The most abundant small cytoplasmic RNA of Saccharomyces cerevisiae has an important function required for normal cell growth.

Abstract

The most abundant RNA visible between 5.8S and 18S rRNA on an ethidium bromide-stained gel of total Saccharomyces cerevisiae RNA has an apparent size of about 600 nucleotides. By purifying the band and using it as a probe to screen a genomic library, we isolated and sequenced the unique gene for this RNA. The transcribed sequence, determined to be 519 nucleotides long, contains elements typical of RNA polymerase III transcription. The RNA is predominantly cytoplasmic, so we called it small cytoplasmic RNA 1 (scRl). ScRl is neither 3'-polyadenylated nor 5'-trimethylguanosine capped. We constructed a null mutation of the gene by deleting 252 base pairs from the transcribed region. Haploid strains carrying the scrl-A lesion grew very slowly, segregated cytoplasmic petites ([rho-]) at high frequency, and showed signs of aberrant cell division.A secondary structure model for scRl shows some of the conserved features of the signal recognition particle 7SL RNAs.The types of RNA in living organisms vary greatly from the most abundant classes, directly involved in translation, to a variety of less abundant classes of diverse or unknown function, loosely termed "small RNAs," of which an increasing number are being shown to be involved in aspects of DNA replication or precursor RNA (pre-RNA) processing (14).In eucaryotes, most of the better-characterized small RNAs are located in the nucleus, where they exist as ribonucleoprotein particles (RNPs). Of these, most of the abundant U small nuclear RNPs are involved in pre-mRNA splicing and 3'-end formation (5, 32, 49); nucleolar U3 small nuclear RNP is associated with pre-rRNA, but its function remains obscure (25,27,40); RNase P matures pre-tRNA (1); telomerase is involved in telomere replication (21); and the role of 7SK RNA is unknown (36, 37).In the cytoplasm, the Ro RNPs contain RNA polymerase 111-transcribed "Y" small RNAs; their functions are unknown, and they are defined as a class of autoimmune antigens (la, 24, 28, 42, 55). Prosomes or cylinder particles appear to be heterogeneous in composition, are distributed throughout both nuclei and cytoplasm, and are even less well understood (29). Nuclear-encoded RNAs are required for the functions of the mitochondrial RNPs (MRP), which processes RNA primers of mitochondrial DNA (mtDNA) synthesis (8), and mtDNA primase, which appears to contain cytoplasmic 5.8S rRNA (56).The best-characterized small cytoplasmic RNA is the 7SL RNA component of the signal recognition particle, whose activity is defined in vitro as mediating the translocation of membrane and secretory proteins into the endoplasmic reticulum (53).In this article we report the characterization of an abundant cytoplasmic RNA of Saccharomyces cerev'isiae and of * Corresponding author.

The Alu domain homolog of the yeast signal recognition particle consists of an Srp14p homodimer and a yeast-specific RNA structure

“…The SRP14p and scR1 RNA genes were amplified from genomic DNA of the yeast strain RMY326 (kindly provided by Dr+ Didier Picard) with the polymerase chain reaction (PCR) using Pfu DNA polymerase (Stratagene, La Jolla, California) and the appropriate primers+ The primer sequences were derived from the published sequences of SRP14 and SCR1 (Felici et al+, 1989;Brown et al+, 1994)+ SCR1 was amplified using the primers GGA ATT CTA ATA CGA CTC ACT ATA AGG CTG TAA TGG CTT T and GCT TAA AAA ATA TGG TTC AGG AC, which add the T7 RNA polymerase promoter at the 59 end of the gene and a DraI restriction site at the 39 end+ The fragment was cloned into the pUC plasmid linearized with HincII (pURy)+ SRP14 was amplified from genomic DNA with the primers GCT TTC TTA CCA CC TAA AAC and CAA ATT GAT CAC GCC GGT ACC+ The amplified fragment of the expected size was cloned into the plasmid SP64 linearized with HincII (pSY2)+ Several clones were isolated from independent amplification reactions and the inserts sequenced+ In all these clones, the genes contained the same few nucleotide changes when compared to the published sequences+ In SRP14, nucleotide changes in the coding region altered amino acid residues S69 into G69 and G137 into A137+ The scR1 gene we isolated contained an additional G at positions 50 and 99 as shown in Figure 3+ Truncations and mutations in both genes were introduced using PCR technology and the changes were verified by sequence analysis+ Rycom188 comprises nt 1-160 and 491-516 at the 59 and 39 ends, respectively, of scR1 RNA linked by two adenosine nucleotides (see Fig+ 3 for the exact sequence)+ Rycom71 RNA comprises the first 43 and the last 16 nucleotides of scR1 RNA linked by the sequence ACAUUUCUU+ Proteins were expressed using wheat-germ lysate prepared according to Erickson and Blobel (1983)+ Uncapped synthetic transcripts were produced from the linearized plasmids with SP6 RNA polymerase (Promega, Madison, Wisconsin)+ Proteins were labeled with [ 35 S]-methionine (1,500 Ci/mmol; Amersham Pharmacia Biotech, Zürich, Switzerland) as described in Strub and Walter (1990)+ For expression in E. coli, the f-epitope (12 amino acid residues recognized by T7 Tag antibodies; Novagen, Madison, Wisconsin) was added to the N-terminus of Srp14p using PCR, and the new gene was inserted into the pEt9a vector (Studier et al+, 1990; Novagen, Madison, Wisconsin)+ Lysis of the bacteria and purification of the recombinant protein on an 18-mL heparin column (BioRad Laboratories, Hercules, California) was done as described in Thomas et al+ (1997)+ For further purification of the protein, we used hydroxyapatite gel chromatography (Bio-Gel HTP, Bio-Rad Laboratories) and the buffers described in Siegel and Walter (1985)+ The protein eluted with a 400-mM sodium phosphate buffer and was at least 95% pure based on Coomassie staining+ It was quantified by comparing it to a Coomassie-stained lysozyme standard+…”

