The role of Limch1 alternative splicing in skeletal muscle function

  1. Matthew S. Penna
  2. George G. Rodney
  3. Rong-Chi Hu
  4. Thomas A. Cooper

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Abstract

Postnatal skeletal muscle development is a highly dynamic period associated with extensive transcriptome remodeling. A significant aspect of postnatal development is widespread alternative splicing changes, required for the adaptation of tissues to adult function. These splicing events have significant implications since the reversion of adult mRNA isoforms to fetal isoforms is observed in forms of muscular dystrophy. LIM and Calponin Homology Domains 1 (LIMCH1) is a stress fiber associated protein that is alternative spliced to generate uLIMCH1, a ubiquitously expressed isoform, and mLIMCH1, a skeletal muscle-specific isoform. mLIMCH1 contains 454 in-frame amino acids which are encoded by six contiguous exons simultaneously included after birth in mouse. The developmental regulation and tissue specificity of this splicing transition is conserved in mice and humans. To determine the physiologically relevant functions of mLIMCH1 and uLIMCH1, CRISPR-Cas9 was used to delete the genomic segment containing the six alternatively spliced exons of LIMCH1 in mice, thereby forcing the constitutive expression of the predominantly fetal isoform, uLIMCH1 in adult skeletal muscle. mLIMCH1 knockout mice had significant grip strength weakness in vivo and maximum force generated was decreased ex vivo . Calcium handling deficits were observed during myofiber stimulation that could explain the mechanism by which mLIMCH1 knockout leads to muscle weakness. Additionally, LIMCH1 is mis-spliced in myotonic dystrophy type 1 with the muscle blind-like (MBNL) family of proteins acting as the likely major regulator of Limch1 alternative splicing in skeletal muscle.

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    Reply to the reviewers

    1. General Statements

    We thank the reviewers from Review Commons for their thorough reviews of our manuscript entitled, “The role of Limch1 alternative splicing in skeletal muscle function.” We were delighted by the many supportive comments of all three reviewers calling our study a “definite advance in our understanding of developmentally-regulated splice isoform transitions that are disease relevant”, “good comprehensive study with convincing results, the design of the experiments is good, and the conclusions are solid”, and “The article is well written, and I favor the publication of this [article] with minor revisions.”

    The reviews include comments on the interest in identifying the mechanism of action of mLIMCH1 in skeletal muscle function such as “ [The study] presents multiple new tools to study mLimch1 and identifies a possible role for mLIMCH1 in calcium regulation, but stops short of identifying the mechanism by which this regulation occurs.” While we agree that how the skeletal muscle-specific isoform of LIMCH1 affects calcium handling is of interest, we respectfully suggest that this manuscript describe previously unknown biology that will be of interest to investigators in different fields including muscle physiology, alternative splicing regulation, and skeletal muscle pathology in myotonic dystrophy. All experiments in this manuscript are performed in vivo using skeletal muscle tissues from animals lacking the isoform of Limch1 that is expressed only in skeletal muscle and is normally induced after birth. Comparisons were made to age-matched wild-type control animals, often litter mates. The results establish the functional significance of the LIMCH1 protein and particularly the muscle-specific isoform in skeletal muscle through extensive analysis of LIMCH1 localization and the impact of mLIMCH1 knockout on muscle strength, force generation, calcium handling and the disease relevance of this splicing transition in myotonic dystrophy type 1. Please review the comments of all three reviewers who were quite favorable to the significance of the work and overall favorable to its publication. Below, we clarify and describe additional data that has, and will be added to the manuscript to address all comments of the reviewers.

    2. Description of the planned revisions

    Reviewer 1

    “Page 6 - data not shown. The point of conservation is not essential to this story, but the authors should either include a table or panel with that data, or remove the data not shown statement. Given the putative relevance to DM1, it might be preferable to include data to support the developmental transition in human data.”

    We have removed the “data not shown” statement as suggested and we highlighted the importance of conservation of the induction of a skeletal muscle isoform of LIMCH1 after birth as a strong indication of functional importance for the isoform. We agree that data showing the conserved LIMCH1 splicing transition in human skeletal muscle development will support this point. We will include RT-PCR analysis of LIMCH1 splicing in fetal and adult human skeletal muscle RNA in Figure 6 to support the reversion of splicing to the fetal pattern observed in DM1. The results will complement the normal Limch1 splicing transition in mice (Figure 1) and the normal and aberrant fetal splicing patterns shown for unaffected and DM1 adult skeletal muscle, respectively (Figure 6).

