Chromatin architect of muscle expression (Charme) is a muscle-restricted long noncoding RNA (lncRNA) that plays an important role in myogenesis. Earlier evidence indicates that the nuclear Charme isoform, named pCharme, acts on the chromatin by assisting the formation of chromatin domains where myogenic transcription occurs. By combining RNA antisense purification (RAP) with mass spectrometry and loss-of-function analyses, we have now identified the proteins that assist these chromatin activities. These proteins-which include a sub-set of splicing regulators, principally PTBP1 and the multifunctional RNA/DNA binding protein MATR3-bind to sequences located within the alternatively spliced intron-1 to form nuclear aggregates. Consistent with the functional importance of pCharme interactome in vivo, a targeted deletion of the intron-1 by a CRISPR-Cas9 approach in mouse causes the release of pCharme from the chromatin and results in cardiac defects similar to what was observed upon knockout of the full-length transcript.

Center for Life Nano Istituto Italiano di Tecnologia Viale Regina Elena 291 00161 Rome Italy

Czech Centre of Phenogenomics and Laboratory of Transgenic Models of Diseases Institute of Molecular Genetics of the Czech Academy of Sciences v v i Prumyslova 595 252 50 Vestec Czech Republic

Department of Biochemical Sciences A Rossi Fanelli Sapienza University of Rome P le A Moro 5 00185 Rome Italy

Department of Biology and Biotechnology Charles Darwin Sapienza University of Rome P le A Moro 5 00185 Rome Italy

Department of Biology and Biotechnology Charles Darwin Sapienza University of Rome P le A Moro 5 00185 Rome Italy; Center for Life Nano Istituto Italiano di Tecnologia Viale Regina Elena 291 00161 Rome Italy

Zobrazit více v PubMed

Allhoff M., Seré K., Pires J.F., Zenke M., Costa I.G. Differential peak calling of ChIP-seq signals with replicates with THOR. Nucleic Acids Res. 2016;44:e153. PubMed PMC

Ames E.G., Lawson M.J., Mackey A.J., Holmes J.W. Sequencing of mRNA identifies re-expression of fetal splice variants in cardiac hypertrophy. J. Mol. Cell. Cardiol. 2013;62:99–107. PubMed PMC

Ballarino M., Cazzella V., D’Andrea D., Grassi L., Bisceglie L., Cipriano A., Santini T., Pinnarò C., Morlando M., Tramontano A., Bozzoni I. Novel long noncoding RNAs (lncRNAs) in myogenesis: a miR-31 overlapping lncRNA transcript controls myoblast differentiation. Mol. Cell. Biol. 2015;35:728–736. PubMed PMC

Ballarino M., Cipriano A., Tita R., Santini T., Desideri F., Morlando M., Colantoni A., Carrieri C., Nicoletti C., Musarò A. Deficiency in the nuclear long noncoding RNA Charme causes myogenic defects and heart remodeling in mice. EMBO J. 2018;37:e99697. PubMed PMC

Banerjee A., Vest K.E., Pavlath G.K., Corbett A.H. Nuclear poly(A) binding protein 1 (PABPN1) and Matrin3 interact in muscle cells and regulate RNA processing. Nucleic Acids Res. 2017;45:10706–10725. PubMed PMC

Batista P.J., Chang H.Y. Long noncoding RNAs: cellular address codes in development and disease. Cell. 2013;152:1298–1307. PubMed PMC

Bland C.S., Wang E.T., Vu A., David M.P., Castle J.C., Johnson J.M., Burge C.B., Cooper T.A. Global regulation of alternative splicing during myogenic differentiation. Nucleic Acids Res. 2010;38:7651–7664. PubMed PMC

Bolger A.M., Lohse M., Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. PubMed PMC

Castle J.C., Zhang C., Shah J.K., Kulkarni A.V., Kalsotra A., Cooper T.A., Johnson J.M. Expression of 24,426 human alternative splicing events and predicted cis regulation in 48 tissues and cell lines. Nat. Genet. 2008;40:1416–1425. PubMed PMC

Cerase A., Armaos A., Neumayer C., Avner P., Guttman M., Tartaglia G.G. Phase separation drives X-chromosome inactivation: a hypothesis. Nat. Struct. Mol. Biol. 2019;26:331–334. PubMed

Chaudhary R., Gryder B., Woods W.S., Subramanian M., Jones M.F., Li X.L., Jenkins L.M., Shabalina S.A., Mo M., Dasso M. Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3. eLife. 2017;6:e23244. PubMed PMC

Chen L.L. Linking Long Noncoding RNA Localization and Function. Trends Biochem. Sci. 2016;41:761–772. PubMed

