Bulk and single-cell alternative splicing analyses reveal roles of TRA2B in myogenic differentiation

Abstract

Alternative splicing (AS) disruption has been linked to disorders of muscle development, as well as muscular atrophy. However, the precise changes in AS patterns that occur during myogenesis are not well understood. Here, we employed isoform long-reads RNA-seq (Iso-seq) and single-cell RNA-seq (scRNA-seq) to investigate the AS landscape during myogenesis. Our Iso-seq data identified 61,146 full-length isoforms representing 11,682 expressed genes, of which over 52% were novel. We identified 38,022 AS events, with most of these events altering coding sequences and exhibiting stage-specific splicing patterns. We identified AS dynamics in different types of muscle cells through scRNA-seq analysis, revealing genes essential for the contractile muscle system and cytoskeleton that undergo differential splicing across cell types. Gene-splicing analysis demonstrated that AS acts as a regulator, independent of changes in overall gene expression. Two isoforms of splicing factor TRA2B play distinct roles in myogenic differentiation by triggering AS of TGFBR2 to regulate canonical TGF-β signalling cascades differently. Our study provides a valuable transcriptome resource for myogenesis and reveals the complexity of AS and its regulation during myogenesis.

References

[1] Wosczyna MN, Rando TA. A muscle stem cell support group: coordinated cellular responses in muscle regeneration. Dev Cell. 2018;46:135-143.

[2] Bren-Mattison Y, Hausburg M, Olwin BB. Growth of limb muscle is dependent on skeletal-derived Indian hedgehog. Dev Biol. 2011;356:486-495.

[3] Chal J, Pourquié O. Making muscle: skeletal myogenesis in vivo and in vitro. Development. 2017;144:2104-2122.

[4] Geiger AE, Daughtry MR, Gow CM, Siegel PB, Shi H, Gerrard DE. Long-term selection of chickens for body weight alters muscle satellite cell behaviors. Poult Sci. 2018;97:2557-2567.

[5] Bland CS, Wang ET, Vu A, et al. Global regulation of alternative splicing during myogenic differentiation. Nucleic Acids Res. 2010;38:7651-7664.

[6] Chen M, Manley JL. Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. Nat Rev Mol Cell Biol. 2009;10:741-754.

[7] Kim E, Magen A, Ast G. Different levels of alternative splicing among eukaryotes. Nucleic Acids Res. 2007;35:125-131.

[8] Takeda J, Suzuki Y, Nakao M, et al. Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56 419 completely sequenced and manually annotated full-length cDNAs. Nucleic Acids Res. 2006;34:3917-3928.

[9] Liu Y, Gonzàlez-Porta M, Santos S, et al. Impact of alternative splicing on the human proteome. Cell Rep. 2017;20:1229-1241.

[10] Shargunov AV, Krasnov GS, Ponomarenko EA, et al. Tissue-specific alternative splicing analysis reveals the diversity of chromosome 18 transcriptome. J Proteome Res. 2014;13:173-182.

[11] Sebastian S, Faralli H, Yao Z, et al. Tissue-specific splicing of a ubiquitously expressed transcription factor is essential for muscle differentiation. Genes Dev. 2013;27:1247-1259.

[12] Pagliarini V, La Rosa P, Sette C. Faulty RNA splicing: consequences and therapeutic opportunities in brain and muscle disorders. Hum Genet. 2017;136:1215-1235.

[13] Nikonova E, Kao S-Y, Ravichandran K, Wittner A, Spletter ML. Conserved functions of RNA-binding proteins in muscle. Int J Biochem Cell Biol. 2019;110:29-49.

[14] Jeong S. SR proteins: binders, regulators, and connectors of RNA. Mol Cells. 2017;40:1-9.

[15] Geuens T, Bouhy D, Timmerman V. The hnRNP family: insights into their role in health and disease. Hum Genet. 2016;135:851-867.

[16] Yee BA, Pratt GA, Graveley BR, Van Nostrand EL, Yeo GW. RBP-Maps enables robust generation of splicing regulatory maps. RNA. 2019;25:193-204.

[17] Fu X-D, Ares M. Context-dependent control of alternative splicing by RNA-binding proteins. Nat Rev Genet. 2014;15:689-701.

[18] Denoeud F, Aury J-M, Da Silva C, et al. Annotating genomes with massive-scale RNA sequencing. Genome Biol. 2008;9:R175.

[19] Wang B, Tseng E, Regulski M, et al. Unveiling the complexity of the maize transcriptome by single-molecule long-read sequencing. Nat Commun. 2016;7:11708.