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Reviewer 1

    “Figure 4 - The authors do a nice experiment to show the localization of Limch1 and raise an antibody to detect the muscle specific isoform. The data seem to show that the muscle-specific isoform localizes to the sarcolemma, and this staining is largely lost in the mutant mice. By contrast, one could infer that the cytoplasmic signal in the WT comes from the ubiquitous isoform (which accounts for 30-40% of the Limch1 expression). This is consistent with the validation in Fig. 2. However, the authors in the text claim this experiment reveals an increased distribution throughout the myofiber, or a more even signal distribution in the cytoplasm, and that the uLimch1 cannot recapitulate mLimch1 localization. Fig. 2 suggests that total levels of Limch1 are increased (as noted by the authors in the discussion). Given that the muscle-specific isoform localizes to the sarcolemma, and the ubiquitous isoform is presumably sarcoplasmic, it isn't clear to me that there is any change in localization per se. What the authors show is just that the signal at the sarcolemma is lost, and if one compares the intensity in the right-hand plots in Fig. 4B, they are comparable in the sarcoplasmic region. It seems likely there is more of the ubiquitous isoform, and what is seen here is just how that isoform localizes. The quantification the authors perform in D would likely show this strong difference in the localization of the muscle isoform. If the authors redo this quantification, exclude the signal at the sarcolemma and normalize to the average pixel intensity in the fiber, do they still see a difference? I am not convinced that the "clustering" of the signal of the ubiquitous, cytoplasmic isoform is in any way changed. Given the difference in the two proteins, I also would not expect that the ubiquitous isoform could compensate for loss of the muscle isoform, and would not expect it to "recapitulate" the muscle-isoform localization.”

    We agree that Figure 4 and the explanation in the text was not clear and we thank the reviewer for pointing this out. We have addressed this concern by modifying the figure as suggested by the reviewer and clarifying the description in the results section. The main point, that is recognized by the reviewer but needed clarification, is that the mLIMCH1 isoform preferentially localizes to the sarcolemma and the uLIMCH1 isoform is preferentially cytoplasmic. In the HOM Limch1 6exKO myofibers, the increased cytoplasmic signal is due to the increased level of uLIMCH1 as shown by the western blot in Figure 2. The reviewer is correct that there is not a “change in localization of isoforms per se”. We clarified this point to highlight the differential localization of the uLIMCH1 and mLIMCH1 isoforms within the sarcolemma vs. the sarcoplasm. The revision of the plot profile in Figure 4B and the analysis of the standard deviation of signal in Figure 4D demonstrates the stark difference in staining observed between the HOM Limch1 6exKO and WT myofibers when stained with a pan-LIMCH1 antibody. The signal intensity plot profile from sarcolemma to sarcolemma (Figure 4B) indicates that the uLIMCH1 isoform is not “mis-localized” upon mLIMCH1 knockout as we originally (mis)-stated. Upon mLIMCH1 knockout, there is increased uLIMCH1 expression compared to WT myofibers. Considering this in combination with the sarcolemma preference of mLIMCH1 (Figure 4E) and the significant loss of signal in the sarcolemma region in Limch1 6exKO myofibers, we conclude that in HOM Limch1 6exKO myofibers, uLIMCH1 is primarily localized throughout the sarcoplasm.

    Reviewer 1 (optional)

    “Experiments looking more closely at LIMCH1 co-localization with other proteins at the sarcolemma or the sufficiency of the muscle-specific region to localize would also be useful (for example, can the muscle-specific region localize GFP to the membrane in cells?).”

