Alternative splicing regulation of membrane trafficking genes during myogenesis

Hinkle, Emma R.; Giudice, Jimena; Torres, Eddie; Jackson, M Stutts; Black, Adam J.; Blue, R. Eric; Harris, Sarah E.; Guzman, Bryan B.; Gentile, Gabrielle M.; Lee, Eunice; Tsai, Yi-Hsuan; Parker, Joel S.; Domínguez, Daniel; Giudice, Jimena

doi:10.1261/rna.078993.121

Cited by 4 publications

(3 citation statements)

References 77 publications

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“…Myoblasts are undifferentiated muscle cells that mimic early muscle development or muscle that has atrophied due to disease; while differentiated muscle cells more closely mimic normal, and more adult muscle. Notably, differentiation of C2C12 myoblasts into multinucleated myotubes in culture reproduces the transcriptional and posttranscriptional transitions observed during skeletal muscle development in vivo 2 , 18 , 19 which establishes these cells as a good cell culture model for understanding mechanotransduction 18 – 20 . We stretched undifferentiated cells (myoblasts) and cells after four days of differentiation (differentiated cells).…”

Section: Resultsmentioning

confidence: 56%

Stretching muscle cells induces transcriptional and splicing transitions and changes in SR proteins

et al. 2022

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Alternative splicing is an RNA processing mechanism involved in skeletal muscle development and pathology. Muscular diseases exhibit splicing alterations and changes in mechanobiology leading us to investigate the interconnection between mechanical forces and RNA processing. We performed deep RNA-sequencing after stretching muscle cells. First, we uncovered transcriptional changes in genes encoding proteins involved in muscle function and transcription. Second, we observed that numerous mechanosensitive genes were part of the MAPK pathway which was activated in response to stretching. Third, we revealed that stretching skeletal muscle cells increased the proportion of alternatively spliced cassette exons and their inclusion. Fourth, we demonstrated that the serine and arginine-rich proteins exhibited stronger transcriptional changes than other RNA-binding proteins and that SRSF4 phosphorylation is mechanosensitive. Identifying SRSF4 as a mechanosensitive RNA-binding protein that might contribute to crosstalk between mechanotransduction, transcription, and splicing could potentially reveal novel insights into muscular diseases, particularly those with unknown etiologies.

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Section: Resultsmentioning

confidence: 56%

Stretching muscle cells induces transcriptional and splicing transitions and changes in SR proteins

et al. 2022

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“…1A ). C2C12 cell differentiation recapitulates the extensive transitions in gene expression and alternative splicing programs occurring in early skeletal muscle development in vivo [ 36 , 37 , 38 , 39 ]. Therefore, this is a suitable system to investigate the role of SETD2 in transcriptional and posttranscriptional programs in the context of muscle.…”

Section: Resultsmentioning

confidence: 99%

SET domain containing 2 (SETD2) influences metabolism and alternative splicing during myogenesis

et al. 2022

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Epigenetic regulatory mechanisms are increasingly recognized as crucial determinants of cellular specification and differentiation. During muscle cell differentiation (myogenesis), extensive remodelling of histone acetylation and methylation occurs. Several of these histone modifications aid in the expression of muscle‐specific genes and the silencing of genes that block lineage commitment. Therefore, the identification of new epigenetic regulatory mechanisms is of high interest. Still, the functional relevance of numerous histone modifications during myogenesis remain completely uncertain. In this study, we focus on the function of H3K36me3 and its epigenetic writer, SET domain containing 2 (SETD2), in the context of muscle cell differentiation. We first observed that SETD2 expression increases during myogenesis. Targeted depletion of SETD2 in undifferentiated (myoblasts) and differentiated (myotubes) muscle cells reduced H3K36me3 levels and induced profound changes in gene expression and slight alterations in alternative splicing, as determined by deep RNA‐sequencing analysis. Enzymes that function in metabolic pathways were upregulated in response to SETD2 depletion. Furthermore, we demonstrated that upregulation of several glycolytic enzymes was associated with an increase in intracellular pyruvate levels in SETD2‐depleted cells, indicating a novel role for SETD2 in metabolic programming during myogenesis. Together, our results provide new insight into the signalling pathways controlled by chromatin‐modifying enzymes and their associated histone modifications during muscle cell differentiation.

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“…We performed our qPCR-based binding assay as previously described 112 with a few modifications. Dynabeads MyOne Streptavidin T1 magnetic beads (Thermo Fisher) were washed in blocking buffer (25 mM Tris pH 8.0, 150 mM KCl, 3 mM MgCl 2 , 1 mg/mL BSA, 2 units/1 μL SUPERase-In (Invitrogen), and 1 mg/mL yeast tRNA (Fisher Scientific)), and then in qPCR binding buffer (25 mM Tris pH 8.0, 150 mM KCl, 3 mM MgCl 2 , 1 mg/mL BSA, 2 units/1 mL SUPERase-In, and 50 nM random sequence RNA).…”

Section: Methodsmentioning

confidence: 99%

Understanding species-specific and conserved RNA-protein interactionsin vivoandin vitro

Harris,

Alexis,

Giri

et al. 2024

Preprint

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While evolution is often considered from a DNA- and protein-centric view, RNA-based regulation can also impact gene expression and protein sequences. Here we examined interspecies differences in RNA-protein interactions using the conserved neuronal RNA binding protein, Unkempt (UNK) as model. We find that roughly half of mRNAs bound in human are also bound in mouse. Unexpectedly, even when transcript-level binding was conserved across species differential motif usage was prevalent. To understand the biochemical basis of UNK-RNA interactions, we reconstituted the human and mouse UNK-RNA interactomes using a high-throughput biochemical assay. We uncover detailed features driving binding, show that in vivo patterns are captured in vitro, find that highly conserved sites are the strongest bound, and associate binding strength with downstream regulation. Furthermore, subtle sequence differences surrounding motifs are key determinants of species-specific binding. We highlight the complex features driving protein-RNA interactions and how these evolve to confer species-specific regulation.

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Alternative splicing regulation of membrane trafficking genes during myogenesis

Cited by 4 publications

References 77 publications

Stretching muscle cells induces transcriptional and splicing transitions and changes in SR proteins

Stretching muscle cells induces transcriptional and splicing transitions and changes in SR proteins

SET domain containing 2 (SETD2) influences metabolism and alternative splicing during myogenesis

Understanding species-specific and conserved RNA-protein interactionsin vivoandin vitro

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