[20] Abdel-Ghany SE, Hamilton M, Jacobi JL, et al. A survey of the sorghum transcriptome using single-molecule long reads. Nat Commun. 2016;7:11706.

[21] Tanay A, Regev A. Scaling single-cell genomics from phenomenology to mechanism. Nature. 2017;541:331-338.

[22] Chen G, Ning B, Shi T. Single-cell RNA-seq technologies and related computational data analysis. Front Genet. 2019;10:317.

[23] Patrick R, Humphreys DT, Janbandhu V, et al. Sierra: discovery of differential transcript usage from polyA-captured single-cell RNA-seq data. Genome Biol. 2020;21:167.

[24] Wen WX, Mead AJ, Thongjuea S. MARVEL: an integrated alternative splicing analysis platform for single-cell RNA sequencing data. Nucleic Acids Res. 2023;51:e29.

[25] Anders S, Reyes A, Huber W. Detecting differential usage of exons from RNA-seq data. Genome Res. 2012;22:2008-2017.

[26] Steijger T, Abril JF, Engström PG, et al. Assessment of transcript reconstruction methods for RNA-seq. Nat Methods. 2013;10:1177-1184.

[27] Ouyang H, Yu J, Chen X, Wang Z, Nie Q. A novel transcript of MEF2D promotes myoblast differentiation and its variations associated with growth traits in chicken. PeerJ. 2020;8:e8351.

[28] Mukherjee K, Ishii K, Pillalamarri V, et al. Actin capping protein CAPZB regulates cell morphology, differentiation, and neural crest migration in craniofacial morphogenesis. Hum Mol Genet. 2016;25:1255-1270.

[29] Yang X, Ren H, Shao Y, et al. Chaperonin-containing T-complex protein 1 subunit 8 promotes cell migration and invasion in human esophageal squamous cell carcinoma by regulating α-actin and β-tubulin expression. Int J Oncol. 2018;52:2021-2030.

[30] Li H, Randall WR, Du S-J. skNAC (skeletal Naca), a muscle-specific isoform of Naca (nascent polypeptide-associated complex alpha), is required for myofibril organization. FASEB J. 2009;23:1988-2000.

[31] Roberts JM, Ennajdaoui H, Edmondson C, Wirth B, Sanford JR, Chen B. Splicing factor TRA2B is required for neural progenitor survival: TRA2B is required for cortical development. J Comp Neurol. 2014;522:372-392.

[32] Yang L, Tao T, Wang Y, Bao Z, He X, Cui G. Knocking down the expression of TRA2β inhibits the proliferation and migration of human glioma cells. Pathol Res Pract. 2015;211:731-739.

[33] Vander Ark A, Cao J, Li X. TGF-β receptors: In and beyond TGF-β signaling. Cell Signal. 2018;52:112-120.

[34] Dube DK, Dube S, Abbott L, et al. Sarcomeric TPM3 expression in human heart and skeletal muscle. Cytoskeleton. 2020;77:313-328.

[35] Krcmery J, Camarata T, Kulisz A, Simon H-G. Nucleocytoplasmic functions of the PDZ-LIM protein family: new insights into organ development. Bioessays. 2010;32:100-108.

[36] Wu Z, Ge L, Ma L, et al. TPM2 attenuates progression of prostate cancer by blocking PDLIM7-mediated nuclear translocation of YAP1. Cell Biosci. 2023;13:39.

[37] Baralle FE, Giudice J. Alternative splicing as a regulator of development and tissue identity. Nat Rev Mol Cell Biol. 2017;18:437-451.

[38] Bentzinger CF, Wang YX, Rudnicki MA. Building muscle: molecular regulation of myogenesis. Cold Spring Harb Perspect Biol. 2012;4:a008342.

[39] Reyes A, Huber W. Alternative start and termination sites of transcription drive most transcript isoform differences across human tissues. Nucleic Acids Res. 2018;46:582-592.

[40] Chaudhary S, Khokhar W, Jabre I, et al. Alternative splicing and protein diversity: plants versus animals. Front Plant Sci. 2019;10:708.

[41] Brinegar AE, Xia Z, Loehr JA, Li W, Rodney GG, Cooper TA. Extensive alternative splicing transitions during postnatal skeletal muscle development are required for calcium handling functions. eLife. 2017;6:e27192.

[42] Mukund K, Subramaniam S. Skeletal muscle: a review of molecular structure and function, in health and disease. Wiley Interdiscip Rev Syst Biol Med. 2020;12:e1462.

