CELF Family RNA–Binding Protein UNC-75 Regulates Two Sets of Mutually Exclusive Exons of the Gene in Neuron-Specific Manners in
An enormous number of alternative pre–mRNA splicing patterns in multicellular organisms are coordinately defined by a limited number of regulatory proteins and cis elements. Mutually exclusive alternative splicing should be strictly regulated and is a challenging model for elucidating regulation mechanisms. Here we provide models of the regulation of two sets of mutually exclusive exons, 4a–4c and 7a–7b, of the Caenorhabditis elegans uncoordinated (unc)-32 gene, encoding the a subunit of V0 complex of vacuolar-type H+-ATPases. We visualize selection patterns of exon 4 and exon 7 in vivo by utilizing a trio and a pair of symmetric fluorescence splicing reporter minigenes, respectively, to demonstrate that they are regulated in tissue-specific manners. Genetic analyses reveal that RBFOX family RNA–binding proteins ASD-1 and FOX-1 and a UGCAUG stretch in intron 7b are involved in the neuron-specific selection of exon 7a. Through further forward genetic screening, we identify UNC-75, a neuron-specific CELF family RNA–binding protein of unknown function, as an essential regulator for the exon 7a selection. Electrophoretic mobility shift assays specify a short fragment in intron 7a as the recognition site for UNC-75 and demonstrate that UNC-75 specifically binds via its three RNA recognition motifs to the element including a UUGUUGUGUUGU stretch. The UUGUUGUGUUGU stretch in the reporter minigenes is actually required for the selection of exon 7a in the nervous system. We compare the amounts of partially spliced RNAs in the wild-type and unc-75 mutant backgrounds and raise a model for the mutually exclusive selection of unc-32 exon 7 by the RBFOX family and UNC-75. The neuron-specific selection of unc-32 exon 4b is also regulated by UNC-75 and the unc-75 mutation suppresses the Unc phenotype of the exon-4b-specific allele of unc-32 mutants. Taken together, UNC-75 is the neuron-specific splicing factor and regulates both sets of the mutually exclusive exons of the unc-32 gene.
Vyšlo v časopise:
CELF Family RNA–Binding Protein UNC-75 Regulates Two Sets of Mutually Exclusive Exons of the Gene in Neuron-Specific Manners in. PLoS Genet 9(2): e32767. doi:10.1371/journal.pgen.1003337
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1003337
Souhrn
An enormous number of alternative pre–mRNA splicing patterns in multicellular organisms are coordinately defined by a limited number of regulatory proteins and cis elements. Mutually exclusive alternative splicing should be strictly regulated and is a challenging model for elucidating regulation mechanisms. Here we provide models of the regulation of two sets of mutually exclusive exons, 4a–4c and 7a–7b, of the Caenorhabditis elegans uncoordinated (unc)-32 gene, encoding the a subunit of V0 complex of vacuolar-type H+-ATPases. We visualize selection patterns of exon 4 and exon 7 in vivo by utilizing a trio and a pair of symmetric fluorescence splicing reporter minigenes, respectively, to demonstrate that they are regulated in tissue-specific manners. Genetic analyses reveal that RBFOX family RNA–binding proteins ASD-1 and FOX-1 and a UGCAUG stretch in intron 7b are involved in the neuron-specific selection of exon 7a. Through further forward genetic screening, we identify UNC-75, a neuron-specific CELF family RNA–binding protein of unknown function, as an essential regulator for the exon 7a selection. Electrophoretic mobility shift assays specify a short fragment in intron 7a as the recognition site for UNC-75 and demonstrate that UNC-75 specifically binds via its three RNA recognition motifs to the element including a UUGUUGUGUUGU stretch. The UUGUUGUGUUGU stretch in the reporter minigenes is actually required for the selection of exon 7a in the nervous system. We compare the amounts of partially spliced RNAs in the wild-type and unc-75 mutant backgrounds and raise a model for the mutually exclusive selection of unc-32 exon 7 by the RBFOX family and UNC-75. The neuron-specific selection of unc-32 exon 4b is also regulated by UNC-75 and the unc-75 mutation suppresses the Unc phenotype of the exon-4b-specific allele of unc-32 mutants. Taken together, UNC-75 is the neuron-specific splicing factor and regulates both sets of the mutually exclusive exons of the unc-32 gene.
Zdroje
1. WangET, SandbergR, LuoS, KhrebtukovaI, ZhangL, et al. (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456: 470–476.
2. HammondSM, WoodMJ (2011) Genetic therapies for RNA mis-splicing diseases. Trends Genet 27: 196–205.
3. BlackDL (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 72: 291–336.
4. NilsenTW, GraveleyBR (2010) Expansion of the eukaryotic proteome by alternative splicing. Nature 463: 457–463.
5. ChenM, ManleyJL (2009) Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. Nat Rev Mol Cell Biol 10: 741–754.
6. KalsotraA, CooperTA (2011) Functional consequences of developmentally regulated alternative splicing. Nat Rev Genet 12: 715–729.
