RNA Polymerase I-Promoted HIS4 Expression Yields Uncapped, Polyadenylated mRNA That Is Unstable and Inefficiently Translated in Saccharomyces cerevisiae

Abstract

The HIS4 gene in Saccharomyces cerevisiae was put under the transcriptional control of RNA polymerase I to determine the in vivo consequences on mRNA processing and gene expression. This gene, referred to as rhis4, was substituted for the normal HIS4 gene on chromosome III. The rhis4 gene transcribes two mRNAs, of which each initiates at the polymerase (pol) I transcription initiation site. One transcript, rhis4s, is similar in size to the wild-type HIS4 mRNA. Its 3 end maps to the HIS4 3 noncoding region, and it is polyadenylated. The second transcript, rhis4l, is bicistronic. It encodes the HIS4 coding region and a second open reading frame, YCL184, that is located downstream of the HIS4 gene and is predicted to be transcribed in the same direction as HIS4 on chromosome III. The 3 end of rhis4l maps to the predicted 3 end of the YCL184 gene and is also polyadenylated. Based on in vivo labeling experiments, the rhis4 gene appears to be more actively transcribed than the wild-type HIS4 gene despite the near equivalence of the steady-state levels of mRNAs produced from each gene. This finding indicated that rhis4 mRNAs are rapidly degraded, presumably due to the lack of a cap structure at the 5 end of the mRNA. Consistent with this interpretation, a mutant form of XRN1, which encodes a 5-3 exonuclease, was identified as an extragenic suppressor that increases the half-life of rhis4 mRNA, leading to a 10-fold increase in steady-state mRNA levels compared to the wild-type HIS4 mRNA level. This increase is dependent on pol I transcription. Immunoprecipitation by anticap antiserum suggests that the majority of rhis4 mRNA produced is capless. In addition, we quantitated the level of His4 protein in a rhis4 xrn1⌬ genetic background. This analysis indicates that capless mRNA is translated at less than 10% of the level of translation of capped HIS4 mRNA. Our data indicate that polyadenylation of mRNA in yeast occurs despite HIS4 being transcribed by RNA polymerase I, and the 5 cap confers stability to mRNA and affords the ability of mRNA to be translated efficiently in vivo.RNA transcribed by RNA polymerase (pol) II undergoes a number of covalent modifications before being exported to the cytoplasm as mature mRNA and subsequently translated. Two such modifications, capping at the 5Ј end and polyadenylation at the 3Ј end of mRNA, are believed to be limited to the RNA pol II transcriptional machinery. The addition of the unique cap structure to the 5Ј end of all mature eukaryotic mRNAs is tightly coupled to RNA pol II transcription as the cap can be detected when the 5Ј end of mRNA emerges from the pol II transcriptional machinery (22,32,49). It has been shown that the cap structure is important for RNA transport, pre-RNA splicing, and mRNA stability (16,23,30,40).One of the best-understood functions of the cap structure is its role in translation initiation. According to the ribosomal scanning model (34), the eukaryotic initiation factor eIF-4F complex is required for the binding of the ribosomal preinitiation complex to ...

On the importance of being co-transcriptional

“…Thus, polyadenylation targeting by Pol II can be overridden by other processing signals. Indeed, polyadenylation signals in nascent yeast RNAs support partial polyadenylation of mRNAs transcribed by either Pol I, T7 RNA polymerase or Pol II lacking the CTD (Licatosi, 2002;Lo et al, 1998;McNeil et al, 1998;Dower and Rosbash, 2002), which confirms that a strict coupling between Pol II and polyadenylation is not required. Another case of modulation of polyadenylation function occurs in alternative terminal exon usage, in which polyadenylation sites in upstream exons are not used in favor of those sites found in downstream alternative exons.…”

Inhibition of 5′ to 3′ mRNA degradation under stress conditions in Saccharomyces cerevisiae: from GCN4 to MET16

“…It has been observed that decapped and non-poly(A) mRNAs accumulate under these conditions (Hsu and Stevens 1993). The expression of uncapped mRNA, which is albeit 10-fold lower than capped species, and the presence of non-poly(A) mRNAs in the polysome fraction has already been described (Proweller and Butler 1994;Lo et al 1998), but because the cap is required for efficient translation initiation, the uncapped mRNAs are not likely to be as competent for translation as other mRNA species. Therefore, stress-regulated 5Ј degradation may not lead to a major modification of the general protein pattern.…”

Hepatitis C Virus Internal Ribosome Entry Site-Dependent Translation in Saccharomyces cerevisiae Is Independent of Polypyrimidine Tract-Binding Protein, Poly(rC)-Binding Protein 2, and La Protein

“…5). If low levels of fragmented RNAs are present, these RNAs would lack a 5Ј-cap structure, and perhaps a poly(A) tail, and would be poorly translated in wildtype yeast (8,42,60). Uncapped cellular mRNAs are found only in mitochondria in yeast and are translated in a prokaryotic-like manner with formylmet tRNA i as the initiating codon (18).…”