Pre–mRNAs are often processed in complex patterns in tissue-specific manners to produce a variety of protein isoforms from single genes. However, mechanisms orchestrating the processing of the entire transcript are not well understood. Muscle-specific alternative pre–mRNA processing of the unc-60 gene in Caenorhabditis elegans, encoding two tissue-specific isoforms of ADF/cofilin with distinct biochemical properties in regulating actin organization, provides an excellent in vivo model of complex and tissue-specific pre–mRNA processing; it consists of a single first exon and two separate series of downstream exons. Here we visualize the complex muscle-specific processing pattern of the unc-60 pre–mRNA with asymmetric fluorescence reporter minigenes. By disrupting juxtaposed CUAAC repeats and UGUGUG stretch in intron 1A, we demonstrate that these elements are required for retaining intron 1A, as well as for switching the processing patterns of the entire pre–mRNA from non-muscle-type to muscle-type. Mutations in genes encoding muscle-specific RNA–binding proteins ASD-2 and SUP-12 turned the colour of the unc-60 reporter worms. ASD-2 and SUP-12 proteins specifically and cooperatively bind to CUAAC repeats and UGUGUG stretch in intron 1A, respectively, to form a ternary complex in vitro. Immunohistochemical staining and RT–PCR analyses demonstrate that ASD-2 and SUP-12 are also required for switching the processing patterns of the endogenous unc-60 pre-mRNA from UNC-60A to UNC-60B in muscles. Furthermore, systematic analyses of partially spliced RNAs reveal the actual orders of intron removal for distinct mRNA isoforms. Taken together, our results demonstrate that muscle-specific splicing factors ASD-2 and SUP-12 cooperatively promote muscle-specific processing of the unc-60 gene, and provide insight into the mechanisms of complex pre-mRNA processing; combinatorial regulation of a single splice site by two tissue-specific splicing regulators determines the binary fate of the entire transcript.
Vyšlo v časopise: Muscle-Specific Splicing Factors ASD-2 and SUP-12 Cooperatively Switch Alternative Pre-mRNA Processing Patterns of the ADF/Cofilin Gene in. PLoS Genet 8(10): e32767. doi:10.1371/journal.pgen.1002991 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1002991
1. LicatalosiDD, DarnellRB (2010) RNA processing and its regulation: global insights into biological networks. Nat Rev Genet 11 : 75–87.
2. NilsenTW, GraveleyBR (2010) Expansion of the eukaryotic proteome by alternative splicing. Nature 463 : 457–463.
3. PanQ, ShaiO, LeeLJ, FreyBJ, BlencoweBJ (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet 40 : 1413–1415.
4. WangET, SandbergR, LuoS, KhrebtukovaI, ZhangL, et al. (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456 : 470–476.
5. MatlinAJ, ClarkF, SmithCW (2005) Understanding alternative splicing: towards a cellular code. Nat Rev Mol Cell Biol 6 : 386–398.
6. BlackDL (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 72 : 291–336.
7. WittenJT, UleJ (2011) Understanding splicing regulation through RNA splicing maps. Trends Genet 27 : 89–97.
8. McManusCJ, GraveleyBR (2011) RNA structure and the mechanisms of alternative splicing. Curr Opin Genet Dev 21 : 373–379.
9. ChenM, ManleyJL (2009) Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. Nat Rev Mol Cell Biol 10 : 741–754.
10. SchiaffinoS, ReggianiC (1996) Molecular diversity of myofibrillar proteins: gene regulation and functional significance. Physiol Rev 76 : 371–423.
11. LlorianM, SmithCW (2011) Decoding muscle alternative splicing. Curr Opin Genet Dev 21 : 380–387.
12. KalsotraA, CooperTA (2011) Functional consequences of developmentally regulated alternative splicing. Nat Rev Genet 12 : 715–729.
13. GunningPW, SchevzovG, KeeAJ, HardemanEC (2005) Tropomyosin isoforms: divining rods for actin cytoskeleton function. Trends Cell Biol 15 : 333–341.
14. WeiB, JinJP (2010) Troponin T isoforms and posttranscriptional modifications: Evolution, regulation and function. Arch Biochem Biophys 505 : 144–154.
15. KalsotraA, XiaoX, WardAJ, CastleJC, JohnsonJM, et al. (2008) A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart. Proc Natl Acad Sci U S A 105 : 20333–20338.
16. BlandCS, WangET, VuA, DavidMP, CastleJC, et al. (2010) Global regulation of alternative splicing during myogenic differentiation. Nucleic Acids Res 38 : 7651–7664.
