Prp43: An RNA helicase-like factor involved in spliceosome disassembly

Abstract

The Saccharomyces cerevisiae genes PRP2, PRP16, and PRP22 encode pre-mRNA splicing factors that belong to the highly conserved "DEAH" family of putative RNA helicases. We previously identified two additional members of this family, JA1 and JA2. To investigate its biological function, we cloned the JA1 gene and generated alleles carrying mutations identical to those found in highly conserved regions of other members of the DEAH family. A ja1 allele carrying a mutation identical to that in the temperature-sensitive (ts) prp22-1 gene conferred ts phenotype when integrated into the genome of a wild-type strain by gene replacement. Northern analysis of RNA obtained from the ts strain shifted to a nonpermissive temperature revealed accumulation of unspliced pre-mRNAs and excised intron lariats. Furthermore, analysis of splicing complexes showed that intron lariats accumulated in spliceosomes. The results presented indicate that JA1 encodes a pre-mRNA processing factor (Prp) involved in disassembly of spliceosomes after the release of mature mRNA. We have therefore renamed this gene PRP43.

Figure 1

Alignment of DEAH family proteins, including the helicase and C-terminal domains. The alignment includes residues 96–736 of Prp43. The complete sequences of Prp43 and JA2 have been deposited in GenBank, accession numbers U41851 and AF005090, respectively. The seven highly conserved motifs (I–VI) of the putative helicase domain are boxed. The positions of single amino acid changes introduced by mutagenesis are indicated with black boxes. Highly conserved motifs of the C-terminal domain are also boxed.

Figure 2

(a) Complementation of the prp43–1 ts phenotype. The prp43–1 and prp22–1 strains were transformed with the following plasmids: pYCP50, control vector; pYCP.ja1, genomic pYCP50 clone containing an ≈15-kb fragment including PRP43; pRS316, control vector; pRSJA1, pRS316 carrying PRP43; and pRSJA1sn, pRS316 carrying a truncated PRP43 gene. From each transformation, eight colonies were randomly selected and arranged in rows for ts testing. The upper panel shows the growth of prp43–1 transformants. The lower panel shows growth of prp22–1 transformants at the indicated temperatures. (b) Constructs containing the PRP43 gene under control of the GAL1 promotor were subcloned into the low-copy-number CEN plasmid pRS416 and the high-copy-number 2μ plasmid pRS426. Identical clones were also prepared for PRP43 alleles carrying the mutations G194D and S247L. All constructs were used to transform wild-type yeast cells. Uracil-independent (URA⁺) transformants were isolated and tested for growth on glucose and galactose. The plasmid and PRP43 allele harbored by the transformants is shown in the schematic below.

Figure 3

(a) Northern blot analysis of total RNA prepared from yeast strains grown at room temperature (−) and after shift to 37°C for 2 hr (+). WT, wild type. The blot was probed with a radiolabeled DNA fragment complementary to sequences of the intron of the actin gene. (b) The indicated strains and prp43–1 transformed with various complementing plasmids were grown at room temperature and shifted to 37°C. Total RNA was prepared after temperature shift and analyzed by Northern blotting as in a. A schematic representation of each splicing product is shown to the right.

Figure 4

(a) Splicing extracts from wild type (WT), prp43–1, and prp22–1 were heated for 60 min at 23°C or 30°C as indicated. Standard splicing reactions were then set with radiolabeled actin pre-mRNA and the previously heated extracts. Splicing complexes formed were resolved by nondenaturing PAGE. See text for description of complexes. (b) Splicing complexes were sliced off the gel in a and their eluted RNA contents were resolved by denaturing PAGE.

Figure 5

Seven putative RNA helicases required during spliceosome assembly, catalytic steps, and spliceosome disassembly are shown. While some limiting second-step splicing factors may leave the spliceosome at the time of mRNA release, many splicing factors, including snRNPs, are not recycled until the excised intron lariat is released.