    We performed immunofluorescence microscopy of LIMCH1 with several skeletal muscle-relevant proteins but did not observe: (1) disruptions of normal structures in HOM Limch1 6exKO compared to WT myofibers or (2) colocalization that helped clarify any mechanistic role of mLIMCH1 or uLIMCH1. Therefore this data was not included in the original manuscript. In regard to the suggestion on the sufficiency of the muscle-specific region to localize to the sarcolemma region, we had previously generated a plasmid to express a fluorescent protein fused to the protein encoded by the six skeletal muscle-specific exons of LIMCH1 but it failed to localize to the sarcolemma. In collaboration with protein structural experts at Baylor College of Medicine, we analyzed the skeletal muscle-specific region of LIMCH1 and found it to be entirely disordered without known homologs. It appears that this region has no secondary structure but when expressed within the entire LIMCH1 protein which has conserved domains (calponin homology, LIM, coiled-coil regions) and upon protein binding, it is possible for the region to adopt a structure facilitating its binding in the sarcolemma region. Therefore we believe that regions common to both isoforms are required in combination with the muscle-specific region for preferential localization to the sarcolemma.

    Reviewer 1 (minor comments)

    “In the Figure 3 legend, the order of the descriptions for B-C and D-E is switched. The order of the panels matches the text, but the legend switches the description of the force-frequency curves (shown in B & C but labeled as D & E), with the description of the rate of relaxation and contraction plots (shown in D and E but labeled as B and C in the legend).”

    We fixed this error and thank the reviewer for pointing it out.

    “The scale in Figure 4, panel B between the top and bottom plots is not the same, so it is difficult to compare, particularly for the panels on the right. See comment above.”

    In addition to clarifying uLIMCH1’s localization upon mLIMCH1 knockout within the text, we added figure titles above the plot profile which will clarify the different plot profiles for the reader. In regard to the comment about the scale of the plot profile, we have addressed this by re-scaling the two plot profiles on the right in Figure 4B. These plot profiles now share the same scale, which is advantageous because this plot profile better emphasizes the stark difference in signal observed between the sarcolemma and sarcoplasm in WT myofibers that is lacking in HOM Limch1 6exKO myofibers.

    Reviewer 2

    “Figure 6A: There is a discrepancy between gene structures and splicing isoforms shown in Fig. 1 vs Fig. 6. There are differences in spacing between exons, and there appear to be six exons in the differentially regulated region in Fig 1, but seven exons in Fig 6. Perhaps this is a difference between human and mouse genes? Does the human gene actually regulate seven exons in this region, rather than six exons in the mouse? In both figures the gene is labeled as Limchi1, and both figures indicate that the ubiquitous isoform lacks exons 9-14. Please clarify.”

    The reviewer is correct that the human mLIMCH1 isoform contains seven exons that are skeletal muscle-specific compared with the six exons that are skeletal muscle-specific in the mouse. The seven human exons encode 544 amino acids with 65% homology with the mouse segment. We have clarified this in the figure legend and text. Exons 9-14 are shown in Figure 6B since this diagrams the mouse gene.

    “The methods section on RT-qPCR and RNA splicing presumably refers to analysis of mouse tissues. What is the origin of the human DM1 RNA-seq data?”

    We obtained adult human DM1-affected and non-affected skeletal muscle autopsy samples from colleagues and the NDRI and performed RNA-sequencing at Baylor College of Medicine. The RNA-seq has not yet been published, but we include the data for* LIMCH1* to demonstrate the dramatic change in the alternative splicing pattern in DM1 skeletal muscle tissue. This has been clarified in the methods section.

    “Perhaps the word "activity" should be deleted in the following sentence: "The sole study investigating the function of LIMCH1 characterized it as an actin stress fiber associated protein that binds non-muscle myosin 2A (NM2A) activity to regulate focal adhesion formation."

    We thank the reviewer for pointing this out and we have removed this word.

    Reviewer 3

    “The diminution of the muscle force production in Limch16exKO is not correlated with a change in morphology of the myofibers in H&E and picrosirius stainings (Fig S2). Did the authors look at other skeletal muscles, fiber type, size, or different time points? (The age of the mouse and the name of the skeletal muscle used for the histology could be included in the results sections or figure legend).”

    As suggested by Reviewer 3, we have included additional histological data in Supplementary Figure 2. In addition to the histology at 10-12 weeks of age, the new data includes histology of multiple skeletal muscle tissues (quadriceps, EDL, soleus) at one year of age. The histology of Limch1 6exKO tissue at different time points showed no morphological differences (centralized nuclei or fibrosis) consistent with no change in muscle weight which led us to emphasize the significant effect of mLIMCH1 knockout on skeletal muscle function in the absence of muscle loss or overt structural changes. In regard to fiber-type, we have included histology of both the EDL (fast-twitch) and soleus (slow-twitch) and even after one year, we observe no gross morphological differences. Additionally, we analyzed the force production of both the EDL and soleus (Figure 3) with the fiber-type predominance of these tissues in mind and found decreased force generation in both tissues. We included the types of skeletal muscle tissue analyzed and the age of the mice in Supplementary Figure 2 as per the reviewer’s suggestion.