“…Although signal recognition and targeting functions of SRP have been studied in mammalian, yeast, and bacterial organisms, the elongation arrest domain in SRPs of distantly related organisms has not been characterized+ We therefore decided to define a putative elongation arrest domain of Saccharomyces cerevisiae SRP based on the assumption that its components are ancestrally related to the components of the mammalian Alu domain+ The S. cerevisiae SRP appeared to be most suitable for these studies for two reasons+ First, compared to the mammalian species it is the most distantly related organism for which structural components characteristic for the Alu domain have been identified (Strub et al+, 1991;Brown et al+, 1994) and second, the results of these studies should facilitate the genetic analysis of this function+ Saccharomyces cerevisiae SRP (scSRP) has been shown to play a role in the translocation of a number of proteins, but interacts preferentially with proteins bearing strongly hydrophobic signal sequences (Hansen & Walter, 1988;Hann & Walter, 1991;Stirling & Hewitt, 1992;Ng et al+, 1996)+ In contrast to bacteria and other yeast species, scSRP is not essential for the survival of this organism+ Presumably, it can adapt to the loss of SRP by using alternative targeting factors (Ogg et al+, 1992)+ The scSRP comprises at least six proteins and one RNA molecule (Hann & Walter, 1991;Stirling & Hewitt, 1992;Brown et al+, 1994)+ Five of the identified SRP proteins in S. cerevisiae, Srp72p,Srp68p,Srp54p,Srp19p,and Srp14p, are homologous to mammalian SRP proteins+ Another protein, Srp21p, has so far only been identified in yeast+ Yeast SRP RNA, called scR1 RNA, is almost twice as large as other known SRP RNAs+ Its secondary structure remains elusive because computer-generated structures failed to reveal a similarity to the canonical secondary structure of SRP RNAs and to identify the conserved stem VIII (Felici et al+, 1989;Hann & Walter, 1991)+ In contrast, SRP RNAs from Schizosaccharomyces pombe and Yarrowia lipolytica could be folded to resemble the canonical structure of SRP RNAs in the S domain, but lacked the typical two-hairpin structure at their 59 ends (Brennwald et al+, 1988;Poritz et al+, 1988;Ribes et al+, 1988;He et al+, 1989)+ The components of the mammalian Alu domain that are also present in yeast include Srp14p, which shares 30% sequence identity with mammalian SRP14 …”

SRH1 protein, the yeast homologue of the 54 kDa subunit of signal recognition particle, is involved in ER translocation of secretory proteins

“…Considering the good homology to mammalian SRP54, function of Srhlp may well be the recognition of signal sequence and the initiation of the following reactions such as targeting to the ER membrane and translocation promotion. Besides Srhlp, homologues, of 7SL RNA [30] and a-subunit of SRP receptor [31] are found in S. cereuisim, although genetic and biochemical interactions with SRI-II are not precisely revealed. S. pombe homologue of the SRP54 is shown to be associated with the homologue of 7SL RNA in viva [3%].…”

Genome-wide location and regulated recruitment of the RSC nucleosome-remodeling complex

“…3A). In addition, other Pol III-transcribed genes, such as RPR1 (Lee et al 1991) and SCR1 (Felici et al 1989), are clear RSC targets. Quantitative PCR analysis confirms that RSC occupancies at all six tRNA promoters tested and the U6 promoter are three-to sevenfold above the background level (Fig.…”