7. WittenJT, UleJ (2011) Understanding splicing regulation through RNA splicing maps. Trends Genet 27: 89–97.
8. BarashY, CalarcoJA, GaoW, PanQ, WangX, et al. (2010) Deciphering the splicing code. Nature 465: 53–59.
9. SmithCW (2005) Alternative splicing–when two's a crowd. Cell 123: 1–3.
10. HemaniY, SollerM (2012) Mechanisms of Drosophila Dscam mutually exclusive splicing regulation. Biochem Soc Trans 40: 804–809.
11. TakeuchiA, HosokawaM, NojimaT, HagiwaraM (2010) Splicing reporter mice revealed the evolutionally conserved switching mechanism of tissue-specific alternative exon selection. PLoS ONE 5: e10946 doi:10.1371/journal.pone.0010946
12. WarzechaCC, SatoTK, NabetB, HogeneschJB, CarstensRP (2009) ESRP1 and ESRP2 are epithelial cell-type-specific regulators of FGFR2 splicing. Mol Cell 33: 591–601.
13. BaraniakAP, ChenJR, Garcia-BlancoMA (2006) Fox-2 mediates epithelial cell-specific fibroblast growth factor receptor 2 exon choice. Mol Cell Biol 26: 1209–1222.
14. CarstensRP, WagnerEJ, Garcia-BlancoMA (2000) An intronic splicing silencer causes skipping of the IIIb exon of fibroblast growth factor receptor 2 through involvement of polypyrimidine tract binding protein. Mol Cell Biol 20: 7388–7400.
15. OlsonS, BlanchetteM, ParkJ, SavvaY, YeoGW, et al. (2007) A regulator of Dscam mutually exclusive splicing fidelity. Nat Struct Mol Biol 14: 1134–1140.
16. GraveleyBR (2005) Mutually exclusive splicing of the insect Dscam pre-mRNA directed by competing intronic RNA secondary structures. Cell 123: 65–73.
17. Zahler AM (2012) Pre-mRNA splicing and its regulation in Caenorhabditis elegans. WormBook. 2012/04/03 ed. pp. 1–21.
18. RamaniAK, CalarcoJA, PanQ, MavandadiS, WangY, et al. (2011) Genome-wide analysis of alternative splicing in Caenorhabditis elegans. Genome Res 21: 342–348.
19. KuroyanagiH, KobayashiT, MitaniS, HagiwaraM (2006) Transgenic alternative-splicing reporters reveal tissue-specific expression profiles and regulation mechanisms in vivo. Nat Methods 3: 909–915.
20. 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.
21. 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.
22. 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.
23. SyntichakiP, SamaraC, TavernarakisN (2005) The vacuolar H+ -ATPase mediates intracellular acidification required for neurodegeneration in C. elegans. Curr Biol 15: 1249–1254.
24. OkaT, ToyomuraT, HonjoK, WadaY, FutaiM (2001) Four subunit a isoforms of Caenorhabditis elegans vacuolar H+-ATPase. Cell-specific expression during development. J Biol Chem 276: 33079–33085.
25. PujolN, BonnerotC, EwbankJJ, KoharaY, Thierry-MiegD (2001) The Caenorhabditis elegans unc-32 gene encodes alternative forms of a vacuolar ATPase a subunit. J Biol Chem 276: 11913–11921.
26. 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
27. KuroyanagiH (2009) Fox-1 family of RNA-binding proteins. Cell Mol Life Sci 66: 3895–3907.
28. LoriaPM, DukeA, RandJB, HobertO (2003) Two neuronal, nuclear-localized RNA binding proteins involved in synaptic transmission. Curr Biol 13: 1317–1323.
29. DasguptaT, LaddAN (2012) The importance of CELF control: molecular and biological roles of the CUG-BP, Elav-like family of RNA-binding proteins. Wiley Interdiscip Rev RNA 3: 104–121.
30. LeeBJ, CansizogluAE, SuelKE, LouisTH, ZhangZ, et al. (2006) Rules for nuclear localization sequence recognition by karyopherin beta 2. Cell 126: 543–558.
31. SmithCW, Nadal-GinardB (1989) Mutually exclusive splicing of alpha-tropomyosin exons enforced by an unusual lariat branch point location: implications for constitutive splicing. Cell 56: 749–758.
32. LetunicI, CopleyRR, BorkP (2002) Common exon duplication in animals and its role in alternative splicing. Hum Mol Genet 11: 1561–1567.
33. GromakN, MatlinAJ, CooperTA, SmithCW (2003) Antagonistic regulation of alpha-actinin alternative splicing by CELF proteins and polypyrimidine tract binding protein. RNA 9: 443–456.
34. JonesRB, WangF, LuoY, YuC, JinC, et al. (2001) The nonsense-mediated decay pathway and mutually exclusive expression of alternatively spliced FGFR2IIIb and -IIIc mRNAs. J Biol Chem 276: 4158–4167.