17. BrudnoM, GelfandMS, SpenglerS, ZornM, DubchakI, et al. (2001) Computational analysis of candidate intron regulatory elements for tissue-specific alternative pre-mRNA splicing. Nucleic Acids Res 29 : 2338–2348.
18. SugnetCW, SrinivasanK, ClarkTA, O'BrienG, ClineMS, et al. (2006) Unusual intron conservation near tissue-regulated exons found by splicing microarrays. PLoS Comput Biol 2: e4 doi:10.1371/journal.pcbi.0020004.
19. PascualM, VicenteM, MonferrerL, ArteroR (2006) The Muscleblind family of proteins: an emerging class of regulators of developmentally programmed alternative splicing. Differentiation 74 : 65–80.
20. KuroyanagiH (2009) Fox-1 family of RNA-binding proteins. Cell Mol Life Sci 66 : 3895–3907.
21. VlasovaIA, BohjanenPR (2008) Posttranscriptional regulation of gene networks by GU-rich elements and CELF proteins. RNA Biol 5 : 201–207.
22. LlorianM, SchwartzS, ClarkTA, HollanderD, TanLY, et al. (2010) Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB. Nat Struct Mol Biol 17 : 1114–1123.
23. ChenCD, KobayashiR, HelfmanDM (1999) Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat beta-tropomyosin gene. Genes Dev 13 : 593–606.
24. McKimKS, MathesonC, MarraMA, WakarchukMF, BaillieDL (1994) The Caenorhabditis elegans unc-60 gene encodes proteins homologous to a family of actin-binding proteins. Mol Gen Genet 242 : 346–357.
25. OnoS (2007) Mechanism of depolymerization and severing of actin filaments and its significance in cytoskeletal dynamics. Int Rev Cytol 258 : 1–82.
26. OnoK, ParastM, AlbericoC, BenianGM, OnoS (2003) Specific requirement for two ADF/cofilin isoforms in distinct actin-dependent processes in Caenorhabditis elegans. J Cell Sci 116 : 2073–2085.
27. OnoS, BenianGM (1998) Two Caenorhabditis elegans actin depolymerizing factor/cofilin proteins, encoded by the unc-60 gene, differentially regulate actin filament dynamics. J Biol Chem 273 : 3778–3783.
28. YamashiroS, MohriK, OnoS (2005) The two Caenorhabditis elegans actin-depolymerizing factor/cofilin proteins differently enhance actin filament severing and depolymerization. Biochemistry 44 : 14238–14247.
29. OnoK, YamashiroS, OnoS (2008) Essential role of ADF/cofilin for assembly of contractile actin networks in the C. elegans somatic gonad. J Cell Sci 121 : 2662–2670.
30. AnyanfulA, OnoK, JohnsenRC, LyH, JensenV, et al. (2004) The RNA-binding protein SUP-12 controls muscle-specific splicing of the ADF/cofilin pre-mRNA in C. elegans. J Cell Biol 167 : 639–647.
31. KuroyanagiH, KobayashiT, MitaniS, HagiwaraM (2006) Transgenic alternative-splicing reporters reveal tissue-specific expression profiles and regulation mechanisms in vivo. Nat Methods 3 : 909–915.
32. OhnoG, HagiwaraM, KuroyanagiH (2008) STAR family RNA-binding protein ASD-2 regulates developmental switching of mutually exclusive alternative splicing in vivo. Genes Dev 22 : 360–374.
33. KuroyanagiH, OhnoG, SakaneH, MaruokaH, HagiwaraM (2010) Visualization and genetic analysis of alternative splicing regulation in vivo using fluorescence reporters in transgenic Caenorhabditis elegans. Nat Protoc 5 : 1495–1517.
34. VolkT, IsraeliD, NirR, Toledano-KatchalskiH (2008) Tissue development and RNA control: “HOW” is it coordinated? Trends Genet 24 : 94–101.
35. OguraK, WickyC, MagnenatL, ToblerH, MoriI, et al. (1994) Caenorhabditis elegans unc-51 gene required for axonal elongation encodes a novel serine/threonine kinase. Genes Dev 8 : 2389–2400.
36. TomodaT, BhattRS, KuroyanagiH, ShirasawaT, HattenME (1999) A mouse serine/threonine kinase homologous to C. elegans UNC51 functions in parallel fiber formation of cerebellar granule neurons. Neuron 24 : 833–846.
37. OnoS, BaillieDL, BenianGM (1999) UNC-60B, an ADF/cofilin family protein, is required for proper assembly of actin into myofibrils in Caenorhabditis elegans body wall muscle. J Cell Biol 145 : 491–502.
38. KuroyanagiH, OhnoG, MitaniS, HagiwaraM (2007) The Fox-1 family and SUP-12 coordinately regulate tissue-specific alternative splicing in vivo. Mol Cell Biol 27 : 8612–8621.
