Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6587-6591, July 1995 Biochemistry A genetic screen identifies cellular factors involved in retroviral -1 frameshifting (translation/CUP1/IFSI/paromomycin) SUSANNA I. LEE*, JAMES G. UMENt, AND HAROLD E. VARMUS*t# Departments of *Microbiology and Immunology and tBiochemistry and Biophysics, University of California, San Francisco, CA 94143 Contributed by Harold E. Varmus, February 17, 1995 ABSTRACT To identify cellular factors that function in gene causes increased frameshifting and a decrease in trans- -1 ribosomal frameshifting, we have developed assays in the lational fidelity in response to antibiotics that target the 40S yeast Saccharomyces cerevisiae to screen for host mutants in ribosomal subunit. which frameshifting is specifically affected. Expression vec- tors have been constructed in which the mouse mammary tumor virus gag-pro frameshift region is placed upstream of MATERIALS AND METHODS the lacZ gene or the CUPI gene so that the reporters are in the Yeast Strains and Genetic Methods. Saccharomyces cerevi- -1 frame relative to the initiation codon. These vectors have siae strains used in this study are listed in Table 1. been used to demonstrate that -1 frameshifting is recapitu- The numbering of the MMTV sequence is reported here lated in yeast in response to retroviral mRNA signals. Using with the first A residue of the heptanucleotide shifty site these reporters, we have isolated spontaneous host mutants in designated + 1. The CUP1 reporter plasmid contained nt -17 two complementation groups, ifsl and ifs2, in which frame- to +68 of MMTV inserted into the Kpn I site of pGM14 shifting is increased 2-fold. These mutants are also hypersen- (J.G.U., unpublished data). l3-Galactosidase was expressed sitive to antibiotics that target the 40S ribosomal subunit. from a CYCI-lacZ fusion gene cloned from pLGHY2 (12). The We have cloned the IFS1 gene and shown that it encodes a hemagglutinin (HA) epitope tag sequence (GATTACAAG- previously undescribed protein of 1091 aa with clusters of GACGATGACGATAAA) fused to nt -968 to +110 of the acidic residues in the carboxyl-terminal region. Haploid MMTV sequence was inserted into the BamHI site of the cells lacking 82% of the IFS) open reading frame are viable CYCl-lacZ fusion gene. and phenotypically identical to ifsl-l mutants. This ap- Frameshift Assays. The copper-containing media and cop- proach could help identify potential targets for antiretro- per-resistance assay have been described (13). After replica viral agents. plating, growth on copper was scored after 4 days at 30°C. /3-Galactosidase assays of colonies in situ were performed as Ribosomal frameshifting, a mode of translational regulation, described (10). involves a directed change in reading frame at a specific site on Western blotting was carried out on whole cell extracts. the mRNA at a defined frequency. This allows the production Equal amounts of total protein from each lysate were frac- of two or more proteins at fixed ratios from overlapping coding tionated by SDS/PAGE. Monoclonal anti-HA antibodies regions with a single translation initiation site (1, 2). The (12CA5) were obtained from Babco (Richmond, CA). For frequency of -1 frameshifting on retroviral RNA determines quantitation, secondary antibodies conjugated to 1251 were the ratio of gag to pol gene products, a ratio that dramatically used; the amount of gag-pro-1B-galactosidase in each lysate was affects viral assembly (3). Hence, the frameshifting is a step in measured with a Phosphorlmager (Molecular Dynamics) and the retroviral life cycle whose efficiency is thought to be tightly normalized to a scale where the amount of the fusion protein controlled. Experiments using in vitro translation in rabbit expressed from the in-frame construct equaled 100% frame- reticulocyte lysates have defined the cis-acting signals in shifting. retroviral mRNA directing -1 frameshifting. In all cases, the The Screen for Increased Frameshifting Mutants. L5 cells, frameshifting signal is bipartite. Shifting occurs at a hep- a with the UUUUUUA tanucleotide sequence that conforms to the general motif X carrying CUP1 reporter shifty site, XXY YYZ (triplets represent the 0 frame), which allows the which confers resistance to 0.2 mM copper, served as the anticodons of tRNAs at both the P and A ribosomal sites to parental strain. Four pools of 2 x 106 cells each were plated maintain two of three base pairs after shifting into the -1 at a density of 106 cells per plate onto complete synthetic frame (4). Frameshift sites also contain a higher-order RNA medium containing 0.4 mM copper sulfate. Each pool origi- structure, either a stem-loop (5, 6) or a pseudoknot (7, 8), nated from a single colony grown to mid-logarithmic phase in downstream of the shift sequence. complete synthetic