Effects of Mutations in the Saccharomyces cerevisiae RNA14, RNA15, and PAP1 Genes on Polyadenylation In Vivo

Abstract

The RNA14 and RNA15 gene products have been implicated in a variety of cellular processes. Mutations in these genes lead to faster decay of some mRNAs and yield extracts that are deficient in cleavage and polyadenylation in vitro. These results suggest that the RNA14 and RNA15 gene products may be involved in both adenylation and deadenylation in vivo. To explore the roles of these gene products in vivo, we examined the site of adenylation and the rate of deadenylation for individual mRNAs in rna14 and rna15 mutant strains. We observed that the rates of deadenylation are not affected by lesions in either the RNA14 or the RNA15 gene. This result suggests that the proteins encoded by these genes are not involved in regulation of the deadenylation rate. In contrast, we observed that the site of adenylation for the ACT1 transcript can be altered in these mutants. Interestingly, we also observed that mutation of the poly(A) polymerase gene altered the site of ACT1 polyadenylation. These observations suggest that the RNA14, RNA15, and PAP1 proteins are involved in poly(A) site choice. This alteration in poly(A) site choice in the rna14 mutant can be corrected by the ssm4 suppressor, indicating that this suppression acts at the level of polyadenylation and not by slowing mRNA degradation.Most eukaryotic mRNAs have a polyadenylate sequence at the 3Ј end which is added in a posttranscriptional process. This poly(A) tail is important for the proper stability of transcripts (for a review, see reference 9), for efficient initiation of translation (11,17,32), and for efficient nuclear-cytoplasmic transport (25, 34). In addition, the site at which the poly(A) tail is added can be regulated, thereby leading to the production of alternative gene products from a single transcriptional unit.Examination of the process of polyadenylation in mammalian cell extracts has led to substantial progress in understanding the mechanism by which the poly(A) tail is added (for reviews, see references 18, 38, and 40). Addition of a poly(A) tail is initiated by assembly of a complex of proteins on the sequences that specify a polyadenylation site. This complex cleaves the pre-mRNA and then adds a short poly(A) tail. The addition of a nuclear poly(A) binding protein, PABII, then alters the adenylation reaction to allow rapid elongation of the short poly(A) tail to its full length (3). In Saccharomyces cerevisiae, the use of an in vitro system has also shown that mRNA 3Ј end maturation is similar in that the precursor mRNA is also cleaved at a specific site to which the poly(A) tail is added (5). However, there are clearly differences in the sequences that specify a polyadenylation site in S. cerevisiae and mammals (for reviews, see references 18, 38, and 40).To understand the process of 3Ј end formation, the proteins involved in cleavage and polyadenylation need to be identified and their functions need to be determined. In mammalian cells, fractionation of extracts has identified several factors required for adenylation, in addition to the poly(...

3′-End processing of pre-mRNA in eukaryotes

“…The same is true for poly(A) polymerase: pap1 mutants have defects in polyadenylation in vivo and in vitro, but cleavage is normal [111,121,122]. Similar experiments con¢rm that two other subunits of PF I, Pta1p and Fip1p are speci¢cally involved in polyadenylation [117,120].…”

The RNA Polymerase II C-terminal Domain-interacting Domain of Yeast Nrd1 Contributes to the Choice of Termination Pathway and Couples to RNA Processing by the Nuclear Exosome

“…3A). T ACT1 has multiple pA sites, whose usage is greatly affected by mutations in cleavage/pA factors (48,49). In WT cells, RNApII occupancy at ADH1-T ACT1 decreased after T ACT1 (77 and 26% of the promoter RNApII signal at positions 4 and 5, respectively), indicating that T ACT1 successfully terminates ADH1 (Fig.…”

The role of the putative 3′ end processing endonuclease Ysh1p in mRNA and snoRNA synthesis

“…The well-characterized 39-UTR of the ACT1 gene includes five alternative polyadenylation sites and is therefore useful to examine poly(A) site selection in 39 end processing mutants (see scheme in Fig. 3D; Mandart and Parker 1995). Low-resolution Northern blot analysis revealed a change in the ACT1 hybridization pattern in ysh1 mutants compared to wild-type (Fig.…”

“…3D). To corroborate this phenotype, we further analyzed RNA obtained from two mutant strains (ysh1-13 and ysh1-15) on high-resolution Northern blots following RNase H treatment (Mandart and Parker 1995). In these experiments, total RNA was targeted by RNase H and oligonucleotides complementary to the 39 end of ACT1 mRNA and to the poly(A) tail allowing the resolution of the five poly(A) sites (Fig.…”