Open AccessRNA Polymerase I-Promoted HIS4 Expression Yields Uncapped, Polyadenylated mRNA That Is Unstable and Inefficiently Translated in Saccharomyces cerevisiae
Author(s) -
H. Shuen Lo,
Han Huang,
Thomas F. Donahue
Publication year - 1998
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.18.2.665
Subject(s) - biology , polyadenylation , microbiology and biotechnology , rna polymerase ii , transcription (linguistics) , exonuclease , gene , primary transcript , gene expression , messenger rna , genetics , rna polymerase iii , polymerase , rna polymerase , rna , alternative splicing , promoter , linguistics , philosophy
TheHIS4 gene inSaccharomyces 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 asrhis4 , was substituted for the normalHIS4 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 theHIS4 3′ noncoding region, and it is polyadenylated. The second transcript,rhis4l , is bicistronic. It encodes theHIS4 coding region and a second open reading frame,YCL184 , that is located downstream of theHIS4 gene and is predicted to be transcribed in the same direction asHIS4 on chromosome III. The 3′ end ofrhis4l maps to the predicted 3′ end of theYCL184 gene and is also polyadenylated. Based on in vivo labeling experiments, therhis4 gene appears to be more actively transcribed than the wild-typeHIS4 gene despite the near equivalence of the steady-state levels of mRNAs produced from each gene. This finding indicated thatrhis4 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 ofXRN1 , which encodes a 5′-3′ exonuclease, was identified as an extragenic suppressor that increases the half-life ofrhis4 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 ofrhis4 mRNA produced is capless. In addition, we quantitated the level of His4 protein in arhis4 xrn1Δ genetic background. This analysis indicates that capless mRNA is translated at less than 10% of the level of translation of cappedHIS4 mRNA. Our data indicate that polyadenylation of mRNA in yeast occurs despiteHIS4 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.