The splicing of yeast mitochondrial group I and group II introns requires a DEAD-box protein with RNA chaperone function

Abstract

Group I and II introns self-splice in vitro, but require proteins for efficient splicing in vivo, to stabilize the catalytically active RNA structure. Recent studies showed that the splicing of some Neurospora crassa mitochondrial group I introns additionally requires a DEAD-box protein, CYT-19, which acts as an RNA chaperone to resolve nonnative structures formed during RNA folding. Here we show that, in Saccharomyces cerevisiae mitochondria, a related DEAD-box protein, Mss116p, is required for the efficient splicing of all group I and II introns, some RNA end-processing reactions, and translation of a subset of mRNAs, and that all these defects can be partially or completely suppressed by the expression of CYT-19. Results for the aI2 group II intron indicate that Mss116p is needed after binding the intron-encoded maturase, likely for the disruption of stable but inactive RNA structures. Our results suggest that both group I and II introns are prone to kinetic traps in RNA folding in vivo and that the splicing of both types of introns may require DEAD-box proteins that function as RNA chaperones.bakers' yeast ͉ mitochondria ͉ splicing factor D ExH͞D-box proteins are a large, ubiquitous protein family, whose members use the energy of ATP hydrolysis to mediate RNA structural rearrangements in a variety of cellular processes (1, 2). These proteins have a core region containing nine conserved motifs flanked by unique N-and͞or C-terminal extensions, which in some cases target the proteins to their sites of action by specific RNA or protein interactions. The proteins are named for the amino acid sequence of motif II, which in different subfamilies is DEAD, DEAH, or some variant thereof. Experiments with model substrates show that DExH͞D-box proteins can act as RNA helicases (3) or can disrupt ribonucleoprotein (RNP) complexes independently of their helicase activity (4). However, how these proteins function on their natural substrates has remained largely unknown.Group I and II introns self-splice in vitro but require proteins for efficient splicing in vivo to help fold the intron RNA into the catalytically active structure (5). In the fungus Neurospora crassa, the splicing of a subset of mitochondrial (mt) group I introns depends on two proteins encoded by nuclear genes, the mt tyrosyl-tRNA synthetase (CYT-18 protein), which stabilizes the catalytically active RNA structure, and the DEAD-box protein 7). Recently, CYT-19 was shown to function as an ATP-dependent RNA chaperone to destabilize nonnative structures that constitute kinetic traps in the CYT-18-assisted RNA folding pathway (7). A mutation in the cyt-19 gene did not affect the splicing of non-CYT-18-dependent group I introns or a group II intron, but did inhibit some 5Ј and 3Ј end processing reactions and, possibly, mt translation (7,8).The Saccharomyces cerevisiae nuclear genome encodes three DExH͞D-box proteins (Suv3p, Mrh4p, and Mss116p) that function in mitochondria (9-11). Of these, Mss116p is the most closely related to CYT-19. The two proteins have 32...

The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

“…Examples are the splicing helicases Cyt-19 and Mss116 (Mohr et al, 2002;Huang et al, 2005;Halls et al, 2007) that are general RNA chaperones, and Ded1p, a helicase involved in translation (Iost et al, 1999). These proteins bind unspecifically to structured RNA via their basic Cterminal extensions, and their helicase cores then unwind loosely attached neighboring double helical regions (Grohman et al, 2007;Mohr et al, 2008).…”

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

“…The discovery of DEAD-box proteins that promote splicing in the well-characterized group I and II introns opened the door for recent mechanistic breakthroughs [18,[37][38][39]. Interestingly, these DEAD-box proteins were shown to be interchangeable to a substantial extent, demonstrating that they function rather promiscuously.…”

“…Interestingly, these DEAD-box proteins were shown to be interchangeable to a substantial extent, demonstrating that they function rather promiscuously. Deletion of Mss116p, a mitochondrial DEAD-box protein from Saccharomyces cerevisiae, adversely affected the splicing of group I and II introns in vivo; these deleterious effects could be largely rescued by the expression of a related DEAD-box protein, CYT-19 from Neurospora crassa [38]. Further, Mss116p and CYT-19 were shown to promote group I and group II intron splicing in vitro, as was the cytosolic DEAD-box protein, Ded1p [37][38][39].…”

“…Deletion of Mss116p, a mitochondrial DEAD-box protein from Saccharomyces cerevisiae, adversely affected the splicing of group I and II introns in vivo; these deleterious effects could be largely rescued by the expression of a related DEAD-box protein, CYT-19 from Neurospora crassa [38]. Further, Mss116p and CYT-19 were shown to promote group I and group II intron splicing in vitro, as was the cytosolic DEAD-box protein, Ded1p [37][38][39]. Each of these proteins was also shown to unwind RNA helices non-specifically in an ATP-dependent manner [36,37,[39][40][41].…”

“…These results led to the proposal of a model for CYT-19's activity: non-specific binding of the chaperone, followed by structural disruption via local duplex unwinding [38] -The authors demonstrate the promiscuity of DEAD-box proteins CYT-19 (from Neurospora crassa) and Mss116p (from Saccharomyces cerevisiae) by showing that splicing reactions in Mss116p-deletion mutants can be rescued by expression of CYT-19 in vivo.…”