Mutations in the yeast RNA14 and RNA15 genes result in an abnormal mRNA decay rate; sequence analysis reveals an RNA-binding domain in the RNA15 protein.

Abstract

In Saccharomyces cerevisiae, temperature-sensitive mutations in the genes RNA14 and RNA15 correlate with a reduction of mRNA stability and poly(A) tail length. Although mRNA transcription is not abolished in these mutants, the transcripts are rapidly deadenylated as in a strain carrying an RNA polymerase B(II) temperature-sensitive mutation. This suggests that the primary defect could be in the control of the poly(A) status of the mRNAs and that the fast decay rate may be due to the loss of this control. By complementation of their temperature-sensitive phenotype, we have cloned the wild-type genes. They are essential for cell viability and are unique in the haploid genome. The RNA14 gene, located on chromosome II, is transcribed as three mRNAs, one major and two minor, which are 2.2, 1.5, and 1.1 kb in length. The RNA15 gene gives rise to a single 1.2-kb transcript and maps to chromosome XVI. Sequence analysis indicates that RNA14 encodes a 636-amino-acid protein with a calculated molecular weight of 75,295. No homology was found between RNA14 and RNA15 or between RNA14 and other proteins contained in data banks. The RNA15 DNA sequence predicts a protein of 296 amino acids with a molecular weight of 32,770. Sequence comparison reveals an N-terminal putative RNA-binding domain in the RNA15-encoded protein, followed by a glutamine and asparagine stretch similar to the opa sequences. Both RNA14 and RNA15 wild-type genes, when cloned on a multicopy plasmid, are able to suppress the temperature-sensitive phenotype of strains bearing either the rnal4 or the rnal5 mutation, suggesting that the encoded proteins could interact with each other.In yeast cells, all known mRNAs terminate in a poly(A) tail that is probably added to the 3' end of the pre-mature RNA in the nucleus in a posttranscriptional manner (18), as was previously shown to be the case in higher eucaryotes (57). One of the major roles for the poly(A) tail was suggested by its involvement in protein biosynthesis (47,63), and in yeast cells the poly(A)-binding protein (PABP [1,68]) was shown to be required for translation initiation (69). More recently, in vitro data have supported the idea that the poly(A) tail enhances translation through the interaction of an initiation factor or the ribosomal subunit with the PABP (59).Besides the implication of the poly(A) tail in translation (reviewed in reference 46), its role in mRNA half-life has been evoked as many times (30,44,82,83,86), sometimes in association with the PABP (11, 12, 17), as refuted (41,49,69,72). Nevertheless, it appears that the poly(A) tail is not solely responsible for determination of the stability of mRNA in vivo, but that it acts in association with other factors.To obtain mutants of Saccharomyces cerevisiae impaired in poly(A) mRNA metabolism, two mutants simultaneously cordycepin (3'-deoxyadenosine) sensitive and temperature sensitive were isolated by using a selective approach (13). The mutations, located at two independent loci, were renamed rnal4 and rnal5 for reasons of homonymy (see...

Inactivation of Cleavage Factor I Components Rna14p and Rna15p Induces Sequestration of Small Nucleolar Ribonucleoproteins at Discrete Sites in the Nucleus

“…Because Pap1p is the major poly(A) polymerase responsible for the synthesis of poly(A) tails of mRNAs in yeast (Patel and Butler, 1992), it is most likely that the poly(A) ϩ RNA detected in wild-type cells predominantly corresponds to mRNA. Total poly(A) ϩ RNA staining was also greatly reduced in the rna14-1 and rna15-2 strains incubated at 37°C for 1 h (Figure 4, h-j), consistent with previous reports (Minvielle-Sebastia et al, 1991;Torchet et al, 2002). By contrast, in strains lacking only Rrp6p (Figure 4, c-e), a strong accumulation of polyadenylated RNA was observed in the nucleus, as reported previously (Allmang et al, 1999a;van Hoof et al, 2000;Mitchell et al, 2003;Fang et al, 2004;Kuai et al, 2004;Carneiro et al, 2007).…”

Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I

“…1C). Yeast whole-cell extracts made from the temperature-sensitive strains rna15-2, rna14-1 (20), or pcf11-2 (21) do not polyadenylate mRNA precursor in vitro when the assay is performed under nonpermissive conditions. However, they are capable of complementing each other when two different mutant extracts are mixed together, presumably by supplying a functional protein in trans.…”

Regulation of the Nuclear Poly(A)-binding Protein by Arginine Methylation in Fission Yeast

“…Poly(A) tail length analyses were based on a previously described method (47). Briefly, 1 g of total fission yeast RNA was 3Ј-end-labeled at 4°C for 18 -20 h with 25 Ci of […”