A DExH/D-box Protein Coordinates the Two Steps of Splicing in a Group I Intron

Abstract

SummaryProteins 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 proteindependent group I intron splicing system that requires such an ATPase for coordinating successive steps in splicing. While genetic analysis in yeast have shown that at least five nuclear-encoded proteins are required for splicing of the mitochondrial aI5β group I intron, we show that efficient in vitro splicing of aI5β 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 aI5β 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.

Splicing of Yeast aI5β Group I Intron Requires SUV3 to Recycle MRS1 via Mitochondrial Degradosome-promoted Decay of Excised Intron Ribonucleoprotein (RNP)

“…2B). Notably, mrs1⌬ showed enhanced accumulation of aI5␤-containing COX1 pre-mRNA, which is consistent with the Mrs1p protein having a role in the first step of splicing (27). Protein cofactors having a role after the first step of splicing, such as Pet54p, are likely to have loss of ligated exon product, with no increase in the amount of pre-mRNA because the partially spliced product is likely to be unstable (28).…”

“…Question marks reflect a gap in our knowledge of the degree to which Suv3p recycles Mrs1p from aI5␤ versus bI3. (27,37,49). The affinity is modest, but as shown for the bI3 intron, other splicing cofactors can increase affinity of Mrs1p for its RNA targets (49).…”

“…Here we show a strong genetic interaction between MRS1 and SUV3 in that overexpression of MRS1 partially suppressed splicing defects in the suv3-1 strain. Recent work has shown that two Mrs1p dimers specifically bind both aI5␤-and bI3-containing pre-mRNAs and, in collaboration with other intron-specific cofactors, facilitate splicing in vitro (27,49). Because the two introns belong to different structural subclasses and do not share significant sequence homology, it seems likely that, like most group I intron splicing cofactors studied thus far, Mrs1p functions to facilitate splicing of both introns by binding the conserved intron core and stabilizing the catalytically active structure.…”

“…Using a reconstituted in vitro system, we have shown that one of the proteins, Mrs1p, binds to the intron and facilitates the first step in splicing, while the efficiency of the exon ligation step is poor (27). Two other proteins, Pet54p and the DEXH/D protein Mss116p, can separately increase the efficiency of exon ligation in the presence of Mrs1p, although the efficiency only approaches ϳ30% in vitro (27,28).…”

Mne1 Is a Novel Component of the Mitochondrial Splicing Apparatus Responsible for Processing of a COX1 Group I Intron in Yeast

“…In an in vitro study on splicing of the aI5␤ intron, Mrs1 was found to promote the first step in splicing, whereas Mss116 acts subsequently in the exon ligation reaction (47). Because Mrs1 functions at an early step in aI5␤ splicing, we sought to assess the functional relationship of Mrs1 and Mne1.…”