Mutations in PRP43 that uncouple RNA-dependent NTPase activity and pre-mRNA splicing function.

Nucleic acid-dependent NTPases of the DExH/D box family are essential for remodeling the large RNP complexes that catalyze pre-mRNA splicing, ribosome biogenesis and translation (1-3). DExH/D proteins are defined by a set of seven or eight conserved peptide motifs including the signature motif II, which can be DEAD, DEAH or DExH (4-6). The repertoire of biochemical activities ascribed to DExH/D NTPases includes unwinding of nucleic acid duplexes, displacement of proteins from RNA, and annealing of RNA strands (6-9). It has been proposed that DExH/D proteins use the energy of NTP hydrolysis to modulate the structure or the composition of RNA-protein assemblies (10-12). However, the molecular mechanisms by which DExH/D proteins accomplish this and the conformational changes elicited by them are poorly understood. The pre-mRNA spliceosome employs at least eight proteins of the DExH/D family (3). Prp5, Prp28, Brr2 and Sub2/UAP56 are involved in assembling snRNPs and proteins onto the precursor RNA (3). Prp2 and Prp16 use NTP hydrolysis to drive the first and second transesterification reaction, respectively (13, 14). Prp22 and Prp43 then act sequentially to release mature RNA and lariat intron from the spliceosome (15-19). Prp2, Prp16, Prp22 and Prp43 belong to the DEAH subgroup (17, 20). They contain a central ATPase/helicase domain encompassing conserved motifs I-VI, and they also share a ∼300 amino acid C-terminal domain not found in other DExH/D proteins (21). Understanding how these DEAH paralogs carry out their distinct functions during the splicing cycle requires a detailed description of the enzymatic activities to highlight similarities and uncover differences. The significance of conserved residues in motifs I (GxGKT), II (DEAH), III (SAT) and VI (QRxGRxGR) for viability and ATP hydrolysis has been tested for each of the spliceosomal DEAH box proteins in Saccharomyces cerevisiae (18, 22-27). The salient point of these studies is that ATPase activity is necessary for the biological function and for splicing activity in each case. Here, we further characterize the biochemical activities and structure-function relationships of S. cerevisiae Prp43, a 767-amino acid RNA-dependent ATPase that releases the excised lariat intron from the spliceosome. We show that Prp43 hydrolyzes all common NTPs and dNTPs and that it disrupts short nucleic acid duplexes in an NTP-dependent fashion. We assess the effects of alanine substitutions at conserved residues in motifs Ia (146TQPRRVAA153), IV (307LLFLTG312) and V (376TNIAETSLT384). We show that lethal mutations at position Thr384 (motif V) uncouple ATPase activity from Prp43's role in releasing lariat intron RNA from the spliceosome. These findings suggest that ATPase activity is not sufficient for the biological function of Prp43. Prior deletion analyses showed that the conserved C-terminal segments are important for the biological functions of the DEAH splicing factors Prp2, Prp16, Prp22 and Prp43 (18, 21, 23, 28, 29). In the case of Prp2, the C-terminal domain is required for spliceosome binding through interaction with Spp2 (30). Here, we introduced single-alanine or alanine-cluster mutations at 97 residues within the C-terminal domain. None of the PRP43 alleles was lethal, suggesting that individual conserved residues within the C-domain are not critical for Prp43 function in vivo.