Chen C.K., Blanco M., Jackson C., Aznauryan E., Ollikainen N., Surka C., Chow A., Cerase A., McDonel P., Guttman M. Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing. Science. 2016;354:468–472. PubMed

Chin A., Lécuyer E. RNA localization: Making its way to the center stage. Biochim. Biophys. Acta, Gen. Subj. 2017;1861(11 Pt B):2956–2970. PubMed

Chorev M., Carmel L. The function of introns. Front. Genet. 2012;3:55. PubMed PMC

Clemson C.M., Hutchinson J.N., Sara S.A., Ensminger A.W., Fox A.H., Chess A., Lawrence J.B. An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles. Mol. Cell. 2009;33:717–726. PubMed PMC

Coelho M.B., Attig J., Bellora N., König J., Hallegger M., Kayikci M., Eyras E., Ule J., Smith C.W. Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB. EMBO J. 2015;34:653–668. PubMed PMC

Coelho M.B., Ascher D.B., Gooding C., Lang E., Maude H., Turner D., Llorian M., Pires D.E., Attig J., Smith C.W. Functional interactions between polypyrimidine tract binding protein and PRI peptide ligand containing proteins. Biochem. Soc. Trans. 2016;44:1058–1065. PubMed

Coelho M.B., Attig J., Ule J., Smith C.W. Matrin3: connecting gene expression with the nuclear matrix. Wiley Interdiscip. Rev. RNA. 2016;7:303–315. PubMed

Cui M., Wang Z., Chen K., Shah A.M., Tan W., Duan L., Sanchez-Ortiz E., Li H., Xu L., Liu N. Dynamic Transcriptional Responses to Injury of Regenerative and Non-regenerative Cardiomyocytes Revealed by Single-Nucleus RNA Sequencing. Dev. Cell. 2020;53:102–116.e8. PubMed PMC

de Hoon M.J., Imoto S., Nolan J., Miyano S. Open source clustering software. Bioinformatics. 2004;20:1453–1454. PubMed

Desmet F.O., Hamroun D., Lalande M., Collod-Béroud G., Claustres M., Béroud C. Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res. 2009;37:e67. PubMed PMC

Du X.J. Gender modulates cardiac phenotype development in genetically modified mice. Cardiovasc. Res. 2004;63:510–519. PubMed

Engreitz J.M., Ollikainen N., Guttman M. Long non-coding RNAs: spatial amplifiers that control nuclear structure and gene expression. Nat. Rev. Mol. Cell Biol. 2016;17:756–770. PubMed

Fatica A., Bozzoni I. Long non-coding RNAs: new players in cell differentiation and development. Nat. Rev. Genet. 2014;15:7–21. PubMed

Gallego-Iradi M.C., Strunk H., Crown A.M., Davila R., Brown H., Rodriguez-Lebron E., Borchelt D.R. N-terminal sequences in matrin 3 mediate phase separation into droplet-like structures that recruit TDP43 variants lacking RNA binding elements. Lab. Invest. 2019;99:1030–1040. PubMed PMC

Garland W., Jensen T.H. Nuclear sorting of RNA. Wiley Interdiscip. Rev. RNA. 2020;11:e1572. PubMed

Gudenas B.L., Wang L. Prediction of LncRNA Subcellular Localization with Deep Learning from Sequence Features. Sci. Rep. 2018;8:16385. PubMed PMC

Guo C.J., Ma X.K., Xing Y.H., Zheng C.C., Xu Y.F., Shan L., Zhang J., Wang S., Wang Y., Carmichael G.G. Distinct Processing of lncRNAs Contributes to Non-conserved Functions in Stem Cells. Cell. 2020;181:621–636.e22. PubMed

Hall M.P., Nagel R.J., Fagg W.S., Shiue L., Cline M.S., Perriman R.J., Donohue J.P., Ares M., Jr. Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation. RNA. 2013;19:627–638. PubMed PMC

Heinz S., Benner C., Spann N., Bertolino E., Lin Y.C., Laslo P., Cheng J.X., Murre C., Singh H., Glass C.K. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell. 2010;38:576–589. PubMed PMC

Hu B., Yang Y.T., Huang Y., Zhu Y., Lu Z.J. POSTAR: a platform for exploring post-transcriptional regulation coordinated by RNA-binding proteins. Nucleic Acids Res. 2017;45(D1):D104–D114. PubMed PMC

Iradi M.C.G., Triplett J.C., Thomas J.D., Davila R., Crown A.M., Brown H., Lewis J., Swanson M.S., Xu G., Rodriguez-Lebron E., Borchelt D.R. Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy. Sci. Rep. 2018;8:4049. PubMed PMC

Jacob A.G., Smith C.W.J. Intron retention as a component of regulated gene expression programs. Hum. Genet. 2017;136:1043–1057. PubMed PMC

