The Yeast RNAI Gene Product Necessary for RNA Processing Is Located in the Cytosol and Apparently Excluded From the Nucleus

The yeast RNA/gene is required for RNA processing and nuclear transport of RNA. The rnal-1 mutation of this locus causes defects in pre-tRNA splicing, processing of the primary pre-rRNA transcript, production of mRNA and export of RNA from the nucleus to the cytosol. To understand how this gene product can pleiotropically affect these processes, we sought to determine the intracellular location of the RNA1 protein. As determined by indirect immunofluorescence localization and organelle fractionation, the RNA1 antigen is found exclusively or primarily in the cytoplasm. Only a tiny fraction of the endogenous protein could be localized to and functional in the nucleus. Furthermore, the RNA1 antigen does not localize differently under stress conditions. These findings suggest that the RNA1 protein is not directly involved in RNA processing but may modify nuclear proteins or otherwise transmit a signal from the cytosol to the nucleus or play a role in maintaining the integrity of the nucleus. t:KAaVOTIC genes are generally transcribed as precursor RNAs (pre-RNAs) that are processed to mature RNA species. Processing consists of removal of extra 5', 3', or intervening sequences as well as addition of nucleotides and modification of nucleosides. With the exceptions of processing 20S pre-rRNA to mature 18S rRNA (for review see Warner, 1989), and addition of some modifications of the anticodon loop of tRNAs (for review see Bjork et al., 1987) all processing steps appear to occur before transport of RNAs from the nuclear to the cytosolic compartments. The gene products that participate in RNA processing are largely undescribed. However, the approaches of purification of enzyme activities (for example Phizicky et al., 1986), use of autoimmune sera (for review see Padgett et al., 1986), and identification of mutations of yeast that block particular processing steps (for review see Woolford, 1989; Culbertson and Winey, 1989) have all contributed to a rapidly growing identification and understanding of these gene products. Our work concerns the characterization of the wild-type counterpart of yeast genes identified by mutation. Many Saccharomyces cerevisiae genes encoding products necessary for the processing of mature RNA species have been identified among collections of conditionally lethal yeast mutants (HartweU et al., 1967; Vijayraghavan et al., 1989). The prp2-prp11 alleles (Rosbash et al., 1981; Fried et al., 1981; previously these alleles were designated rna2rna11), prp14 (Couto et al., 1987) and several of the prpl7prp271esions (Vijayraghavan et al., 1989) affect removal of intervening sequences from pre-mRNAs. Many of the PRP Dr. Dunst's current address is Fotodyne, 16700 West Victor Road, New Berlin, WI 53151-4131. genes encode proteins that either affect the assembly of premRNA onto spliceosomes (Lin et al., 1987) or are components of snRNPs and presumably spliceosomes (Lossky et al., 1987; Chang et al., 1988; Bjorn et al., 1989; Banroques and Abelson, 1989). The protein products of some of these genes have been localized to the nucleus (Chang et al., 1988; Last and Woolford, 1986). The RNA/gene resembles the PRP genes in that a mutation of this gene, rna/-1, affects RNA processing. There is also genetic evidence that relates the RNA/gene to the PRP genes because mutations of the SRN1 locus suppress phenotypes of some of the prp mutations as well as the rna/-1 mutation (Pearson et al., 1982; Nolan, S. L. and A. K. Hopper, unpublished observations). However, there are distinctions between the RNA/gene and the PRP genes. The rna/-1 mutation pleiotropically affects all classes of RNA, tRNA at the step of removal of intervening sequences (Knapp et al., 1978; Hopper et al., 1978), ribosomal RNA at the step of processing the primary transcript (Hopper et al., 1978), and all mRNAs at a step(s) that is not well described (Hutchison et al., 1969; Shiokawa and Pogo, 1974; St. John and Davis, 1981). A recent study shows that the poly(A) tails of mRNAs generated at the nonpermissive temperature are increased in length (Piper and Aamand, 1989). The rna/-1 mutation also affects transport of RNA from the nucleus to the cytosol (Hutchison et al., 1969; Shiokawa and Pogo, 1974), although this may be a secondary consequence of RNA processing defects. Conversely, mutations of PRP loci do not appear to affect pre-tRNA splicing (Hopper, A. K., unpublished resuits) and affect only mRNAs coded for by genes that contain an intron (Rosbash et al., 1981). Because the rna/-1 mutation causes pleiotropic defects in RNA production, it has been © The Rockefeller University Press, 0021-9525/90/08/309/13 $2.00 The Journal of Cell Biology, Volume 111, August 1990 309-321 309 on N ovem er 6, 2017 jcb.rress.org D ow nladed fom thought that RNA/might code for a component common to several