39. LiS, ArmstrongCM, BertinN, GeH, MilsteinS, et al. (2004) A map of the interactome network of the metazoan C. elegans. Science 303 : 540–543.
40. PulakR, AndersonP (1993) mRNA surveillance by the Caenorhabditis elegans smg genes. Genes Dev 7 : 1885–1897.
41. KabatJL, Barberan-SolerS, McKennaP, ClawsonH, FarrerT, et al. (2006) Intronic alternative splicing regulators identified by comparative genomics in nematodes. PLoS Comput Biol 2: e86 doi:10.1371/journal.pcbi.0020086.
42. HollinsC, ZorioDA, MacMorrisM, BlumenthalT (2005) U2AF binding selects for the high conservation of the C. elegans 3′ splice site. RNA 11 : 248–253.
43. Blumenthal T, Steward K (1997) RNA processing and gene structure. In: Riddle D, Blumenthal T, Meyer B, Priess J, editors. C elegans II. Woodbury, N.Y.: Cold Spring Harbor Laboratory Press. pp. 117–145.
44. Barberan-SolerS, MedinaP, EstellaJ, WilliamsJ, ZahlerAM (2011) Co-regulation of alternative splicing by diverse splicing factors in Caenorhabditis elegans. Nucleic Acids Res 39 : 666–674.
45. ZaffranS, AstierM, GratecosD, SemerivaM (1997) The held out wings (how) Drosophila gene encodes a putative RNA-binding protein involved in the control of muscular and cardiac activity. Development 124 : 2087–2098.
46. BaehreckeEH (1997) who encodes a KH RNA binding protein that functions in muscle development. Development 124 : 1323–1332.
47. NirR, GrossmanR, ParoushZ, VolkT (2012) Phosphorylation of the Drosophila melanogaster RNA-binding protein HOW by MAPK/ERK enhances its dimerization and activity. PLoS Genet 8: e1002632 doi:10.1371/journal.pgen.1002632.
48. LobbardiR, LambertG, ZhaoJ, GeislerR, KimHR, et al. (2011) Fine-tuning of Hh signaling by the RNA-binding protein Quaking to control muscle development. Development 138 : 1783–1794.
49. JinD, HidakaK, ShiraiM, MorisakiT (2010) RNA-binding motif protein 24 regulates myogenin expression and promotes myogenic differentiation. Genes Cells 15 : 1158–1167.
50. LiHY, BourdelasA, CarronC, ShiDL (2010) The RNA-binding protein Seb4/RBM24 is a direct target of MyoD and is required for myogenesis during Xenopus early development. Mech Dev 127 : 281–291.
51. MaraghS, MillerRA, BesslingSL, McGaugheyDM, WesselsMW, et al. (2011) Identification of RNA binding motif proteins essential for cardiovascular development. BMC Dev Biol 11 : 62.
52. PoonKL, TanKT, WeiYY, NgCP, ColmanA, et al. (2012) RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility. Cardiovasc Res
53. MiyamotoS, HidakaK, JinD, MorisakiT (2009) RNA-binding proteins Rbm38 and Rbm24 regulate myogenic differentiation via p21-dependent and -independent regulatory pathways. Genes Cells 14 : 1241–1252.
54. MalletJ, HouhouL, PajakF, OdaY, CerviniR, et al. (1998) The cholinergic locus: ChAT and VAChT genes. J Physiol Paris 92 : 145–147.
55. KitamotoT, WangW, SalvaterraPM (1998) Structure and organization of the Drosophila cholinergic locus. J Biol Chem 273 : 2706–2713.
56. AlfonsoA, GrundahlK, McManusJR, AsburyJM, RandJB (1994) Alternative splicing leads to two cholinergic proteins in Caenorhabditis elegans. J Mol Biol 241 : 627–630.
57. CampbellRE, TourO, PalmerAE, SteinbachPA, BairdGS, et al. (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 99 : 7877–7882.
58. MitaniS (1995) Genetic regulation of mec-3 gene expression implicated in the specification of the mechanosensory neuron cell types in Caenorhabditis elegans. Dev Growth & Diff 37 : 551–557.
59. KamathRS, Martinez-CamposM, ZipperlenP, FraserAG, AhringerJ (2001) Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol 2: RESEARCH0002.
60. OnoS (2001) The Caenorhabditis elegans unc-78 gene encodes a homologue of actin-interacting protein 1 required for organized assembly of muscle actin filaments. J Cell Biol 152 : 1313–1319.
Zvýšte si kvalifikáciu online z pohodlia domova
Nemáte účet? Registrujte sa
Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.
