Nuclear m6A reader YTHDC1 promotes muscle stem cell activation/proliferation by regulating mRNA splicing and nuclear export

  1. Yulong Qiao
  2. Qiang Sun
  3. Xiaona Chen
  4. Liangqiang He
  5. Di Wang
  6. Ruibao Su
  7. Yuanchao Xue
  8. Hao Sun
  9. Huating Wang

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    eLife assessment

    This is a valuable study performing elegant experiments making identification of a specific regulator in skeletal muscle regeneration. It will form a foundation for further mechanistic investigation. The work is of importance in the clinical field of muscle injury and regeneration.

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Abstract

Skeletal muscle stem cells (also known as satellite cells [SCs]) are essential for muscle regeneration and the regenerative activities of SCs are intrinsically governed by gene regulatory mechanisms, but the post-transcriptional regulation in SCs remains largely unknown. N(6)-methyladenosine (m6A) modification of RNAs is the most pervasive and highly conserved RNA modification in eukaryotic cells; it exerts powerful impact on almost all aspects of mRNA processing that is mainly endowed by its binding with m6A reader proteins. In this study, we investigate the previously uncharacterized regulatory roles of YTHDC1, an m6A reader in mouse SCs. Our results demonstrate that YTHDC1 is an essential regulator of SC activation and proliferation upon acute injury-induced muscle regeneration. The induction of YTHDC1 is indispensable for SC activation and proliferation; thus, inducible YTHDC1 depletion almost abolishes SC regenerative capacity. Mechanistically, transcriptome-wide profiling using LACE-seq in both SCs and mouse C2C12 myoblasts identifies m6A-mediated binding targets of YTHDC1. Next, splicing analysis defines splicing mRNA targets of m6A-YTHDC1. Furthermore, nuclear export analysis also leads to the identification of potential mRNA export targets of m6A-YTHDC1 in SCs and C2C12 myoblasts;interestingly, some mRNAs can be regulated at both splicing and export levels. Lastly, we map YTHDC1 interacting protein partners in myoblasts and unveil a myriad of factors governing mRNA splicing, nuclear export, and transcription, among which hnRNPG appears to be a bona fide interacting partner of YTHDC1. Altogether, our findings uncover YTHDC1 as an essential factor controlling SC regenerative ability through multifaceted gene regulatory mechanisms in mouse myoblast cells.

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  1. Version published to 10.7554/elife.82703 on eLife
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    Author Response

    Reviewer #1 (Public Review):

    Reviewer 1 confirmed the view that your paper provides new insight into YTHDC1 function in regulating SC activation/proliferation but added that some of the data could be improved to fully support the conclusions. Specifically:

    The title "Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export" seems a bit overstated. Their data are not sufficient to show YTHDC1 regulating nuclear export. From figure 6 we could see some mRNAs export was inhibited upon YTHDC1 loss but intron retention also occurs on these mRNAs, for example, Dnajc14. Since intron retention could lead to mRNA nuclear retention, the mRNA export inhibition may be caused by splicing deficiency. From the data they provided we could not draw the conclusion that YTHDC1 directly affects mRNA export. I think they could not emphasize this point in the title.

    Thanks for the suggestion. It is true that in our initial submission, we had more data to support YTHDC1 regulation of mRNA splicing but not enough on nuclear export. It will take substantial amount of time and efforts to have thorough dissection on both mechanisms. Nevertheless, we argue that our data does provide evidence on YTHDC1 regulation of nuclear export. For example, in Figures 6 C, H, and M, only ~20% of the target mRNAs (such as Dnaj14) showed alteration in both splicing and export upon YTHDC1 loss while the majority of the export targets showed no splicing deficiency. For example, Btbd7 and Tiparp in Figure 6 N showed no intron retention. In addition, we have now performed Co-IP experiments to validate the interaction between YTHDC1 and THOC7 (new result added in Figure 7L), which provides extra evidence to support YTHDC1 function in regulating mRNA nuclear export. We thus would like to keep the original title in order to reflect the multifaceted function of YTHDC1 in muscle stem cells.

    The mechanism of YTHDC1 promoting muscle stem cell activation/proliferation is not solidified. The authors could strengthen their evidence through bioinformatics analysis or give more discussion. Besides, the previous work done by Zhao and colleagues (Zhao et al,. Nature 542, 475-478 (2017).) reported another m6A reader Ythdf2 promotes m6A-dependent maternal mRNA clearance to facilitate zebrafish maternal-to-zygotic transition. Does YTHDC1 regulate mRNA clearance during SC activation/proliferation? The authors should explore this possibility by deep-seq data analysis and give some discussion.

    Thanks for the critical comment. For the first concern, we think YTHDC1 promotes muscle stem cell activation/proliferation through the multi-level gene regulatory capabilities of YTHDC1 on both transcriptional and post-transcriptional processes and the myriads of targets regulated by YTHDC1. In addition, with the newly added data, we believe that YTHDC1’s function is largely dependent on its synergism with hnRNPG (Figure 7 K). We have added the discussion in lines 421-427 of the revised text. For the second question, our data showed that YTHDC1 predominantly localizes in the nucleus of SCs and myoblasts (Figure 1 F&G), thus it may not have a role in regulating mRNA clearance in the cytoplasm like YTHDF2. Nevertheless, there are a few existing reports1, 2 suggesting its possible role in mRNA degradation and stability which may arise from its transient shuttling to cytoplasm of cells. We have now added this point in lines 469-472 of the revised text.