RNA processing reactions, a regulator of genes coding for processing products, or might be involved in the general structure of the nucleus (Hopper et al., 1980; Atkinson et al., 1985). One approach to understanding the role of RNA/in RNA processing pathways is to localize the RNAI protein in yeast. Based upon the proposed models of the RNA1 protein function, we expected this gene product to be found exclusively within the yeast nucleus and perhaps near the inner nuclear membrane. In previous studies we cloned (Atkinson et al., 1985) and sequenced (Traglia et al., 1989) the RNA/gene. This work provided the tools and information to generate anti-RNA1 sera. Using such antisera, we show that the RNA1 protein is present in the cytoplasm and appears to be excluded from the nuclear compartment. These results are unexpected and have forced us to reevaluate the models of RNA1 function in RNA processing. Materials and Methods Strains, Media, and Genetic Methods The yeast strains used in this study are described in Table I. These strains were grown in either YEPD, complete minus uracil or complete minus uracil and leucine, formulated as described previously (Hurt et al., 1987). Bacterial strain RR1 (Fpro leu thi lacY Str r r-k m-k hsdR hsdM endol) was used for all manipulations except for construction of the OmpF-RNA/LacZ fusion. To generate this plasmid, Escherichia ~oli strains MH3000 and TK1046 were used as described by Weinstock et al. (1985). L-broth and YT-amp solid media were formulated as described previously (Maniatis et al., 1982). Yeast cells and bacteria were transformed according to the method of Ito et al. (1983) and Maniatis et al. (1982) respectively. DNA Manipulations and Plasmid DNAs Restriction endonucleases (Bethesda Research Laboratories, Inc., Gaithersburg, MD; New England Biolabs, Inc., Boston, MA; Boehringer Mannhelm Biochemicals, Indianapolis, IN) were used as prescribed by the manufacturers. Ligations using T4 DNA ligase (Bethesda Research Laboratories) were carried out in the buffer supplied by the manufacturer at 23"C for 4 h. A variety of yeast/E, coli shuttle vectors were employed. YEp24 is a UK43-contalning vector that achieves multiple copies in yeast (Botstein et al., 1979). YEpRNA1 and YEprnal-1 are derivatives of YEp24 that contain genomic fragments encoding the wild-type R/CA/and mutant rna/-1 alleles, respectively (Atkinson et al., 1985). YCpRNA1 is a CEN3-containing single or low copy plasmid that harbors genomic RNA/ sequences; YEpcRNA(I.4) was derived from pMac561, a TRPl-containing multicopy vector with a cDNA encoding RNA/regulated by the ADH1 promoter (Atkinson et al., 1985). YEpcRNA (1.4) leads to high expression of RNA/in yeast cens. pFB1-7a and pFB1-67a are multicopy plasmids that contain the yeast LEU2 gene and gene fusions of sequences encoding yeast historic H2B (H2B2) and E. ¢oli fl-galactosidase (Moreland et al., 1987). pFB1-7a contains 7 codons of H2B2 fused in-frame to LacZ and encodes a H2B-/~galactosidase chimeric protein that is located in yeast cytoplasm; pFB1-67a contains 67 codons of H2B2 fused in-frame to LacZ and encodes a chimeric protein that translocates to yeast nuclei (Moreland et al., 1987). YEpRNAI(1-187)/LacZ was derived from pFB1-7a as diagrammed in Fig. 1, A and B. The H2B2-containing fragment of pFB1-7a was removed by digestion with Sma I and Barn HI. A region extending from a Dra I site upstream of the RNA/ORF to a Barn HI site at codon 187 was ligated into the Sma I-Barn HI-digested pFB1-7a. This created an in-frame fusion of RNA/to LacZ. pORF1-RNA1, a derivative of pORF1 (Weinstock et al., 1983) contains in-frame fusion ofE. coli OmpF, an internal portion of the yeast RNA/gene, and LacZ. To generate pORF1-RNA1 a Dpn I-Hint II restriction fragment from an RNA/-containing plasmid was inserted into pORF1 at the Sma I site (Fig. 1, B and C). Protein Extraction and Blot Analysis To purify the OmpF-RNAl-fl-galactosidase trihybrid protein, pORF1-RNA1 was transferred to E. coil strain TK1046 that has the OmplL:s allele. Trihybrid protein production was induced by temperature shift from 25 to 42°C as described by Weinstock et al. (1983). The OmpF-RNA1-/~-galactosidase hybrid protein was extracted from 500 ml of culture as described by Last and Woolford (1986) except that the purification was terminated after resuspension and boiling of the 2 % Triton X-100 washed pellet. As determined by SDS-PAGE analysis the vast majority of protein at this step was the desired fusion protein (data not shown). Yeast cell protein extracts were prepared by modifications of the procedures described by Hopper et al. (1974) or Schultz et al. (1987) as indicated in the figure legends. Protein blot analysis followed the procedure of Towbin (1979). Table L Yeast Strains Strain Genotype Description/Source A364a MATa gall adel ade2 ural his7 tyrl lys2 L. Hartwell; parent ts136: the original strain containing rnal-1 (Hutchison et al., 1969)