    “The authors performed RNAseq analysis in the skeletal muscle of the KO mouse (Fig 2B). What is the result of this experiment? Is the KO muscle transcriptome different or similar to control muscles?”

    We conducted RNA-sequencing on tissue from HOM Limch1 6exKO and WT controls and the results were disappointing showing minor differences that did not contribute to understanding the phenotype. We used this data only to show the loss of the six exons in Fig. 2B, however, we decided that RT-PCR analysis was the better assay since it shows not only that the exons are not included but also that exons 8 and 15 are spliced correctly, which is not apparent using the RNA-seq displayed on the genome browser.

    4. Description of analyses that authors prefer not to carry out

    Reviewer 1 (____Both points listed as optional)

    “If the authors perform TEM, can they see defects in t-tubules or organization of the sarcoplasmic reticulum, that are not visible by light microscopy?”

    We considered conducting TEM to investigate sarcomeric, T-tubule, or sarcolemma changes in myofibers derived from HOM Limch1 6exKO mice, but we concluded that it would most likely be of limited use. We do not think that T-tubule structural changes will be observed via TEM primarily due to the challenges of finding significant changes compared to WT controls in which one can always find abnormal structures. In our experience and the experience of our collaborator (Dr. Rodney) the disruptions must be dramatic to distinguish from the noncanonical structures often observed. Thus, we do not plan on conducting TEM to identify defects in the T-tubules.

    “If the muscle-specific isoform is transfected or transduced into differentiated myotubes, how does this affect calcium dynamics in the culture system?”

    While an interesting idea, we do not plan on conducting this experiment for multiple reasons. One issue is that all of our data is derived from in vivo analysis or from isolated myofibers and our concern is that the relatively immature state of myotubes in culture will provide a poor comparison to isolated myofibers. Therefore, we believe that it will be difficult to add meaningful data to the calcium data presented in Figure 5 through this experiment. Additionally, we have observed mis-localization of the overexpressed uLIMCH1 and mLIMCH1 in C2C12 cells that we believe would add too many caveats for meaningful interpretation of the results, regardless of the effects on calcium dynamics

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, the authors have generated a knockout mouse model of a skeletal muscle-specific splice variant isoform of Limch1. These KO mice present skeletal muscle force production and calcium handling defects. These results could explain why the deficiency in splicing in myotonic dystrophy1 can lead to skeletal muscle defects.

    Overall, this is a good comprehensive study with convincing results, the design of the experiments is good, and the conclusions are solid. The article is well written, and I favor the publication of this article with minor revisions.

    Issues that I think the authors should clarify:

    • The diminution of the muscle force production in Limch16exKO, is not correlated with a change in morphology of the myofibers in H&E and picrosirius stainings (Fig S2). Did the authors look at other skeletal muscles, fiber type, size, or different time points? (The age of the mouse and the name of the skeletal muscle used for the histology could be included in the results sections or figure legend)
    • The authors performed RNAseq analysis in the skeletal muscle of the KO mouse (Fig 2B). What is the result of this experiment? Is the KO muscle transcriptome different or similar to control muscles?

    Significance

    In this manuscript, the authors have generated a knockout mouse model of a skeletal muscle-specific splice variant isoform of Limch1. These KO mice present skeletal muscle force production and calcium handling defects. These results could explain why the deficiency in splicing in myotonic dystrophy1 can lead to skeletal muscle defects.

    Overall, this is a good comprehensive study with convincing results, the design of the experiments is good, and the conclusions are solid. The article is well written, and I favor the publication of this article EMBO journal with minor revisions.

