localization of select neuronal mRNAs
| PRJNA799732 | ENA
Project description:N6-methyladenosine (m6A) plays critical roles in gene expression control by recruiting the cytoplasmic reader proteins YTHDF1,2 and 3. Recently, the function of YTHDF proteins and whether they bind to shared or distinct sets of methylated mRNAs in cells has been the subject of debate. Here, we developed TRIBE-STAMP, an approach for single-molecule detection of the target RNAs of two RNA binding proteins simultaneously in cells. Applying TRIBE-STAMP to the YTHDF proteins revealed that most target mRNAs are shared among the three YTHDF proteins. Surprisingly, we also found that individual mRNA molecules can be bound by more than one YTHDF protein throughout their lifetime. Furthermore, we show that YTHDF proteins bind sequentially to target methylated mRNAs, with YTHDF1 binding before YTHDF2 or YTHDF3. Our data reveal shared molecular interactions among the YTHDF proteins and support a model in which YTHDF1 and YTHDF3 are unlikely to promote rapid mRNA decay.
2022-10-28 | GSE201540 | GEO
Project description:The YTH N6-methyladenosine RNA Binding Proteins (YTHDFs) mediate the functions of N6-methyladenosine (m6A) in RNA. Recently, a report proposed that all YTHDFs work redundantly to facilitate RNA decay, raising questions about the exact functions of individual YTHDFs, especially YTHDF1 and YTHDF2. We show that YTHDF1 and YTHDF2 differ in their low-complexity domains (LCDs), and this causes different behaviors in condensate formation and subsequent physiological functions. Biologically, we find that the global stabilization of RNA after depletion of all YTHDFs is a result of increased P-body formation and is independent of mRNA m6A methylation.
2023-01-12 | GSE201070 | GEO