Affiliation:
1. Department of Biology, Indiana University, Bloomington, Indiana 47405
Abstract
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 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.
Publisher
American Society for Microbiology
Subject
Cell Biology,Molecular Biology
Cited by
42 articles.
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