[43] Ule J, Blencowe BJ. Alternative splicing regulatory networks: functions, mechanisms, and evolution. Mol Cell. 2019;76:329-345.

[44] Van Nostrand EL, Freese P, Pratt GA, et al. A large-scale binding and functional map of human RNA-binding proteins. Nature. 2020;583:711-719.

[45] Wei N, Cheng Y, Wang Z, et al. SRSF10 plays a role in myoblast differentiation and glucose production via regulation of alternative splicing. Cell Rep. 2015;13:1647-1657.

[46] Hall MP, Nagel RJ, Fagg WS, et al. Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation. RNA. 2013;19:627-638.

[47] Yang J, Hung L-H, Licht T, et al. RBM24 is a major regulator of muscle-specific alternative splicing. Dev Cell. 2014;31:87-99.

[48] De Micheli AJ, Laurilliard EJ, Heinke CL, et al. Single-cell analysis of the muscle stem cell hierarchy identifies heterotypic communication signals involved in skeletal muscle regeneration. Cell Rep. 2020;30:3583-3595.e5.

[49] Yaseen W, Kraft-Sheleg O, Zaffryar-Eilot S, et al. Fibroblast fusion to the muscle fiber regulates myotendinous junction formation. Nat Commun. 2021;12:3852.

[50] Kelly NH, Huynh NPT, Guilak F. Single cell RNA-sequencing reveals cellular heterogeneity and trajectories of lineage specification during murine embryonic limb development. Matrix Biol. 2020;89:1-10.

[51] Ma Y, Zhang H, Yang X, et al. Establishment of a human induced pluripotent stem cell line (SDQLCHi004-A) from a patient with nemaline myopathy-4 disease carrying heterozygous mutation in TPM2 gene. Stem Cell Res. 2019;40:101559.

[52] McAdow J, Yang S, Ou T, et al. A pathogenic mechanism associated with myopathies and structural birth defects involves TPM2-directed myogenesis. JCI Insight. 2022;7:e152466.

[53] Mroczek M, Kabzińska D, Chrzanowska KH, Pronicki M, Kochański A. A novel TPM2 gene splice-site mutation causes severe congenital myopathy with arthrogryposis and dysmorphic features. J Appl Genet. 2017;58:199-203.

[54] Akhmanova A, Kapitein LC. Mechanisms of microtubule organization in differentiated animal cells. Nat Rev Mol Cell Biol. 2022;23:541-558.

[55] Wang Z, Grange M, Wagner T, Kho AL, Gautel M, Raunser S. The molecular basis for sarcomere organization in vertebrate skeletal muscle. Cell. 2021;184:2135-2150.e13.

[56] Cléry A, Jayne S, Benderska N, Dominguez C, Stamm S, Allain FH-T. Molecular basis of purine-rich RNA recognition by the human SR-like protein Tra2-β1. Nat Struct Mol Biol. 2011;18:443-450.

[57] Pihlajamäki J, Lerin C, Itkonen P, et al. Expression of the splicing factor gene SFRS10 is reduced in human obesity and contributes to enhanced lipogenesis. Cell Metab. 2011;14:208-218.

[58] Dichmann DS, Walentek P, Harland RM. The alternative splicing regulator Tra2b is required for somitogenesis and regulates splicing of an inhibitory Wnt11b isoform. Cell Rep. 2015;10:527-536.

[59] Grellscheid SN, Dalgliesh C, Rozanska A, et al. Molecular design of a splicing switch responsive to the RNA binding protein Tra2. Nucleic Acids Res. 2011;39:8092-8104.

[60] Singh RK, Kolonin AM, Fiorotto ML, Cooper TA. Rbfox splicing factors maintain skeletal muscle mass by regulating Calpain3 and Proteostasis. Cell Rep. 2018;24:197-208.

[61] Liu D, Kang JS, Derynck R. TGF-β-activated Smad3 represses MEF2-dependent transcription in myogenic differentiation. EMBO J. 2004;23:1557-1566.

[62] Ding Z, Lin J, Sun Y, et al. miR-122-5p negatively regulates the transforming growth factor-β/Smad signaling pathway in skeletal muscle myogenesis. Cell Biochem Funct. 2020;38:231-238.

[63] Chen G, Yin Y, Lin Z, Wen H, Chen J, Luo W. Transcriptome profile analysis reveals KLHL30 as an essential regulator for myoblast differentiation. Biochem Biophys Res Commun. 2021;559:84-91.

[64] Costa ML, Jurberg AD, Mermelstein C. The role of embryonic chick muscle cell culture in the study of skeletal myogenesis. Front Physiol. 2021;12:668600.