    Reviewer #2 (Public Review):

    Reviewer 2 was similarly positive stating that several tour-de-force techniques were used to examine m6A and the biological consequence in satellite cells and that there was a large amount of data supporting the conclusions with only a few minor weaknesses.

    General points: The main body is lengthy, and some content can be reduced or condensed. For example, RNA-seq was used to determine gene expression in WT and cKO cells, but the purpose of this is not well justified given that YTHDC1 mainly functions to regulate splicing and nuclear expert of mRNA rather than controlling their expression levels. Does the RNA-seq data suggest that YTHDC1 may also regulate gene expression independent of m6A reader function?

    Thanks for the comment. We have now revised the entire text to condense the content. Nevertheless, we must point out that the purpose of the RNA-seq is to provide extra evidence for the proliferation defect of the YTHDC1 KO cells but not to search for the underlying mechanism. We have now revised in lines 159-160 to clarify this.

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  3. eLife

    eLife assessment

    This is a valuable study performing elegant experiments making identification of a specific regulator in skeletal muscle regeneration. It will form a foundation for further mechanistic investigation. The work is of importance in the clinical field of muscle injury and regeneration.

  4. eLife

    Reviewer #1 (Public Review):

    As an m6A reader, YTHDC1 is known to affect the processing of RNA post-transcriptionally and this article attempted to relate this function in splicing and nuclear export to defects in muscle regeneration after acute injury using LACE-seq. Mechanistically, they provided evidence on m6A-YTHDC1 participation in modulating splicing and target export in myoblast. Additionally, the authors preliminarily confirmed the interaction of YTHDC1 with several key RNA processing factors such as hnRNPG1 to suggest a possible mechanism for m6A-YTHDC1 regulating splicing. Overall it provides new insight into YTHDC1 function in regulating SC activation/proliferation, although some of the data could be improved to fully support the conclusions.

    1. The title "Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export" seems a bit overstated. Their data are not sufficient to show YTHDC1 regulating nuclear export. From figure 6 we could see some mRNAs export was inhibited upon YTHDC1 loss but intron retention also occurs on these mRNAs, for example, Dnajc14. Since intron retention could lead to mRNA nuclear retention, the mRNA export inhibition may be caused by splicing deficiency. From the data they provided we could not draw the conclusion that YTHDC1 directly affects mRNA export. I think they should not emphasize this point in the title.

    2. The mechanism of YTHDC1 promoting muscle stem cell activation/proliferation is not solidified. The authors could strengthen their evidence through bioinformatics analysis or give more discussion. Besides, the previous work done by Zhao and colleagues (Zhao et al., Nature 542, 475-478 (2017).) reported another m6A reader Ythdf2 promotes m6A-dependent maternal mRNA clearance to facilitate zebrafish maternal-to-zygotic transition. Does YTHDC1 regulate mRNA clearance during SC activation/proliferation? The authors should explore this possibility by deep-seq data analysis and provide some discussion.

  5. eLife

    Reviewer #2 (Public Review):

    Mounting evidence demonstrates that reversible methylation of mRNA (m6A) is a ubiquitous regulator of mRNA splicing, stability, and translation. The biology of m6A involves writer proteins that add a methyl group to mRNA, reader proteins that mediate the function of the methylated mRNA, and eraser proteins that remove the methyl group upon accomplishing the goal. This manuscript reports a key role of the m6A reader protein YTHDC1 in regulating the function of skeletal muscle stem cells that are crucial for postnatal muscle growth and regeneration.

    The strengths of the manuscript include using several tour-de-force techniques to examine m6A and the biological consequence in satellite cells. A large amount of data supports the conclusion. Combining conditional knockout animal models and molecular tools to dissect in vivo functions of YTHDC1 and molecular mechanisms underlying the function.

    There are only a few minor weaknesses. The main body is lengthy, and some content can be reduced or condensed. For example, RNA-seq was used to determine gene expression in WT and cKO cells, but the purpose of this is not well justified given that YTHDC1 mainly functions to regulate splicing and nuclear expert of mRNA rather than controlling their expression levels. Does the RNA-seq data suggest that YTHDC1 may also regulate gene expression independent of m6A reader function?

  6. eLife

    Reviewer #3 (Public Review):

    YTHDC1 has recently been reported as an epigenetic regulator of chromatin. In addition, this protein is known to regulate RNA splicing and export. This manuscript is trying to understand the RNA regulatory mechanism of YTHDC1 in skeleton muscle activation and proliferation. Inactivating YTHDC1 by inducible knockout and protein degradation demonstrates YTHDC1's role in skeleton muscle regulation. Further, the authors applied their LACE-seq, a house-made pipeline suitable for small cell numbers (e.g., activated skeleton muscle stem cells). Together with meRIP, they identified YTHDC1's potential targets in the skeleton muscle stem cells. Moreover, authors have attempted to investigate YTHDC1's RNA splicing and export targets in regulating skeleton muscle regeneration and proliferation. They also discussed the functional specificity of YTHDC1 by identifying its binding partners. These preliminary analyses provide a valuable foundation for further mechanistic investigation. The identification of YTHDC1 as a regulator in skeleton muscle development would be beneficial for the field of muscle injury and regeneration.

  7. Version published to 10.1101/2022.08.07.503064v1 on bioRxiv