Saccharomyces cerevisiae Sen1 as a model for the study of mutations in human Senataxin that elicit cerebellar ataxia

Abstract

The nuclear RNA and DNA helicase Sen1 is essential in the yeast Saccharomyces cerevisiae and is required for efficient termination of RNA polymerase II transcription of many short noncoding RNA genes. However, the mechanism of Sen1 function is not understood. We created a plasmid-based genetic system to study yeast Sen1 in vivo. Using this system, we show that (1) the minimal essential region of Sen1 corresponds to the helicase domain and one of two flanking nuclear localization sequences; (2) a previously isolated terminator readthrough mutation in the Sen1 helicase domain, E1597K, is rescued by a second mutation designed to restore a salt bridge within the first RecA domain; and (3) the human ortholog of yeast Sen1, Senataxin, cannot functionally replace Sen1 in yeast. Guided by sequence homology between the conserved helicase domains of Sen1 and Senataxin, we tested the effects of 13 missense mutations that cosegregate with the inherited disorder ataxia with oculomotor apraxia type 2 on Sen1 function. Ten of the disease mutations resulted in transcription readthrough of at least one of three Sen1-dependent termination elements tested. Our genetic system will facilitate the further investigation of structure-function relationships in yeast Sen1 and its orthologs.

Keywords: cerebellar ataxia; helicase; neurodegeneration; transcription termination; yeast genetics.

Figure 1

A plasmid-based system for testing mutations in yeast Sen1. (A) Comparison of S. cerevisiae Sen1 with human Senataxin. The conserved helicase domains and their subdomains are highlighted by colored boxes. Numbers indicate amino acid residues. The location of missense disease mutations in Sentaxin are shown by colored dots. (B) Anti-Sen1(HD) immunoblotting of cell extracts from strains with either chromosomal (46α) or plasmid-borne (pRS313-SEN1) SEN1. Nrd1 antiserum was used as a loading control (bottom). (C) Schematic genotype of yeast strain DAB206 after transformation with a mutant SEN1 allele and a terminator-reporter construct. Its chromosomal SEN1 and CUP1 genes are disrupted, and the SEN1 deletion is complemented by pRS316-SEN1. Spontaneous loss of pRS316-SEN1 is selected for by plating on medium containing 5-FOA. Terminator readthrough can be assayed by plating on medium containing copper sulfate, which is detoxified by the CUP1 gene product. (D) Schematic of the ACT1-CUP1 construct for measuring terminator activity. (E) Schematic of the CUP1 reporter for measuring attenuator activity.

Figure 2

The essential region of SEN1 closely corresponds to the helicase domain. (A) Schematic of the SEN1 alleles tested in this study and viability of the indicated alleles in the haploid strains. Numbers indicate the amino acid residues present. (B) Recessive growth phenotypes of the truncated Sen1 alleles in a haploid strain. Eightfold serial dilutions of cultures (starting from an OD of 1.0) were spotted onto YEPD plates and incubated at 16°, 23°, 30°, and 37°. (C) Anti-Sen1(HD) immunoblot of cell extracts from haploid strains containing the indicated SEN1 alleles. Bands with the expected apparent molecular mass are indicated with asterisks. (D) Copper resistance assay for function of different terminators. Eightfold serial dilutions of cultures (starting from an OD of 1.0) of strains containing the indicated SEN1 alleles and reporter genes were spotted on synthetic complete medium with copper sulfate at the indicated concentration and incubated at 30°.

Figure 3

N- and C-terminal flanking sequences localize the Sen1 helicase domain to the nucleus. (A–F) Diagrams of the Sen1 constructs are shown at left. The micrographs show fluorescence of Sen1-GFP constructs (green) or Nhp6A-RFP (nuclear marker; red); differential interference contrast (DIC) images of the cells are shown at right. Bar (A–F), 10 µm. (G) The images of representative cells show subnuclear localization of Sen1(1004–1907)-GFP (green) and Nhp6A-RFP (red). The incomplete colocalization of the two proteins is revealed in the merged images. Cells containing Sen1(1–2231)-GFP or Sen1(1089–1929)-GFP are shown on the left for comparison. Bar, 2 µm.

Figure 4

The sen1-E1597K mutation disrupts an intradomain salt bridge. (A) Predicted hydrogen bond between Upf1 residues E579 and R623 in the helicase domain crystal structure (PDB 2XZL, visualized by PyMOL, DeLano Scientific). (B) Growth phenotypes of strains containing the indicated SEN1 alleles at permissive (30°) and semipermissive (33.5°) temperatures after eightfold serial dilution and spotting on YEPD plates. (C) Copper resistance assay for terminator function. Strains containing the indicated SEN1 alleles and either the CYC1 or the SNR47 reporter construct were serially diluted eightfold, spotted onto synthetic complete medium with 0.1 mM copper sulfate, and incubated at 30°.

Figure 5

A subset of AOA2-associated substitutions in Sen1 are recessive lethal and cause dominant terminator readthrough. (A) Locations of the recessive-lethal mutations modeled on the structure of the Upf1 helicase domain. (B) Copper resistance assay for SNR47 terminator function. Cultures of the merodiploid strains containing the chromosomal wild-type SEN1 and the indicated plasmid-borne SEN1 alleles were serially diluted eightfold, spotted onto synthetic complete medium with 0.3 mM copper sulfate, and incubated at 30°.

Figure 6

Some viable AOA2-associated substitutions in Sen1 confer heat sensitivity. (A) Locations of viable AOA2-associated substitutions modeled on the Upf1 helicase domain. The position of W1166 is not shown since a homologous residue could not be identified in Upf1. (B) Growth phenotypes at 16°, 23°, 30°, and 37° of the haploid Sen1 mutants harboring AOA2 substitutions after eightfold serial dilution on YEPD medium.

Figure 7

Terminator readthrough of endogenous NRD1 and SNR47 transcripts in AOA2 mutant strains. (A) Total RNA from haploid strains harboring the indicated SEN1 alleles was resolved on denaturing agarose gels. The amplicons used as probes are indicated by the bars above the gene maps, and the arrows mark the transcription orientation. The numbers to the left of the blots represent the length in nucleotides of the RNA marker bands. The positions of the NRD1, SNR47, and SNR47-YDR042C readthrough transcript (SNR47-extend) bands are indicated. Ethidium bromide stain of the portion of the filter containing the large and small ribosomal RNAs is shown below the blots. (B) Quantification of the NRD1 and SNR47-extended transcripts. The RNA abundance in each sample relative to wild type (100%) was calculated and averaged. Each experiment was performed twice with independent RNA extracts. The error bars indicate the range.