35. GlauserDA, JohnsonBE, AldrichRW, GoodmanMB (2011) Intragenic alternative splicing coordination is essential for Caenorhabditis elegans slo-1 gene function. Proc Natl Acad Sci U S A 108: 20790–20795.
36. 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.
37. GalloJM, SpickettC (2010) The role of CELF proteins in neurological disorders. RNA Biol 7: 474–479.
38. LaddAN, NguyenNH, MalhotraK, CooperTA (2004) CELF6, a member of the CELF family of RNA-binding proteins, regulates muscle-specific splicing enhancer-dependent alternative splicing. J Biol Chem 279: 17756–17764.
39. LaddAN, CharletN, CooperTA (2001) The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing. Mol Cell Biol 21: 1285–1296.
40. YangY, MahaffeyCL, BerubeN, MaddatuTP, CoxGA, et al. (2007) Complex seizure disorder caused by Brunol4 deficiency in mice. PLoS Genet 3: e124 doi:10.1371/journal.pgen.0030124
41. BrimacombeKR, LaddAN (2007) Cloning and embryonic expression patterns of the chicken CELF family. Dev Dyn 236: 2216–2224.
42. WuJ, LiC, ZhaoS, MaoB (2010) Differential expression of the Brunol/CELF family genes during Xenopus laevis early development. Int J Dev Biol 54: 209–214.
43. BarronVA, ZhuH, HinmanMN, LaddAN, LouH (2010) The neurofibromatosis type I pre-mRNA is a novel target of CELF protein-mediated splicing regulation. Nucleic Acids Res 38: 253–264.
44. SinghG, CharletBN, HanJ, CooperTA (2004) ETR-3 and CELF4 protein domains required for RNA binding and splicing activity in vivo. Nucleic Acids Res 32: 1232–1241.
45. KinoY, WashizuC, OmaY, OnishiH, NezuY, et al. (2009) MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1. Nucleic Acids Res 37: 6477–6490.
46. HanJ, CooperTA (2005) Identification of CELF splicing activation and repression domains in vivo. Nucleic Acids Res 33: 2769–2780.
47. FaustinoNA, CooperTA (2005) Identification of putative new splicing targets for ETR-3 using sequences identified by systematic evolution of ligands by exponential enrichment. Mol Cell Biol 25: 879–887.
48. MoriD, SasagawaN, KinoY, IshiuraS (2008) Quantitative analysis of CUG-BP1 binding to RNA repeats. J Biochem 143: 377–383.
49. TakahashiN, SasagawaN, SuzukiK, IshiuraS (2000) The CUG-binding protein binds specifically to UG dinucleotide repeats in a yeast three-hybrid system. Biochem Biophys Res Commun 277: 518–523.
50. MarquisJ, PaillardL, AudicY, CossonB, DanosO, et al. (2006) CUG-BP1/CELF1 requires UGU-rich sequences for high-affinity binding. Biochem J 400: 291–301.
51. TsudaK, KuwasakoK, TakahashiM, SomeyaT, InoueM, et al. (2009) Structural basis for the sequence-specific RNA-recognition mechanism of human CUG-BP1 RRM3. Nucleic Acids Res 37: 5151–5166.
52. TeplovaM, SongJ, GawHY, TeplovA, PatelDJ (2010) Structural insights into RNA recognition by the alternate-splicing regulator CUG-binding protein 1. Structure 18: 1364–1377.
53. AuweterSD, FasanR, ReymondL, UnderwoodJG, BlackDL, et al. (2006) Molecular basis of RNA recognition by the human alternative splicing factor Fox-1. EMBO J 25: 163–173.
54. CampbellRE, TourO, PalmerAE, SteinbachPA, BairdGS, et al. (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 99: 7877–7882.
55. OhnoG, OnoK, TogoM, WatanabeY, OnoS, et al. (2012) Muscle-Specific Splicing Factors ASD-2 and SUP-12 Cooperatively Switch Alternative Pre-mRNA Processing Patterns of the ADF/Cofilin Gene in Caenorhabditis elegans. PLoS Genet 8: e1002991 doi:10.1371/journal.pgen.1002991
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
PLOS Genetics
2013 Číslo 2
- Je „freeze-all“ pro všechny? Odborníci na fertilitu diskutovali na virtuálním summitu
- Gynekologové a odborníci na reprodukční medicínu se sejdou na prvním virtuálním summitu
Najčítanejšie v tomto čísle
- Complex Inheritance of Melanoma and Pigmentation of Coat and Skin in Grey Horses
- Coordination of Chromatid Separation and Spindle Elongation by Antagonistic Activities of Mitotic and S-Phase CDKs
- Autophagy Induction Is a Tor- and Tp53-Independent Cell Survival Response in a Zebrafish Model of Disrupted Ribosome Biogenesis
- Assembly of the Auditory Circuitry by a Genetic Network in the Mouse Brainstem