Identification of Evolutionarily Conserved Exons as Regulated Targets for the Splicing Activator Tra2β in Development
Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10fl/fl; Nestin-Cretg/+). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.
Vyšlo v časopise:
Identification of Evolutionarily Conserved Exons as Regulated Targets for the Splicing Activator Tra2β in Development. PLoS Genet 7(12): e32767. doi:10.1371/journal.pgen.1002390
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1002390
Souhrn
Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10fl/fl; Nestin-Cretg/+). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.
Zdroje
1. KanZGarrett-EngelePWJohnsonJMCastleJC 2005 Evolutionarily conserved and diverged alternative splicing events show different expression and functional profiles. Nucleic Acids Res 33 5659 5666
2. LareauLFGreenREBhatnagarRSBrennerSE 2004 The evolving roles of alternative splicing. Curr Opin Struct Biol 14 273 282
3. StammS 2002 Signals and their transduction pathways regulating alternative splicing: a new dimension of the human genome. Hum Mol Genet 11 2409 2416
4. LareauLFInadaMGreenREWengrodJCBrennerSE 2007 Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements. Nature 446 926 929
5. NiJZGrateLDonohueJPPrestonCNobidaN 2007 Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay. Genes Dev 21 708 718
6. McGlincyNJSmithCW 2008 Alternative splicing resulting in nonsense-mediated mRNA decay: what is the meaning of nonsense? Trends Biochem Sci 33 385 393
7. NilsenTWGraveleyBR 2010 Expansion of the eukaryotic proteome by alternative splicing. Nature 463 457 463
8. GrellscheidSNSmithCW 2006 An apparent pseudo-exon acts both as an alternative exon that leads to nonsense-mediated decay and as a zero-length exon. Mol Cell Biol 26 2237 2246
9. LicatalosiDDDarnellRB 2010 RNA processing and its regulation: global insights into biological networks. Nat Rev Genet 11 75 87
10. WangZBurgeCB 2008 Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA 14 802 813
11. SmithCWValcarcelJ 2000 Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem Sci 25 381 388
12. TackeRTohyamaMOgawaSManleyJL 1998 Human Tra2 proteins are sequence-specific activators of pre-mRNA splicing. Cell 93 139 148
13. BeilBScreatonGStammS 1997 Molecular cloning of htra2-beta-1 and htra2-beta-2, two human homologs of tra-2 generated by alternative splicing. DNA Cell Biol 16 679 690
14. DauwalderBAmaya-ManzanaresFMattoxW 1996 A human homologue of the Drosophila sex determination factor transformer-2 has conserved splicing regulatory functions. Proc Natl Acad Sci U S A 93 9004 9009
15. ShepardPJHertelKJ 2009 The SR protein family. Genome Biol 10 242
16. BeloteJMBakerBS 1983 The dual functions of a sex determination gene in Drosophila melanogaster. Dev Biol 95 512 517
17. CleryAJayneSBenderskaNDominguezCStammS 2011 Molecular basis of purine-rich RNA recognition by the human SR-like protein Tra2-beta1. Nat Struct Mol Biol
18. TsudaKSomeyaTKuwasakoKTakahashiMHeF 2011 Structural basis for the dual RNA-recognition modes of human Tra2-{beta} RRM. Nucleic Acids Res
19. MendeYJakubikMRiesslandMSchoenenFRossbachK 2010 Deficiency of the splicing factor Sfrs10 results in early embryonic lethality in mice and has no impact on full-length SMN/Smn splicing. Hum Mol Genet 19 2154 2167
20. DaoudRDa Penha BerzaghiMSiedlerFHubenerMStammS 1999 Activity-dependent regulation of alternative splicing patterns in the rat brain. Eur J Neurosci 11 788 802
21. NaylerOCapCStammS 1998 Human transformer-2-beta gene (SFRS10): complete nucleotide sequence, chromosomal localization, and generation of a tissue-specific isoform. Genomics 53 191 202
22. StoilovPDaoudRNaylerOStammS 2004 Human tra2-beta1 autoregulates its protein concentration by influencing alternative splicing of its pre-mRNA. Hum Mol Genet 13 509 524
23. MattoxWPalmerMJBakerBS 1990 Alternative splicing of the sex determination gene transformer-2 is sex-specific in the germ line but not in the soma. Genes Dev 4 789 805
