High-copy-number expression of Sub2p, a member of the RNA helicase superfamily, suppresses hpr1-mediated genomic instability - PubMed (original) (raw)

High-copy-number expression of Sub2p, a member of the RNA helicase superfamily, suppresses hpr1-mediated genomic instability

H Y Fan et al. Mol Cell Biol. 2001 Aug.

Abstract

We report on a novel role for a pre-mRNA splicing component in genome stability. The Hpr1 protein, a component of an RNA polymerase II complex and required for transcription elongation, is also required for genome stability. Deletion of HPR1 results in a 1,000-fold increase in genome instability, detected as direct-repeat instability. This instability can be suppressed by the high-copy-number SUB2 gene, which is the Saccharomyces cerevisiae homologue of the human splicing factor hUAP56. Although SUB2 is essential, conditional alleles grown at the permissive temperature complement the essential function of SUB2 yet reveal nonessential phenotypes. These studies have uncovered a role for SUB2 in preventing genome instability. The genomic instability observed in sub2 mutants can be suppressed by high-copy-number HPR1. A deletion mutant of CDC73, a component of a PolII complex, is also unstable for direct repeats. This too is suppressed by high-copy-number SUB2. Thus, defects in both the transcriptional machinery and the pre-mRNA splicing machinery can be sources of genome instability. The ability of a pre-mRNA splicing factor to suppress the hyperrecombination phenotype of a defective PolII complex raises the possibility of integrating transcription, RNA processing, and genome stability or a second role for SUB2.

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Figures

FIG. 1

Suppression of growth defect associated with hpr1Δ, cdc73Δ , and hpr1Δ cdc73Δ strains by elevated-copy-numbers of the SUB2 gene. Wild type (HKY579-10A), hpr1Δ (U768-1C), cdc73Δ (HFY580-104), and hpr1Δ cdc73Δ (HFY2051-1D) carrying either YEp-vector (YEp351) or YEp-SUB2 (hy41) were streaked on leucine dropout plates to select for plasmids as indicated and allowed to grow at 30 or 37°C for 2 days. YEp351 is a 2μm-based vector, and hy41 contains the SUB2 gene in the YEp351 plasmid.

FIG. 2

Plasmid instability in hpr1Δ strains is suppressed by high-copy-number SUB2. Wild-type (wt) and hpr1Δ strains were transformed with plasmid pRM102 and YEp351 with no insert or with a SUB2 insert. pRM102_CYC1_ter contains a CYC1 transcriptional terminator inserted into the _Apa_I restriction site located directly downstream of the ded1 sequence on pRM102. Plasmid loss rates were determined after transfer from selective to nonselective growth conditions for the pRM102 plasmid. Loss rates were determined as described previously (9). Plasmid loss rates were averaged from three independent experiments.

FIG. 3

SUB2 is an essential gene, as illustrated by complementation of the sub2Δ mutants by different plasmids. Shown is the growth of SUB2 (HKY579-10A), sub2Δ 1::TRP1 + YCp2-SUB2 (pHF68-1) (HFY2170-6A) and sub2Δ 2:: HIS3 + YCp2-SUB2 (pHF68-1) (HFY2210-114B) on yeast extract-peptone-dextrose and FOA-containing media. To examine the effects of SUB2 or sub2 mutant alleles (on CEN vectors) in complementing the lethality of the sub2Δ 1 strain, HFY2170-6A and HFY2210-114B were transformed with various plasmids. The transformants were checked for viability on FOA-containing medium. The genotype of each strain is as indicated. Strains streaked on the FOA-containing medium all carried YCp2-SUB2. Since only uracil-auxotrophic cells can grow on media containing FOA, all cells grown on the FOA-containing medium have lost YCp2-SUB2. SUB2 is an essential gene, as sub2 deletion strains failed to grow on FOA-containing medium which selects against YCp2-SUB2. YCp1-SUB2 (pHF127-3) and YCp2-sub9-267 can substitute for YCp2-SUB2 and rescue the sub2Δ strains, indicating that these plasmids contain a functional SUB2 fragment. YCp2-sub2-112, which contains a mutation in the ATP binding domain of the SUB2 gene, was unable to replace YCp2-SUB2, suggesting that the putative ATPase activity is necessary for the Sub2p function.

