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 cerevisiaewas 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. Therhis4 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-typeHIS4 mRNA. Its 3′ end maps to the HIS4 3′ noncoding region, and it is polyadenylated. The second transcript,rhis4l, is bicistronic. It encodes the HIS4coding 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 rhis4mRNAs 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-typeHIS4 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 xrn1Δ 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.

ACKNOWLEDGMENTS

The first two authors contributed equally to this work.

We thank T. Blumenthal, P. Cherbas, J. Jaehning, and N. Pace for helpful discussions during the course of this work. We thank J. Warner for the rDNA promoter/enhancer plasmid, M. Culbertson for the upf1::URA3 plasmid, C. Dykstra for the dst2 (xrn1)::URA3 plasmid, C. Thompson for the pCT3 yeast genomic library, M. Nomura for the rpa-135Δ strains, E. Lund and R. Parker for the anticap antiserum, and R. Parker and C. Decker for advice on immunoprecipitation of mRNA with the anticap antibody.

This work was supported by Public Health Service grant GM32263 from the National Institutes of Health awarded to T.F.D.