24. HazelriggTTuC 1994 Sex-specific processing of the Drosophila exuperantia transcript is regulated in male germ cells by the tra-2 gene. Proc Natl Acad Sci U S A 91 10752 10756
25. MadiganSJEdeenPEsnayraJMcKeownM 1996 att, a target for regulation by tra2 in the testes of Drosophila melanogaster, encodes alternative RNAs and alternative proteins. Mol Cell Biol 16 4222 4230
26. ElliottDJMillarMROgheneKRossAKiesewetterF 1997 Expression of RBM in the nuclei of human germ cells is dependent on a critical region of the Y chromosome long arm. Proc Natl Acad Sci U S A 94 3848 3853
27. VenablesJPElliottDJMakarovaOVMakarovEMCookeHJ 2000 RBMY, a probable human spermatogenesis factor, and other hnRNP G proteins interact with Tra2beta and affect splicing. Hum Mol Genet 9 685 694
28. GrellscheidSNDalglieshCRozanskaAGrellscheidDBourgeoisCF 2011 Molecular design of a splicing switch responsive to the RNA binding protein Tra2β. Nucleic Acids Res 39 8092 104
29. VenablesJPBourgeoisCFDalglieshCKisterLSteveninJ 2005 Up-regulation of the ubiquitous alternative splicing factor Tra2beta causes inclusion of a germ cell-specific exon. Hum Mol Genet 14 2289 2303
30. LicatalosiDDMeleAFakJJUleJKayikciM 2008 HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456 464 469
31. SanfordJRWangXMortMVanduynNCooperDN 2009 Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts. Genome Res 19 381 394
32. WaterstonRHLindblad-TohKBirneyERogersJAbrilJF 2002 Initial sequencing and comparative analysis of the mouse genome. Nature 420 520 562
33. XueYZhouYWuTZhuTJiX 2009 Genome-wide analysis of PTB-RNA interactions reveals a strategy used by the general splicing repressor to modulate exon inclusion or skipping. Mol Cell 36 996 1006
34. MattickJSMakuninIV 2006 Non-coding RNA. Hum Mol Genet 15 Spec No 1 R17 29
35. HafnerMLandthalerMBurgerLKhorshidMHausserJ 2010 Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141 129 141
36. DarnellRB 2006 Developing global insight into RNA regulation. Cold Spring Harb Symp Quant Biol 71 321 327
37. KerenHLev-MaorGAstG 2010 Alternative splicing and evolution: diversification, exon definition and function. Nat Rev Genet 11 345 355
38. RuppertSColeTJBoshartMSchmidESchutzG 1992 Multiple mRNA isoforms of the transcription activator protein CREB: generation by alternative splicing and specific expression in primary spermatocytes. EMBO J 11 1503 1512
39. ZhangLYZengMChenPSunHQTaoDC 2009 Identification of messenger RNA substrates for mouse T-STAR. Biochemistry (Mosc) 74 1270 1277
40. RayDKazanHChanETPena CastilloLChaudhryS 2009 Rapid and systematic analysis of the RNA recognition specificities of RNA-binding proteins. Nat Biotechnol 27 667 670
41. SorekRShamirRAstG 2004 How prevalent is functional alternative splicing in the human genome? Trends Genet 20 68 71
42. YeoGWVan NostrandEHolsteDPoggioTBurgeCB 2005 Identification and analysis of alternative splicing events conserved in human and mouse. Proc Natl Acad Sci U S A 102 2850 2855
43. RichardsonRTAlekseevOMGrossmanGWidgrenEEThresherR 2006 Nuclear autoantigenic sperm protein (NASP), a linker histone chaperone that is required for cell proliferation. J Biol Chem 281 21526 21534
44. ColesJLHalleggerMSmithCW 2009 A nonsense exon in the Tpm1 gene is silenced by hnRNP H and F. RNA 15 33 43
45. ZhangXHChasinLA 2004 Computational definition of sequence motifs governing constitutive exon splicing. Genes Dev 18 1241 1250
46. ZhangXHKangsamaksinTChaoMSBanerjeeJKChasinLA 2005 Exon inclusion is dependent on predictable exonic splicing enhancers. Mol Cell Biol 25 7323 7332
47. TroncheFKellendonkCKretzOGassPAnlagK 1999 Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat Genet 23 99 103
48. AlekseevOMRichardsonRTO'RandMG 2009 Linker histones stimulate HSPA2 ATPase activity through NASP binding and inhibit CDC2/Cyclin B1 complex formation during meiosis in the mouse. Biol Reprod 81 739 748
