A DExH/D-box protein coordinates the two steps of splicing in a group I intron.
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.
Bifano
Abby L
AL
Center for RNA Molecular Biology, Department of Biochemistry, Case Western Reserve University, School of Medicine, Cleveland, OH 44106-4960, USA.
Caprara
Mark G
MG
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*
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
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