    Issues that I think the authors should clarify:

    • The diminution of the muscle force production in Limch16exKO, is not correlated with a change in morphology of the myofibers in H&E and picrosirius stainings (Fig S2). Did the authors look at other skeletal muscles, fiber type, size, or different time points? (The age of the mouse and the name of the skeletal muscle used for the histology could be included in the results sections or figure legend)
    • The authors performed RNAseq analysis in the skeletal muscle of the KO mouse (Fig 2B). What is the result of this experiment? Is the KO muscle transcriptome different or similar to control muscles?
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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    This manuscript continues the Cooper lab's analysis of the role of alternative splicing in muscle development and function. Here they report an intriguing alternative splicing difference between fetal and adult tissues involving 6 consecutive exons in the LIMCHI1 gene that are included predominantly in adult muscle to encode a longer isoform of the protein. Moreover, by CRISPR/Cas9-mediated deletion of these exons in mouse models, they show that muscle deficient in the longer LIMCHI1 protein isoform exhibits grip strength weakness in vivo and decreased force generation ex vivo. The mechanistic details remain to be investigated, but evidence so far suggests an intrinsic defect in muscle contraction, perhaps related to aberrant calcium handling, without obvious histopathology or muscle loss. Finally, these new findings may have important implications for human patients with myotonic dystrophy type 1 that typically exhibit defects in MBNL-regulated splicing events, because the authors show (1) that patient muscle poorly expresses the muscle isoform of LIMCHI1, due to inappropriate skipping of the exons, and (2) that mice with knockout of MBNL proteins also predominantly skip these exons.

    Major comments

    1. The major conclusions of the manuscript are clear and convincing -a muscle-specific cluster of 6 exons in the LIMCHI1 gene whose splicing is regulated directly or indirectly by MBNL splicing factor(s); loss of these exons compromises muscle strength; and these exons are poorly spliced in muscle of myotonic dystrophy patients. The data for these conclusions is strong.
    2. The authors do consider alternative explanations where appropriate. For example, they speculate in the discussion that muscle defects could be due not only to loss of the muscle-specific isoform, but possibly also due to the corresponding increase in expression of the non-muscle-specific isoform.
    3. Figure 6A: There is a discrepancy between gene structures and splicing isoforms shown in Fig. 1 vs Fig. 6. There are differences in spacing between exons, and there appear to be six exons in the differentially regulated region in Fig 1, but seven exons in Fig 6. Perhaps this is a difference between human and mouse genes? Does the human gene actually regulate seven exons in this region, rather than six exons in the mouse? In both figures the gene is labeled as Limchi1, and both figures indicate that the ubiquitous isoform lacks exons 9-14. Please clarify.

    Minor comments

    1. The methods section on RT-qPCR and RNA splicing presumably refers to analysis of mouse tissues. What is the origin of the human DM1 RNA-seq data?
    2. p. 4: Perhaps the word "activity" should be deleted in the following sentence: "The sole study investigating the function of LIMCH1 characterized it as an actin stress fiber associated protein that binds non-muscle myosin 2A (NM2A) activity to regulate focal adhesion formation."
    3. Other than the issue raised above regarding LIMCHI1 gene structure, the figures are clearly presented.

    Significance

    The results in this study could have important implications both regarding muscle function and regulation of alternative splicing. The demonstration of a muscle-specific isoform of LIMCHI1 is a novel finding that suggests previously unknown functions of the protein in muscle contraction. This raises intriguing questions as to how this alternative domain impacts muscle function through cooperation with other domains previously predicted (or shown) to interact with actin and non-muscle myosin. Regarding splicing, co-regulation of exon clusters is a poorly understood phenomenon that could be the subject of future interesting studies. Both issues could be relevant to understanding defects in human patients with myotonic dystrophy type I.

    The work would be of interest to scientists studying muscle function as well as those studying alternative splicing. Both groups would probably be intrigued by these results but might consider the results to be relatively preliminary, need more mechanistic details in the future.

    Expertise: I have extensive experience in analysis of alternative splicing regulation. My knowledge of specific techniques to evaluate muscle function is more limited. Although the experiments on muscle function seem clear and convincing to me, I admit that I am not an expert on those methods and could have missed an important point.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    In their paper "The role of Limch1 alternative splicing in skeletal muscle function," Penna and colleagues report a muscle-specific isoform of Limch1 and investigate its function in skeletal muscle. They show that a muscle-specific isoform of Limch1 is expressed preferentially in mature muscle, and demonstrate that animals mutant for this isoform have reduced grip strength and force generation. Notably, although muscle structure and T-tubules are structurally not affected, mutant muscle shows evidence of disrupted calcium handling. Limch1 is also misspliced in DM1 and Mbnl1/2 double mutant mice, suggesting the muscle isoform is disease relevant and regulated by MBNL.