Johnson J.O., Pioro E.P., Boehringer A., Chia R., Feit H., Renton A.E., Pliner H.A., Abramzon Y., Marangi G., Winborn B.J., ITALSGEN Mutations in the Matrin 3 gene cause familial amyotrophic lateral sclerosis. Nat. Neurosci. 2014;17:664–666. PubMed PMC

Kopp F., Mendell J.T. Functional Classification and Experimental Dissection of Long Noncoding RNAs. Cell. 2018;172:393–407. PubMed PMC

Langmead B., Trapnell C., Pop M., Salzberg S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10:R25. PubMed PMC

Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R., 1000 Genome Project Data Processing Subgroup The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25:2078–2079. PubMed PMC

Lubelsky Y., Ulitsky I. Sequences enriched in Alu repeats drive nuclear localization of long RNAs in human cells. Nature. 2018;555:107–111. PubMed PMC

Martone J., Mariani D., Desideri F., Ballarino M. Non-coding RNAs Shaping Muscle. Front. Cell Dev. Biol. 2020;7:394. PubMed PMC

Masuda A., Andersen H.S., Doktor T.K., Okamoto T., Ito M., Andresen B.S., Ohno K. CUGBP1 and MBNL1 preferentially bind to 3′ UTRs and facilitate mRNA decay. Sci. Rep. 2012;2:209. PubMed PMC

McHugh C.A., Chen C.K., Chow A., Surka C.F., Tran C., McDonel P., Pandya-Jones A., Blanco M., Burghard C., Moradian A. The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature. 2015;521:232–236. PubMed PMC

Miyagawa R., Tano K., Mizuno R., Nakamura Y., Ijiri K., Rakwal R., Shibato J., Masuo Y., Mayeda A., Hirose T., Akimitsu N. Identification of cis- and trans-acting factors involved in the localization of MALAT-1 noncoding RNA to nuclear speckles. RNA. 2012;18:738–751. PubMed PMC

Moloney C., Rayaprolu S., Howard J., Fromholt S., Brown H., Collins M., Cabrera M., Duffy C., Siemienski Z., Miller D. Analysis of spinal and muscle pathology in transgenic mice overexpressing wild-type and ALS-linked mutant MATR3. Acta Neuropathol. Commun. 2018;6:137. PubMed PMC

Müller T.J., Kraya T., Stoltenburg-Didinger G., Hanisch F., Kornhuber M., Stoevesandt D., Senderek J., Weis J., Baum P., Deschauer M., Zierz S. Phenotype of matrin-3-related distal myopathy in 16 German patients. Ann. Neurol. 2014;76:669–680. PubMed

Naganuma T., Nakagawa S., Tanigawa A., Sasaki Y.F., Goshima N., Hirose T. Alternative 3′-end processing of long noncoding RNA initiates construction of nuclear paraspeckles. EMBO J. 2012;31:4020–4034. PubMed PMC

Palazzo A.F., Lee E.S. Sequence Determinants for Nuclear Retention and Cytoplasmic Export of mRNAs and lncRNAs. Front. Genet. 2018;9:440. PubMed PMC

Pandya-Jones A., Markaki Y., Serizay J., Chitiashvili T., Mancia Leon W.R., Damianov A., Chronis C., Papp B., Chen C.K., McKee R. A protein assembly mediates Xist localization and gene silencing. Nature. 2020;587:145–151. PubMed PMC

Quinlan A.R., Hall I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010;26:841–842. PubMed PMC

Quintero-Rivera F., Xi Q.J., Keppler-Noreuil K.M., Lee J.H., Higgins A.W., Anchan R.M., Roberts A.E., Seong I.S., Fan X., Lage K. MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus. Hum. Mol. Genet. 2015;24:2375–2389. PubMed PMC

Rayaprolu S., D’Alton S., Crosby K., Moloney C., Howard J., Duffy C., Cabrera M., Siemienski Z., Hernandez A.R., Gallego-Iradi C. Heterogeneity of Matrin 3 in the developing and aging murine central nervous system. J. Comp. Neurol. 2016;524:2740–2752. PubMed PMC

Regitz-Zagrosek V., Oertelt-Prigione S., Seeland U., Hetzer R. Sex and gender differences in myocardial hypertrophy and heart failure. Circ. J. 2010;74:1265–1273. PubMed

Ribeiro D.M., Zanzoni A., Cipriano A., Delli Ponti R., Spinelli L., Ballarino M., Bozzoni I., Tartaglia G.G., Brun C. Protein complex scaffolding predicted as a prevalent function of long non-coding RNAs. Nucleic Acids Res. 2018;46:917–928. PubMed PMC

