Whole genome sequencing data and analyses of the underlying SUP35 transcriptional regulation for a Saccharomyces cerevisiae nonsense suppressor mutant

Termination of translation in eukaryotes is governed by two release factors encoded by the SUP45 and SUP35 genes in Saccharomyces cerevisiae. Previously, a set of mutations in these genes had been obtained. However, the exact sequence change associated with one mutation, sup35-222, was not identified by Sanger sequencing of the SUP35 region. Presented here are whole-genome sequencing data for the sup35-222 strain, data on copy number variation in its genome along with supporting pulse-field gel electrophoresis experiment data, and the list of single-nucleotide variations that differentiate this strain and its wild-type ancestor. One substitution upstream the SUP35 gene was located in a sequence corresponding to the Abf1-binding site. Data obtained from the introduction of this variation from sup35-222 strain into a different wild-type strain, specifically, detection of a nonsense-suppressor phenotype accompanied by a decrease in the Sup35 protein level, are also presented in this article.


a b s t r a c t
Termination of translation in eukaryotes is governed by two release factors encoded by the SUP45 and SUP35 genes in Saccharomyces cerevisiae. Previously, a set of mutations in these genes had been obtained. However, the exact sequence change associated with one mutation, sup35-222, was not identified by Sanger sequencing of the SUP35 region. Presented here are whole-genome sequencing data for the sup35-222 strain, data on copy number variation in its genome along with supporting pulse-field gel electrophoresis experiment data, and the list of single-nucleotide variations that differentiate this strain and its wild-type ancestor. One substitution upstream the SUP35 gene was located in a sequence corresponding to the Abf1-binding site. Data  Data on the whole genome sequencing of S. cerevisiae strain with nonsense-suppressor phenotype can be useful for the studies of translation, specifically, identification of mutations that accumulate in cells with high level of translational readthrough.
Genome coverage data, which include the duplication of the region containing SUP35, can be used in studies of chromosomal rearrangements and their adaptive role.
The mutation in the SUP35 promoter identified here is valuable for studies of transcription of the release factor genes as well as for the mechanisms of Abf1-derived regulation of transcription.
The data on the phenotypic analysis of strains with substitutions in the SUP35 promoter can be useful for studies of [PSI þ ] prion and its maintenance during a decrease in the Sup35 protein level. It also can be used for the studies of termination of translation.

Data
Raw whole-genome sequencing data for the sup35-222 Saccharomyces cerevisiae mutant strain was produced with Ion Torrent PGM. The genome was then assembled with the obtained reads (assembly statistics are present in Table 1 and Table S1). Genome coverage analysis performed by aligning short reads to the reference S288C genome showed a duplicated region of the chromosome IV that included the SUP35 ORF (Fig. 1). Pulse field gel electrophoresis (PFGE) analysis was then performed to compare chromosome lengths in the mutant strain and its ancestor and thus find if the duplication was located on the same chromosome (Fig. 2). As the ancestor strain, 1B-D1606, had been sequenced previously, single nucleotide variations (SNVs) between 1B-D1606, 222-1B-D1606 and reference strain were compared (Table S2). One single-nucleotide variation upstream the SUP35 coding sequence was identified. This substitution destroys potential Abf1-binding site in the SUP35 promoter (Fig. 3). Introducing the same variation, as well as deletion of the Abf1-binding site, in the SUP35 promoter into another strain led to detection of nonsense suppressor phenotype (Fig. 4A), accompanied by a decrease in the Sup35 protein level (Fig. 4B).