[65] Luo W, Wu H, Ye Y, et al. The transient expression of miR-203 and its inhibiting effects on skeletal muscle cell proliferation and differentiation. Cell Death Dis. 2014;5:e1347.

[66] McGinnis CS, Murrow LM, Gartner ZJ. DoubletFinder: doublet detection in single-cell RNA sequencing data using artificial nearest neighbors. Cell Syst. 2019;8:329-337.e4.

[67] Butler A, Hoffman P, Smibert P, Papalexi E, Satija R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol. 2018;36:411-420.

[68] Hafemeister C, Satija R. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol. 2019;20:296.

[69] Hu C, Li T, Xu Y, et al. CellMarker 2.0: an updated database of manually curated cell markers in human/mouse and web tools based on scRNA-seq data. Nucleic Acids Res. 2023;51:D870-D876.

[70] Cao J, Spielmann M, Qiu X, et al. The single-cell transcriptional landscape of mammalian organogenesis. Nature. 2019;566:496-502.

[71] Bergen V, Lange M, Peidli S, Wolf FA, Theis FJ. Generalizing RNA velocity to transient cell states through dynamical modeling. Nat Biotechnol. 2020;38:1408-1414.

[72] Wang G, Wen WX, Mead AJ, Roy A, Psaila B, Thongjuea S. Processing single-cell RNA-seq datasets using SingCellaR. STAR Protoc. 2022;3:101266.

[73] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25:402-408.

[74] Köster T, Staiger D. RNA-binding protein immunoprecipitation and high-throughput sequencing. In: Sanchez-Serrano JJ, Salinas J, eds. Arabidopsis Protocols. Springer US; 2021:453-461.

[75] Naftaly AS, Pau S, White MA. Long-read RNA sequencing reveals widespread sex-specific alternative splicing in threespine stickleback fish. Genome Res. 2021;31:1486-1497.

[76] Tardaguila M, de la Fuente L, Marti C, et al. SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification. Genome Res. 2018;28:396-411.

[77] Shen S, Park JW, Lu Z, et al. rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data. Proc Natl Acad Sci U S A. 2014;111:E5593-E5601.

[78] Park JW, Jung S, Rouchka EC, Tseng Y-T, Xing Y. rMAPS: RNA map analysis and plotting server for alternative exon regulation. Nucleic Acids Res. 2016;44:W333-W338.

[79] Trincado JL, Entizne JC, Hysenaj G, et al. SUPPA2: fast, accurate, and uncertainty-aware differential splicing analysis across multiple conditions. Genome Biol. 2018;19:40.

[80] Kumar L, Futschik ME. Mfuzz: a software package for soft clustering of microarray data. Bioinformation. 2007;2:5-7.

[81] Koterniak B, Pilaka PP, Gracida X, et al. Global regulatory features of alternative splicing across tissues and within the nervous system of C. elegans. Genome Res. 2020;30:1766-1780.

[82] Yu G, Wang L-G, Han Y, He Q-Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012;16:284-287.

[83] Zhou Y, Zhou B, Pache L, et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun. 2019;10:1523.

[84] Hänzelmann S, Castelo R, Guinney J. GSVA: gene set variation analysis for microarray and RNA-seq data. BMC Bioinform. 2013;14:7.

[85] Luo W, Lin Z, Chen J, et al. TMEM182 interacts with integrin beta 1 and regulates myoblast differentiation and muscle regeneration. J Cachexia Sarcopenia Muscle. 2021;12:1704-1723.

[86] Li Z, Cai B, Abdalla BA, et al. LncIRS1 controls muscle atrophy via sponging miR-15 family to activate IGF1-PI3K/AKT pathway. J Cachexia Sarcopenia Muscle. 2019;10:391-410.

[87] Xiong Y, Fan L, Hao Y, et al. Physiological and genetic convergence supports hypoxia resistance in high-altitude songbirds. PLoS Genet. 2020;16:e1009270.

[88] Yin Y, Chen G, Lin Z, Zhang D, Lin W, Luo W. Natural antisense transcript of MYOG regulates development and regeneration in skeletal muscle by shielding the binding sites of MicroRNAs of MYOG mRNA 3′UTR. Biochem Biophys Res Commun. 2023;662:93-103.

[89] Chen T, Chen X, Zhang S, et al. The genome sequence archive family: toward explosive data growth and diverse data types. Genom Proteom Bioinform. 2021;19:578-583.

RIGHTS & PERMISSIONS

2023 2023 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.