FIG. 4

Sequence alignment of Sub2p and related proteins. Shown are sequence alignments of the S. cerevisiae Sub2p (GenBank accession number Z74132) with related proteins from S. pombe (Z99162), Drosophila (WM6/HEL, X79802), human 1 (UAP56/BAT1, Z37166), and human 2 (U90426). Sequences were aligned according to the Hotun Hein algorithm method with a PAM250 weight table using Lasergene sequence analysis software. Shaded regions contain amino acid identity. Boxed sequences represent the conserved helicase motifs.

FIG. 5

Interaction of Sub2p with Mud2p in vitro and Rpb3p in vivo. (A) Sub2-HA and Mud2-Flag proteins were synthesized in vitro using a coupled transcription and translation system. Sub2-HA and Mud2-Flag proteins were mixed and immunoprecipitated with monoclonal antibodies as indicated. Immune complexes were resolved on an SDS-polyacrylamide gel electrophoresis gel which was transferred and probed with polyclonal anti-HA antibody. Lane 4 indicated that the Sub2-HA and Mud2-Flag proteins coimmunoprecipitated. Lane 1, positive control; lanes 2 and 3, negative controls. The positions of protein size markers are indicated. (B) Coimmunoprecipitation of Sub2p and the RNA polymerase II component Rpb3. Lanes 1 and 2, Western blots of extracts from HPR1 (wt) + YEp351-SUB2-FLAG and hpr1 + YEp351_-SUB2-FLAG_ cells. Extracts were electrophoresed on a 9% SDS-polyacrylamide gel and subjected to Western blot analysis with anti-FLAG antibodies. Lanes 4 and 6 show extracts from the strains used in lanes 1 and 2 after immunoprecipitation using mouse anti-Rpb3 antibody, goat anti-mouse with a biotin conjugate, and then streptavidin-coated beads. Lanes 3 and 5 are controls in which only the goat anti-mouse biotin conjugate and streptavidin beads were added to the same extracts. The immunoprecipitated proteins were electrophoresed on a 9% SDS-polyacrylamide gel and subjected to Western blot analysis with anti-FLAG antibodies.

FIG. 6

Suppression of hpr1Δ growth defect is MUD2 independent. Wild type (HKY579-10A), hpr1Δ (HFY824-1A), mud2Δ (RMY161-2C), and hpr1Δ mud2Δ (RMY159-11A) carrying either YEp-vector (YEp351) or YEp-SUB2 (pHF80-4) were streaked on leucine dropout plates to select for plasmids as indicated and allowed to grow at 30 or 37°C for 2 days. YEp351 is a 2μm-based vector, and hy41 contains the SUB2 gene in the YEp351 plasmid.

FIG. 7

Involvement of Sub2p in transcriptional silencing at telomeres. Serial dilutions of yeast strains were plated on a leucine dropout plate (A) or an FOA-containing leucine-dropout medium (B). Leucine dropout medium selects for cells containing plasmids, and FOA selects against Ura+ cells. Lanes 1 and 2, wild-type cells containing YCp1-vector (YCplac111) and YCp1-SUB2 (pHF127-3), respectively. Lane 3, sub2Δ cells carrying YCp1-SUB2. All the above strains contain the telomere-silencing assay system adh4::URA3-TEL. The ratio of the number of cells grown on panel B to the number of cells grown on panel A is slightly decreased in lane 3 compared to lanes 1 and 2.

References

1. Abovich N, Liao X C, Rosbash M. The yeast MUD2 protein: an interaction with PRP11 defines a bridge between commitment complexes and U2 snRNP addition. Genes Dev. 1994;8:843–854. - PubMed
1. Aguilera A, Klein H L. Genetic control of intrachromosomal recombination in Saccharomyces cerevisiae. I. Isolation and genetic characterization of hyper-recombination mutations. Genetics. 1988;119:779–790. - PMC - PubMed
1. Aguilera A, Klein H L. HPR1, a novel yeast gene that prevents intrachromosomal excision recombination, shows carboxy-terminal homology to the Saccharomyces cerevisiae TOP1 gene. Mol Cell Biol. 1990;10:1439–1451. - PMC - PubMed
1. Aparicio O M, Billington B L, Gottschling D E. Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae. Cell. 1991;66:1279–1287. - PubMed
1. Chang M, Jaehning J A. A multiplicity of mediators: alternative forms of transcription complexes communicate with transcriptional regulators. Nucleic Acids Res. 1997;25:4861–4865. - PMC - PubMed

High-copy-number expression of Sub2p, a member of the RNA helicase superfamily, suppresses hpr1-mediated genomic instability - PubMed (original) (raw)