A novel genetic screen for snRNP assembly factors in yeast identifies a conserved protein, Sad1p, also required for pre-mRNA splicing - PubMed (original) (raw)
A novel genetic screen for snRNP assembly factors in yeast identifies a conserved protein, Sad1p, also required for pre-mRNA splicing
Z Lygerou et al. Mol Cell Biol. 1999 Mar.
Abstract
The assembly pathway of spliceosomal snRNPs in yeast is poorly understood. We devised a screen to identify mutations blocking the assembly of newly synthesized U4 snRNA into a functional snRNP. Fifteen mutant strains failing either to accumulate the newly synthesized U4 snRNA or to assemble a U4/U6 particle were identified and categorized into 13 complementation groups. Thirteen previously identified splicing-defective prp mutants were also assayed for U4 snRNP assembly defects. Mutations in the U4/U6 snRNP components Prp3p, Prp4p, and Prp24p led to disassembly of the U4/U6 snRNP particle and degradation of the U6 snRNA, while prp17-1 and prp19-1 strains accumulated free U4 and U6 snRNA. A detailed analysis of a newly identified mutant, the sad1-1 mutant, is presented. In addition to having the snRNP assembly defect, the sad1-1 mutant is severely impaired in splicing at the restrictive temperature: the RP29 pre-mRNA strongly accumulates and splicing-dependent production of beta-galactosidase from reporter constructs is abolished, while extracts prepared from sad1-1 strains fail to splice pre-mRNA substrates in vitro. The sad1-1 mutant is the only splicing-defective mutant analyzed whose mutation preferentially affects assembly of newly synthesized U4 snRNA into the U4/U6 particle. SAD1 encodes a novel protein of 52 kDa which is essential for cell viability. Sad1p localizes to the nucleus and is not stably associated with any of the U snRNAs. Sad1p contains a putative zinc finger and is phylogenetically highly conserved, with homologues identified in human, Caenorhabditis elegans, Arabidospis, and Drosophila.
Figures
FIG. 1
Assay for U4/U6 assembly. (A) Total cell RNA was extracted from a wild-type yeast strain (lanes 1 and 2) or a strain in which the U4 snRNA gene was replaced by U4* (lanes 3 and 4). After RNA extraction at 4°C, RNA was incubated for 10 min at either 20°C (lanes 1 and 3) or 70°C (lanes 2 and 4) prior to being loaded on a native polyacrylamide gel. After transfer to nylon membranes, identical filters were hybridized with an oligonucleotide probe specific for the wild-type U4 snRNA (left panel) or the U4* snRNA (right panel). (B) Total cell RNA was extracted from strain BSY295 grown for 0, 0.5, 1, 2, and 4 h in the presence of galactose. After native gel electrophoresis and transfer to a nylon membrane, the expression of the U4* snRNA was detected with a radioactively labelled oligonucleotide complementary to U4*. A labelled U1-specific oligonucleotide probe was included, as U1 snRNA served as a loading control.
FIG. 2
U4/U6 assembly defects of splicing-defective mutants. The prp3-1, prp4-1, and prp24-1 strains and a wild-type (wt) strain (A), the prp17-1 strain (B), and the prp19-1 strain (C), all transformed with the galactose-inducible U4* gene, were grown for 30 min at 37°C (lanes 1, 2, 4, 5, 7, 8, 10, 11, and 13) or left at 23°C (lanes 3, 6, 9, 12, and 14). Production of U4* was subsequently induced by addition of galactose (Gal) (except in lanes 1, 4, and 7), and growth was continued at the same temperature for another 2 h. Total cell RNA was extracted, separated on a native polyacrylamide gel, transferred to a nylon membrane, and hybridized with radioactively labelled oligonucleotides complementary to U4* and U1 (loading control) snRNAs (upper panels). The same filters (after removal of the U1 and U4* probes) were hybridized with an oligonucleotide probe specific to the wild-type U4 snRNA (middle panels) and subsequently with a U6-specific probe (lower panels).
FIG. 3
Procedure used to identify strains defective in U4/U6 particle assembly.
