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Single-molecule analysis of RNA:protein interactions reveals sequential binding of different YTHDF proteins to the same mRNA molecules [Polysome]

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE201538 | GEO | 2022/10/28

REPOSITORIES: GEO

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Single-molecule analysis of RNA:protein interactions reveals sequential binding of different YTHDF proteins to the same mRNA molecules [RIP-Seq]

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.

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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.

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2016-08-18 | GSE85724 | GEO

Expression data from YTHDF1 or YTHDF2 down-regulated PC9

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YTHDF2/3 are required for somatic reprogramming through different RNA deadenylation pathways

Project description:N6-methyladenosine (m6A), the most abundant reversible modification on eukaryote messenger RNA, is recognized by a series of readers, including the YT521-B homology domain family (YTHDF) proteins, which are coupled to perform physiological functions. Here, we report that YTHDF2 and YTHDF3, but not YTHDF1, are required for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). Mechanistically, we found that YTHDF3 recruits the PAN2-PAN3 deadenylase complex and conduces to reprogramming by promoting mRNA clearance of somatic genes, including Tead2 and Tgfb1, which parallels the activity of the YTHDF2-CCR4-NOT deadenylase complex. Ythdf2/3 deficiency further represses mesenchymal-to-epithelial transition (MET) and chromatin silencing at loci containing the TEAD motif, contributing to decreased reprogramming efficiency. Moreover, RNA interference of Tgfb1 or the Hippo signaling effectors Yap1, Taz, and Tead2 rescues Ythdf2/3-defective reprogramming. Overall, YTHDF2/3 couple RNA deadenylation and regulation with the clearance of somatic genes provides insights into iPSC reprogramming at the posttranscriptional level.

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Project description:YTH domain family proteins Ythdf1, Ythdf2, Ythdf3 are three readers with highly similar structure, which were shown to have different roles in the cell. However, their similarity and the fact that they tend to bind the same targets suggest that in some cases they may have an overlapping role. In this paper, we systematically knocked-out (KO) each of the three readers and the three together (triple-KO), and estimated the effect in-vitro in mouse embryonic stem cells (mESCs), and in-vivo, in viability and gametogenesis. We show that in mESCs there is a compensation between the three readers, with a dramatic hyper-pluripotent phenotype only in the triple-KO. However, in-vivo, Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, both in viability of the mouse pups and in gametogenesis. We suggest that compensation is dosage-dependent, and in-vivo we cannot detect it due to difference in the readers’ expression, both in level and in spatial location. In addition, we found that Ythdf1 and Ythdf3 specifically down-regulate 2-cell genes, that Ythdf2-KO in gametogenesis affects microtubuline related genes, and that Mettl3-KO severity is increased as the deletion occurs earlier in gametogenesis.

2020-06-04 | GSE151788 | GEO

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