49. WelchJEO'RandMG 1990 Characterization of a sperm-specific nuclear autoantigenic protein. II. Expression and localization in the testis. Biol Reprod 43 569 578
50. OsakabeATachiwanaHMatsunagaTShigaTNozawaRS 2010 Nucleosome formation activity of human somatic nuclear autoantigenic sperm protein (sNASP). J Biol Chem 285 11913 11921
51. McGuffinMEChandlerDSomaiyaDDauwalderBMattoxW 1998 Autoregulation of transformer-2 alternative splicing is necessary for normal male fertility in Drosophila. Genetics 149 1477 1486
52. GabutMChaudhrySBlencoweBJ 2008 SnapShot: The splicing regulatory machinery. Cell 133 192 e191
53. HofmannYLorsonCLStammSAndrophyEJWirthB 2000 Htra2-beta 1 stimulates an exonic splicing enhancer and can restore full-length SMN expression to survival motor neuron 2 (SMN2). Proc Natl Acad Sci U S A 97 9618 9623
54. JiangZTangHHavliogluNZhangXStammS 2003 Mutations in tau gene exon 10 associated with FTDP-17 alter the activity of an exonic splicing enhancer to interact with Tra2 beta. J Biol Chem 278 18997 19007
55. StammSCasperDHansonVHelfmanDM 1999 Regulation of the neuron-specific exon of clathrin light chain B. Brain Res Mol Brain Res 64 108 118
56. YamadaTGotoISakakiY 1993 Neuron-specific splicing of the Alzheimer amyloid precursor protein gene in a mini-gene system. Biochem Biophys Res Commun 195 442 448
57. DissetABourgeoisCFBenmalekNClaustresMSteveninJ 2006 An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements. Hum Mol Genet 15 999 1013
58. YoungPJDiDonatoCJHuDKotharyRAndrophyEJ 2002 SRp30c-dependent stimulation of survival motor neuron (SMN) exon 7 inclusion is facilitated by a direct interaction with hTra2 beta 1. Hum Mol Genet 11 577 587
59. CartegniLKrainerAR 2002 Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1. Nat Genet 30 377 384
60. KashimaTManleyJL 2003 A negative element in SMN2 exon 7 inhibits splicing in spinal muscular atrophy. Nat Genet 34 460 463
61. ElliottDJGrellscheidSN 2006 Alternative RNA splicing regulation in the testis. Reproduction 132 811 819
62. VenablesJPKlinckRKohCGervais-BirdJBramardA 2009 Cancer-associated regulation of alternative splicing. Nat Struct Mol Biol 16 670 676
63. WangZKayikciMBrieseMZarnackKLuscombeNM 2010 iCLIP predicts the dual splicing effects of TIA-RNA interactions. PLoS Biol 8 e1000530 doi:10.1371/journal.pbio.1000530
64. HofmannYWirthB 2002 hnRNP-G promotes exon 7 inclusion of survival motor neuron (SMN) via direct interaction with Htra2-beta1. Hum Mol Genet 11 2037 2049
65. SergeantKABourgeoisCFDalglieshCVenablesJPSteveninJ 2007 Alternative RNA splicing complexes containing the scaffold attachment factor SAFB2. J Cell Sci 120 309 319
66. LangmeadBTrapnellCPopMSalzbergSL 2009 Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10 R25
67. MignoneJLKukekovVChiangASSteindlerDEnikolopovG 2004 Neural stem and progenitor cells in nestin-GFP transgenic mice. J Comp Neurol 469 311 324
68. BourgeoisCFPopielarzMHildweinGSteveninJ 1999 Identification of a bidirectional splicing enhancer: differential involvement of SR proteins in 5′ or 3′ splice site activation. Mol Cell Biol 19 7347 7356
69. FujitaPARheadBZweigASHinrichsASKarolchikD 2011 The UCSC Genome Browser database: update 2011. Nucleic Acids Res 39 D876 882
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
PLOS Genetics
2011 Číslo 12
- Gynekologové a odborníci na reprodukční medicínu se sejdou na prvním virtuálním summitu
- Je „freeze-all“ pro všechny? Odborníci na fertilitu diskutovali na virtuálním summitu
Najčítanejšie v tomto čísle
- Targeted Proteolysis of Plectin Isoform 1a Accounts for Hemidesmosome Dysfunction in Mice Mimicking the Dominant Skin Blistering Disease EBS-Ogna
- An Assessment of the Individual and Collective Effects of Variants on Height Using Twins and a Developmentally Informative Study Design
- Genomes Reveal Transition of Bacteria from Aquatic to Terrestrial Environments
- A Complex Genomic Rearrangement Involving the Locus Causes Dermal Hyperpigmentation in the Chicken