FIG. 4
The sad1-1 mutant is defective in the assembly of newly synthesized U4* snRNA. The sad1-1 strain and the unrelated temperature-sensitive and splicing-defective prpx-1 strain were grown for 0.5, 1, 2, and 4 h at 37°C. Subsequently, galactose was added and growth was continued for a further 2 h at 37°C. Following RNA extraction, separation on a native acrylamide gel, and transfer, labelled oligonucleotide probes were used to detect the U4* snRNA and U1 snRNA, which serves as a loading control (A). The same filter (after removal of the U1 and U4* probes) was hybridized with an oligonucleotide specific for the U4 snRNA (B).
FIG. 5
The sad1-1 mutant is defective in splicing. (A) Total cell RNAs extracted from the sad1-1 strain (lane 3), three unrelated temperature-sensitive strains (lanes 1, 2, and 4) and the prp2-1 strain (lane 5) grown for 2.5 h at the nonpermissive temperature were resolved on an agarose-formaldehyde gel, transferred to nitrocellulose, and hybridized with a radioactively labelled fragment of the RP29 gene. As a control, RNAs isolated from the prp2-1 strain grown at the permissive temperature (23°C) are presented (lane 6). The positions of migration of the RP29 pre-mRNA and mRNA are indicated. (B) The sad1-1 strain or an isogenic wild-type strain was transformed with either vector alone, a plasmid containing the coding region of the β-galactosidase gene under the control of the GAL10 promoter, the same plasmid in which an intron deriving from the RP51A gene was introduced in the lacZ coding region (intron 1) (62), or the same plasmid in which a synthetic intron was introduced in the lacZ coding region (intron 2) (28). The strains were either grown continually at 23°C or grown for 30 min at 37°C. In either case, galactose was added subsequently and growth was continued for 2 h at the same temperature. Levels of β-galactosidase (bgal) activity produced in these cells are shown (in duplicate).
FIG. 6
Extracts made from sad1-1 cells do not splice a pre-mRNA substrate in vitro. Extracts were prepared from two sad1-1 isolates, from a prp2-1 strain, and from a wild-type (wt) strain grown either at the permissive temperature (lanes 1 to 7) or for 2.5 h at the nonpermissive temperature. Four microliters of extract was incubated with a radioactive actin pre-mRNA for 30 min at 25°C. In lanes 5, 6, 7, 12, 13, and 14, 2 μl each of the indicated extracts was used. Subsequently, RNA was deproteinized and analyzed on a denaturing polyacrylamide gel. The positions of migration of the pre-mRNA substrate, the exon 1 and lariat intermediates, and the mRNA and intron lariat products of the reaction are indicated.
FIG. 7
Cloning and sequence analysis of SAD1. (A) Restriction map of the chromosomal locus containing the SAD1 gene. The extents of the subclones constructed are shown below the map. Complementation of the temperature sensitivity of the sad1-1 strain by the subclones is indicated on the right. (B) Schematic representation of the SAD1 protein. The position of the putative nuclear localization sequence (NLS) and the position and sequence of the putative zinc finger motif are indicated. (C) Alignment of the region surrounding the putative zinc finger in Sad1p and its homologues from C. elegans, Drosophila, Arabidopsis, and human. The positions of the conserved cysteine and histidine residues are marked by arrows. The two mouse ESTs identified encode a peptide identical to the first 40 amino acids deduced from the human ESTs and are not shown. Regions outside this N-terminal block are less well conserved. Identical residues are presented in white letters in a black background, while conservative substitutions are denoted by gray shading.
FIG. 8
Subcellular localization of SAD1p. A strain expressing ProtA-Sad1 and a wild-type strain (inset) were fixed for immunolocalization. Staining of the same cells with an anti-ProtA rabbit antibody (A), an anti-Nop1 monoclonal antibody (B), and DAPI (C) is shown.
References
- Bordonne R, Tarassov I. The yeast SME1 gene encodes the homologue of the human E core protein. Gene. 1996;176:111–117. - PubMed
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