title: Comprehensive in vivo RNA-binding site analyses reveal a role of Prp8 in spliceosomal assembly.
abstract: Prp8 stands out among hundreds of splicing factors as a protein that is intimately involved in spliceosomal activation and the catalytic reaction. Here, we present the first comprehensive in vivo RNA footprints for Prp8 in budding yeast obtained using CLIP (cross-linking and immunoprecipitation)/CRAC (cross-linking and analyses of cDNAs) and next-generation DNA sequencing. These footprints encompass known direct Prp8-binding sites on U5, U6 snRNA and intron-containing pre-mRNAs identified using site-directed cross-linking with in vitro assembled small nuclear ribonucleoproteins (snRNPs) or spliceosome. Furthermore, our results revealed novel Prp8-binding sites on U1 and U2 snRNAs. We demonstrate that Prp8 directly cross-links with U2, U5 and U6 snRNAs and pre-mRNA in purified activated spliceosomes, placing Prp8 in position to bring the components of the active site together. In addition, disruption of the Prp8 and U1 snRNA interaction reduces tri-snRNP level in the spliceosome, suggesting a previously unknown role of Prp8 in spliceosomal assembly through its interaction with U1 snRNA.
authors: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA.
authors: Xueni
authors: X
authors: Li
authors: Wenzheng
authors: W
authors: Zhang
authors: Tao
authors: T
authors: Xu
authors: Jolene
authors: J
authors: Ramsey
authors: Lingdi
authors: L
authors: Zhang
authors: Ryan
authors: R
authors: Hill
authors: Kirk C
authors: KC
authors: Hansen
authors: Jay R
authors: JR
authors: Hesselberth
authors: Rui
authors: R
authors: Zhao
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D007438:Introns; D008969:Molecular Sequence Data; D012322:RNA Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000378:metabolism*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism* / Q000502:physiology; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000378:metabolism* / Q000502:physiology; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism* / Q000502:physiology; D017381:Spliceosomes / Q000378:metabolism*
chemical_list: C077018:PRP8 protein, S cerevisiae; D012322:RNA Precursors; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C405485:U5 small nuclear RNA
references: Mol Cell. 2003 Jul;12(1):5-14
references: 12887888
references: Mol Cell. 2007 Feb 23;25(4):615-24
references: 17317632
references: Methods Enzymol. 2002;350:380-92
references: 12073325
references: PLoS Biol. 2007 Apr;5(4):e90
references: 17388687
references: Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):13817-22
references: 18779563
references: Nat Struct Mol Biol. 2008 Nov;15(11):1199-205
references: 18836455
references: Methods. 2005 Dec;37(4):376-86
references: 16314267
references: Cell. 1988 Oct 7;55(1):41-8
references: 3048702
references: Methods Enzymol. 1988;164:287-309
references: 3071669
references: Curr Opin Chem Biol. 2005 Dec;9(6):603-8
references: 16242989
references: Photochem Photobiol. 1984 Feb;39(2):125-33
references: 6709718
references: RNA. 1998 Dec;4(12):1675-86
references: 9848662
references: Mol Cell Biol. 1998 Nov;18(11):6756-66
references: 9774689
references: Nature. 2009 Mar 26;458(7237):475-80
references: 19325628
references: RNA. 2001 Nov;7(11):1543-53
references: 11720284
references: Nucleic Acids Res. 2004 Jan 1;32(Database issue):D311-4
references: 14681421
references: Nucleic Acids Res. 1993 Sep 25;21(19):4645-6
references: 8233807
references: EMBO J. 2008 Nov 5;27(21):2929-40
references: 18843295
references: Yeast. 1998 Jul;14(10):953-61
references: 9717241
references: Nucleic Acids Res. 1991 Dec 11;19(23):6367-72
references: 1754372
references: Genes Dev. 2012 Nov 1;26(21):2408-21
references: 23124065
references: Genetics. 2000 Aug;155(4):1667-82
references: 10924465
references: RNA Biol. 2010 May-Jun;7(3):345-53
references: 20458185
references: Nat Struct Biol. 2000 Oct;7(10):850-4
references: 11017191
references: J Biol Chem. 2000 Dec 29;275(52):41458-68
references: 11006293
references: Nat Biotechnol. 2011 Jun 01;29(7):607-14
references: 21633356
references: Nature. 2003 Oct 16;425(6959):737-41
references: 14562106
references: Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9613-8
references: 19482942
references: Mol Cell Biol. 1994 Mar;14(3):2180-90
references: 8114748
references: RNA. 1996 Sep;2(9):879-94
references: 8809015
references: Mol Cell Proteomics. 2005 Dec;4(12):1948-58
references: 16150870
references: Proc Natl Acad Sci U S A. 2002 Jul 9;99(14):9145-9
references: 12087126
references: Mol Cell Biol. 1996 Mar;16(3):818-28
references: 8622683
references: Protein Sci. 2007 Jun;16(6):1024-31
references: 17473007
references: J Proteome Res. 2012 Oct 5;11(10):4894-905
references: 22897585
references: Proc Natl Acad Sci U S A. 2011 Jun 14;108(24):10010-5
references: 21610164
references: Mol Cell. 2009 Nov 25;36(4):593-608
references: 19941820
references: Genes Dev. 1990 Dec;4(12B):2264-77
references: 2149118
references: RNA. 2007 Dec;13(12):2252-65
references: 17925343
references: Genes Dev. 1999 Aug 1;13(15):1970-82
references: 10444595
references: Genetics. 1996 Jun;143(2):723-39
references: 8725222
references: RNA. 1999 Nov;5(11):1470-81
references: 10580475
references: RNA. 2005 May;11(5):533-57
references: 15840809