    Major comments

    Page 6 - data not shown. The point of conservation is not essential to this story, but the authors should either include a table or panel with that data, or remove the data not shown statement. Given the putative relevance to DM1, it might be preferable to include data to support the developmental transition in human data.

    Figure 4 - The authors do a nice experiment to show the localization of Limch1, and raise an antibody to detect the muscle specific isoform. The data seem to show that the muscle-specific isoform localizes to the sarcolemma, and this staining is largely lost in the mutant mice. By contrast, one could infer that the cytoplasmic signal in the WT comes from the ubiquitous isoform (which accounts for 30-40% of the Limch1 expression). This is consistent with the validation in Fig. 2. However, the authors in the text claim this experiment reveals an increased distribution throughout the myofiber, or a more even signal distribution in the cytoplasm, and that the uLimch1 cannot recapitulate mLimch1 localization. Fig. 2 suggests that total levels of Limch1 are increased (as noted by the authors in the discussion). Given that the muscle specific isoform localizes to the sarcolemma, and the ubiquitous isoform is presumably sarcoplasmic, it isn't clear to me that there is any change in localization per se. What the authors show is just that the signal at the sarcolemma is lost, and if one compares the intensity in the right-hand plots in Fig. 4B, they are comparable in the sarcoplasmic region. It seems likely there is more of the ubiquitous isoform, and what is seen here is just how that isoform localizes. The quantification the authors perform in D would likely show this strong difference in the localization of the muscle isoform. If the authors redo this quantification, exclude the signal at the sarcolemma and normalize to the average pixel intensity in the fiber, do they still see a difference? I am not convinced that the "clustering" of the signal of the ubiquitous, cytoplasmic isoform is in any way changed. Given the difference in the two proteins, I also would not expect that the ubiquitous isoform could compensate for loss of the muscle isoform, and would not expect it to "recapitulate" the muscle-isoform localization.

    OPTIONAL: It would be interesting to examine how loss of the muscle-specific Limch1 isoform results in disrupted calcium handling. This is the mechanism that is not addressed in the paper, as the authors note in the discussion. If the authors perform TEM, can they see defects in t-tubules or organization of the sarcoplasmic reticulum, that are not visible by light microscopy? Experiments looking more closely at LIMCH1 co-localization with other proteins at the sarcolemma or the sufficiency of the muscle-specific region to localize would also be useful (for example, can the muscle-specific region localize GFP to the membrane in cells?). If the muscle-specific isoform is transfected or transduced into differentiated myotubes, how does this affect calcium dynamics in the culture system? As the authors note in the discussion, identification of mLimch1 versus uLimch1 interactors would be particularly interesting, and provide insight into how this protein can affect calcium handling without impacting structure.

    Minor comments

    • a. In the Figure 3 legend, the order of the descriptions for B-C and D-E is switched. The order of the panels matches the text, but the legend switches the description of the force-frequence curves (shown in B & C but labeled as D & E), with the description of the rate of relaxation and contraction plots (shown in D and E but labeled as B and C in the legend).
    • b. The scale in Figure 4, panel B between the top and bottom plots is not the same, so it is difficult to compare, particularly for the panels on the right. See comment above.

    Significance

    This is a well-written study identifying the function of a muscle-specific isoform of LIMCH1, as well as implicating a switch in Limch1 isoform expression in DM1 models as a target of MBNL regulation. It presents multiple new tools to study mLimch1, and identifies a possible role for mLIMCH1 in calcium regulation, but stops short of identifying the mechanism by which this regulation occurs. The study is a definite advance in our understanding of developmentally-regulated splice isoform transitions that are disease relevant. The work would be of interest to scientists with specialized interests in muscle development and isoform-specific function in myogenesis, as well as more broadly of interest to clinical scientists for the possible connection to DM1.

    I am an expert in RNA regulation and muscle development.

  5. Version published to 10.1101/2022.10.05.510856v1 on bioRxiv