Rinn J.L., Kertesz M., Wang J.K., Squazzo S.L., Xu X., Brugmann S.A., Goodnough L.H., Helms J.A., Farnham P.J., Segal E., Chang H.Y. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell. 2007;129:1311–1323. PubMed PMC

Robinson F., Smith C.W. A splicing repressor domain in polypyrimidine tract-binding protein. J. Biol. Chem. 2006;281:800–806. PubMed

Rother M., Rother K., Puton T., Bujnicki J.M. ModeRNA: a tool for comparative modeling of RNA 3D structure. Nucleic Acids Res. 2011;39:4007–4022. PubMed PMC

Saldanha A.J. Java Treeview--extensible visualization of microarray data. Bioinformatics. 2004;20:3246–3248. PubMed

Sali A., Blundell T.L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 1993;234:779–815. PubMed

Santini T., Martone J., Ballarino M. Visualization of Nuclear and Cytoplasmic Long Noncoding RNAs at Single-Cell Level by RNA-FISH. Methods Mol. Biol. 2021;2157:251–280. PubMed

Schmitz S.U., Grote P., Herrmann B.G. Mechanisms of long noncoding RNA function in development and disease. Cell. Mol. Life Sci. 2016;73:2491–2509. PubMed PMC

Senderek J., Garvey S.M., Krieger M., Guergueltcheva V., Urtizberea A., Roos A., Elbracht M., Stendel C., Tournev I., Mihailova V. Autosomal-dominant distal myopathy associated with a recurrent missense mutation in the gene encoding the nuclear matrix protein, matrin 3. Am. J. Hum. Genet. 2009;84:511–518. PubMed PMC

Shen H., Kan J.L., Ghigna C., Biamonti G., Green M.R. A single polypyrimidine tract binding protein (PTB) binding site mediates splicing inhibition at mouse IgM exons M1 and M2. RNA. 2004;10:787–794. PubMed PMC

Sunwoo H., Colognori D., Froberg J.E., Jeon Y., Lee J.T. Repeat E anchors Xist RNA to the inactive X chromosomal compartment through CDKN1A-interacting protein (CIZ1) Proc. Natl. Acad. Sci. USA. 2017;114:10654–10659. PubMed PMC

Szklarczyk D., Gable A.L., Lyon D., Junge A., Wyder S., Huerta-Cepas J., Simonovic M., Doncheva N.T., Morris J.H., Bork P. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res. 2019;47(D1):D607–D613. PubMed PMC

Taegtmeyer H., Sen S., Vela D. Return to the fetal gene program: a suggested metabolic link to gene expression in the heart. Ann. N Y Acad. Sci. 2010;1188:191–198. PubMed PMC

Tripathi V., Ellis J.D., Shen Z., Song D.Y., Pan Q., Watt A.T., Freier S.M., Bennett C.F., Sharma A., Bubulya P.A. The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Mol. Cell. 2010;39:925–938. PubMed PMC

Uemura Y., Oshima T., Yamamoto M., Reyes C.J., Costa Cruz P.H., Shibuya T., Kawahara Y. Matrin3 binds directly to intronic pyrimidine-rich sequences and controls alternative splicing. Genes Cells. 2017;22:785–798. PubMed

Wagner E.J., Garcia-Blanco M.A. Polypyrimidine tract binding protein antagonizes exon definition. Mol. Cell. Biol. 2001;21:3281–3288. PubMed PMC

Xue Y., Zhou Y., Wu T., Zhu T., Ji X., Kwon Y.S., Zhang C., Yeo G., Black D.L., Sun H. Genome-wide analysis of PTB-RNA interactions reveals a strategy used by the general splicing repressor to modulate exon inclusion or skipping. Mol. Cell. 2009;36:996–1006. PubMed PMC

Yang Y.C., Di C., Hu B., Zhou M., Liu Y., Song N., Li Y., Umetsu J., Lu Z.J. CLIPdb: a CLIP-seq database for protein-RNA interactions. BMC Genomics. 2015;16:51. PubMed PMC

Yin Y., Lu J.Y., Zhang X., Shao W., Xu Y., Li P., Hong Y., Cui L., Shan G., Tian B. U1 snRNP regulates chromatin retention of noncoding RNAs. Nature. 2020;580:147–150. PubMed

Yu Y., Chi B., Xia W., Gangopadhyay J., Yamazaki T., Winkelbauer-Hurt M.E., Yin S., Eliasse Y., Adams E., Shaw C.E., Reed R. U1 snRNP is mislocalized in ALS patient fibroblasts bearing NLS mutations in FUS and is required for motor neuron outgrowth in zebrafish. Nucleic Acids Res. 2015;43:3208–3218. PubMed PMC

Zuckerman B., Ulitsky I. Predictive models of subcellular localization of long RNAs. RNA. 2019;25:557–572. PubMed PMC