Sequencing and in silico analysis
The 1B-D1606 and 222-1B-D1606 strains were obtained and described previously [1,2]. Wholegenome sequencing was conducted with Ion Torrent PGM. Next generation sequencing and data analysis were performed as described earlier [3] with minor modifications. Genomic DNA was prepared with mechanical disruption of yeast cells [4]. SPAdes [5] and Quast [6] were used for the de This work [3] novo genome assembly and estimation of assembly quality, respectively. Bowtie [7], qualimap [8], and UGENE [9,10] were used to generate and analyse alignment of reads with the reference genome. The ggplot2 package [11] for R [12] was also used for coverage visualization. Samtools [13], vcftools v1.0 [14] and snpEff v4.1 [15] were used for SNV calling and annotation. Positions with low quality (q o 30) and low coverage (DP o 3), as well as heterozygous indels and variations in the repeat regions, were filtered out. The difference between the two strains by SNVs relative to the S288C genome was assessed with bedtools-intersect [16]. Complete genome sequence of 1B-D1606 and its assembly statistics have been published previously [3]. The mutation in the SUP35 promoter in 222-1B-D1606 was confirmed by Sanger sequencing of the PCR fragment amplified from genomic DNA of the strain with primers sup35_f (5 0 -CCAACCCTACGG-TAGAAAA-3 0 ) and SUP35_R (5 0 -GGATTGAATTGCTGCTGATAAC-3 0 ). Sanger sequencing was performed with ABI Prism 3500xl. All sequencing reactions were performed at the Research Resource Center "Molecular and Cell Technologies" of the Saint Petersburg State University.

Pulse field gel electrophoresis
Genomic DNA extraction for pulse field gel electrophoresis (PFGE) was performed in low melting point agarose according to the described method [18]. PFGE was run with the CHEF Mapper XA pulse field electrophoresis system (Bio-Rad) according to the manufacturer's recommendations.

Yeast plasmid and strain construction
Standard media and yeast cultivation techniques were used [19]. The pRSU1 and pRSU2 plasmids [20] were used for expression of the SUP35 gene under its wild-type promoter. For expression of SUP35 under control of the mutant promoter, the pRSU1-222 plasmid was constructed by cloning PstI-MluI-restricted PCR fragment, which was obtained by amplifying the SUP35 5 0 -UTR and a part of ORF from 222-1B-D1606 genomic DNA, into the same sites of pRSU1, thus substituting the wild-type promoter sequence with the mutant one. Correct fragment insertion was confirmed by Sanger sequencing of the plasmid. SUP35 regulated by its promoter with deletion of Abf1-binding site was introduced on the pNR-ΔABF1 plasmid [21]. The pRSU2 plasmid was substituted with pRSU1, pRSU1-222, or pNR-ΔABF1 via plasmid shuffling on 5-FOA medium [19] in the U-P S -A-GT671, or U-1-A-GT671 strains. These strains are derivatives of the GT671 strain (MATα ade1-14 his3 lys2 ura3-2 leu3,112 trp1 sup35::HIS3 [CEN LEU2 SUP35] [psi -][pin -]), which is a kind gift from Y.O. Chernoff. U-P S -A-GT671 strain was selected after substituting SUP35-bearing plasmid for pRSU2 in L-P-A-GT671, a   Fig. 4. The data on the effects of the P sup35-222 and P sup35ΔAbf1 promoter variants on nonsense suppression and eRF3 abundance. A. SUP35 regulated by the wild-type promoter , in the initial strain (WT*) was replaced with SUP35 under control of either wildtype (WT, as a control) or mutant (222 or ΔAbf1) promoter. Shown are tenfold serial dilutions of representative clones spotted onto full (YEPD, 1/4YEPD) or selective (SC-Ade) media after 4 or 10 days of incubation, respectively. [PSI þ ] and [psi À ] are U-P S -A-GT671 and U-1-A-GT671 strains, respectively. B. Sup35 (eRF3) protein level in wild-type and mutant strains. Proteins were extracted from clones with sup35-222 or wild-type promoter variants and processed as described in Protein analysis section.
MATa segregant which was obtained after mating GT671 with GT81-1C strain [22]. U-1-A-GT671 was selected in U-P-A-GT671 progeny as a clone that had spontaneously lost the [PSI þ ] prion.