title: A DExH/D-box protein coordinates the two steps of splicing in a group I intron.
abstract: Proteins of the DExH/D family are ATPases that can unwind duplex RNA in vitro. Individual members of this family coordinate many steps in ribonucleoprotein enzyme assembly and catalysis in vivo, but it is largely unknown how the action of these co-factors is specified and precisely timed. As a first step to address this question biochemically, we describe the development of a new protein-dependent group I intron splicing system that requires such an ATPase for coordinating successive steps in splicing. While genetic analysis in yeast has shown that at least five nuclear-encoded proteins are required for splicing of the mitochondrial aI5beta group I intron, we show that efficient in vitro splicing of aI5beta occurs with only two of these co-factors and, furthermore, they fulfill distinct functions in vitro. The Mrs1p protein stabilizes RNA structure and promotes the first step in splicing. In contrast, a DExH/D protein, Mss116p, acts after the first step and, utilizing ATP hydrolysis, specifically enhances the efficiency of exon ligation. An analysis of Mss116p variants with mutations that impair its RNA-stimulated ATP hydrolysis activity or reduce its ability to unwind duplexes show that the efficiency of ATP hydrolysis is a major determinant in promoting exon ligation. These observations suggest that Mss116p acts in aI5beta splicing by catalyzing changes in the structure of the RNA/protein splicing intermediate that promote the second step. More broadly, these observations are consistent with a model in which the "functional-timing" of DExH/D-box protein action can be specified by a specific conformation of its substrate due to the "upstream" activity of other co-factors.
authors.affiliation: Center for RNA Molecular Biology, Department of Biochemistry, Case Western Reserve University, School of Medicine, Cleveland, OH 44106-4960, USA.
authors.forename: Abby L
authors.initials: AL
authors.lastname: Bifano
authors.forename: Mark G
authors.initials: MG
authors.lastname: Caprara
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D007438:Introns / Q000235:genetics*; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D020871:RNA Stability; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C078505:MRS1 protein, S cerevisiae; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C058150:MSS116 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases
references.citation: Science. 2004 Apr 30;304(5671):730-4
references.pmid: 15118161
references.citation: Biochemistry. 1998 Dec 22;37(51):18056-63
references.pmid: 9922174
references.citation: Cell. 2006 Apr 21;125(2):287-300
references.pmid: 16630817
references.citation: Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1460-5
references.pmid: 9465037
references.citation: Biochemistry. 1996 Mar 26;35(12):3754-63
references.pmid: 8619996
references.citation: Cell. 1998 Feb 6;92(3):315-26
references.pmid: 9476892
references.citation: J Mol Biol. 2001 Jun 22;309(5):1087-100
references.pmid: 11399081
references.citation: J Mol Biol. 1989 Jan 20;205(2):275-89
references.pmid: 2538624
references.citation: RNA. 2005 Apr;11(4):437-46
references.pmid: 15769873
references.citation: Nature. 1989 Jan 5;337(6202):84-7
references.pmid: 2535893
references.citation: Curr Genet. 1990 Aug;18(2):117-24
references.pmid: 1699677
references.citation: Mol Cell. 2006 Feb 17;21(4):543-53
references.pmid: 16483935
references.citation: Genes Dev. 2007 Apr 1;21(7):811-20
references.pmid: 17403781
references.citation: J Mol Biol. 1990 Dec 5;216(3):585-610
references.pmid: 2258934
references.citation: EMBO J. 1992 Dec;11(13):5033-9
references.pmid: 1464325
references.citation: J Mol Biol. 2008 Mar 14;377(1):193-205
references.pmid: 18237742
references.citation: RNA. 2006 May;12(5):903-12
references.pmid: 16556937
references.citation: J Mol Biol. 2006 Aug 25;361(4):771-84
references.pmid: 16872630
references.citation: Mol Cell. 2006 Nov 17;24(4):611-7
references.pmid: 17188036
references.citation: Q Rev Biophys. 2005 Nov;38(4):397-403
references.pmid: 16934171
references.citation: Nucleic Acids Res. 2008 May;36(9):2958-68
references.pmid: 18388132
references.citation: Curr Opin Struct Biol. 2002 Jun;12(3):296-300
references.pmid: 12127447
references.citation: Gene. 1989 Apr 15;77(1):51-9
references.pmid: 2744487
references.citation: Genes Dev. 2007 Apr 1;21(7):821-34
references.pmid: 17403782
references.citation: Biochemistry. 2002 Mar 19;41(11):3693-702
references.pmid: 11888286
references.citation: EMBO J. 1998 May 15;17(10):2926-37
references.pmid: 9582286
references.citation: Mol Cell. 2001 Aug;8(2):251-62
references.pmid: 11545728
references.citation: Nucleic Acids Res. 2006;34(15):4168-80
references.pmid: 16936318
references.citation: Genes Dev. 2005 Oct 1;19(19):2255-60
references.pmid: 16204176
references.citation: Nat Rev Mol Cell Biol. 2004 Nov;5(11):908-19
references.pmid: 15520810
references.citation: Nat Struct Mol Biol. 2005 Jan;12(1):82-9
references.pmid: 15580277
references.citation: Curr Opin Struct Biol. 1999 Jun;9(3):339-45
references.pmid: 10361090
references.citation: Curr Opin Struct Biol. 2005 Jun;15(3):349-54
references.pmid: 15922593
references.citation: RNA. 2007 Feb;13(2):211-22
references.pmid: 17164477
references.citation: J Mol Biol. 2007 Jan 19;365(3):835-55
references.pmid: 17081564
references.citation: Curr Biol. 1998 Apr 9;8(8):441-51
references.pmid: 9550699
references.citation: Proc Natl Acad Sci U S A. 2003 Mar 4;100(5):2300-5
references.pmid: 12591943
references.citation: Biochemistry. 1996 Oct 15;35(41):13469-77
references.pmid: 8873616
references.citation: Biochemistry. 1999 Mar 23;38(12):3468-77
references.pmid: 10090733
references.citation: Biochemistry. 1998 Feb 24;37(8):2180-93
references.pmid: 9485364
references.citation: Proc Natl Acad Sci U S A. 2002 Jan 8;99(1):128-33
references.pmid: 11773622
references.citation: Mol Cell. 2004 Nov 5;16(3):351-62
references.pmid: 15525509
references.citation: EMBO J. 2004 Jul 7;23(13):2478-87
references.pmid: 15201868
references.citation: Mol Cell. 2004 May 7;14(3):343-54
references.pmid: 15125837
references.citation: Cell. 2002 Jun 14;109(6):769-79
references.pmid: 12086675
references.citation: Proc Natl Acad Sci U S A. 2005 Jan 4;102(1):163-8
references.pmid: 15618406
references.citation: J Mol Biol. 1996 Mar 29;257(2):330-41
references.pmid: 8609627
references.citation: Gene. 2006 Feb 15;367:17-37
references.pmid: 16337753
references.citation: EMBO J. 1987 Jul;6(7):2123-9
references.pmid: 2443348
references.citation: RNA. 2004 Dec;10(12):1867-87
references.pmid: 15547134
references.citation: J Biol Chem. 2000 Aug 4;275(31):23725-8
references.pmid: 10825168
references.citation: Mol Cell. 2007 Oct 12;28(1):159-66
references.pmid: 17936712
references.citation: Nat Struct Biol. 2000 Oct;7(10):831-3
references.pmid: 11017186