minimal medium under appropriate selec- Here we report the development and use of a genetic tion. Twenty to 30 copper-resistant colonies from each pool approach to identify host factors involved in retroviral frame- were isolated and mated to L5 cells to determine whether the shifting. Using both a selectable (CUP1) and an enzymatic copper-resistance phenotype was dominant or recessive. (lacZ) reporter, we demonstrate that yeast recapitulates ret- Cloning and Sequencing ofIFS). A centromere-based yeast roviral -1 frameshifting. Using the mouse mammary tumor genomic library (14) was used to transform Hi cells, and virus (MMTV) gag-pro frameshift site as a model substrate, we individual transformants were assayed for loss of nonsense identified yeast mutants in which -1 frameshifting levels were codon suppression in paromomycin (Sigma) at 1 mg/ml. Two specifically elevated. The gene defective in one of these mutants encodes a previously undescribed protein, Ifslp.§ Abbreviations: MMTV, mouse mammary tumor virus; HA, hem- Though the IFSI gene product is nonessential, deletion of the magglutinin. *To whom reprint requests should be addressed at present address: National Institutes of Health, Building 1, Room 126, 9000 Rockville The publication costs of this article were defrayed in part by page charge Pike, Bethesda, MD 20892. payment. This article must therefore be hereby marked "advertisement" in §The sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U28158). 6587 Downloaded by guest on October 2, 2021 6588 Biochemistry: Lee et al. Proc. Natl. Acad. Sci. USA 92 (1995) Table 1. Yeast strains frameshift site Strain Genotype Source GPD actinintron L5* MATa cup]A::ura3At ura3-52 his3-A200 K. Yamamoto trp] leu2 lys2 ade2 rAUG; ~~~CUPl 2g BJ2168 MATa leu2 trpl ura3-52 prbl-1122 Ref. 9 pep4-3 prcl-407 gal2 Hi Same as L5 except ifsl-l This study OFRAME H2 Same as L5 except ifs2-1 This study -1 FRAME H3 Same as L5 except MATa ifs]-] ifs2-1 This study H4 Same as L5 except MA Ta This study HA frameshift as This tag site P9 Same L4 except pep4A::LEU2 study CEN/ARS PlO Same as HI except pep4A::LEU2 This study CYC 1 for colony D6 Diploid resulting from Hi x H4 This study |AU_ 4, lacZ screens D7, D8 Same as D6 except ifs]A::URA3/ifs]-1 This study 2,u-for *Derived from two backcrosses of K3aAcupl (10) to YPH252a (11). enzymatic tThe ura3 gene in the cupl locus has a 17-bp deletion. Hence this strain assays requires uracil for growth. O FRAME -1 FRAME independent clones which complemented both the paromo- mycin hypersensitivity and the increased frameshifting phe- FIG. 1. Yeast frameshifting reporters. The copper-resistance re- I DNA porter contains the S. cerevisiae CUP] sequence on a high-copy 2,u notypes were isolated. A common 5.5-kb Xba I-Xho vector. The mRNA is transcribed from the glyceraldehyde-3- fragment, found sufficient for full complementing activity, was phosphate dehydrogenase (GPD) promoter and spliced (ss = splice sequenced entirely on both DNA strands from a double- site) at the actin intron (13). The ,B-galactosidase reporter contains the stranded plasmid template. The IFS1 open reading frame was E. coli lacZ sequence transcribed from the iso-1-cytochrome c (CYC]) identified by testing nested 5' and 3' deletion mutants of the promoter (12) on a CEN/ARS plasmid (for colony screens on filters) 5.5-kb fragment for complementation. The 5' end of the or on a 2,u vector (for quantitation of frameshift levels in whole cell mRNA was mapped by RNase protection analysis to within a extracts). The HA epitope tag was introduced adjacent to the start 100-nt region containing a single in-frame AUG. codon. Disruption of IFSJ. A fragment of the IFS1 open reading frame corresponding to nt 103-542 (with respect to the ATG tion extracts (16). We first introduced mutations into the start codon) was amplified by PCR and inserted into the Not AAAAAAC shifty site of the MMTV gag-pro overlap (Fig. I and BamHI sites of the URA3-marked integrating vector 2A). Substitution with the UUUUUUA shifty sequence, which pRS306 (15) to generate pIFSA. This construct was linearized had previously been reported to serve as an efficient shifty site with Spe I and used to transform diploid strain D6 (IFSI! in yeast (13), resulted in even higher frameshifting frequencies. ifs]-]) to generate diploids D7 and D8. In contrast, introduction of a scrambled heptanucleotide se- quence (AAAUCGA) that does not fit the XXXYYYZ con- figuration (Fig. 2A; scrambled) abrogated /-galactosidase RESULTS activity or copper resistance. Yeast Frameshifting Reporters. To assay -1 frameshifting Mutational studies were also used to demonstrate the re- in yeast, the MMTV gag-pro frameshift region was placed quirement for the downstream pseudoknot structure in yeast upstream of the S. cerevisiae CUP] or the Escherichia coli lacZ frameshifting (Fig.