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Molecular and Cellular Biology, April 2000, p. 2505-2516, Vol. 20, No. 7
Laboratory of Eukaryotic Gene Regulation,
National Institute of Child Health and Human Development, Bethesda,
Maryland 208921; Department of
Microbiology, Umeå University, Umeå, Sweden2;
and Department of Biochemistry and Molecular Biology,
Pennsylvania State University College of Medicine, Hershey,
Pennsylvania 170333
Received 13 October 1999/Returned for modification 24 November
1999/Accepted 30 December 1999
Induction of GCN4 translation in amino acid-starved
cells involves the inhibition of initiator tRNAMet
binding to eukaryotic translation initiation factor 2 (eIF2) in
response to eIF2 phosphorylation by protein kinase GCN2. It was shown
previously that GCN4 translation could be induced
independently of GCN2 by overexpressing a mutant
tRNAAACVal (tRNAVal*) or the RNA
component of RNase MRP encoded by NME1. Here we show that
overexpression of the tRNA pseudouridine 55 synthase encoded by
PUS4 also leads to translational derepression of
GCN4 (Gcd
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Defects in tRNA Processing and Nuclear Export Induce
GCN4 Translation Independently of Phosphorylation of the
Subunit of Eukaryotic Translation Initiation Factor 2
phenotype) independently of eIF2
phosphorylation. Surprisingly, the Gcd
phenotype of
high-copy-number PUS4 (hcPUS4) did not require
PUS4 enzymatic activity, and several lines of evidence indicate that PUS4 overexpression did not diminish functional initiator
tRNAMet levels. The presence of hcPUS4 or
hcNME1 led to the accumulation of certain tRNA
precursors, and their Gcd
phenotypes were reversed by
overexpressing the RNA component of RNase P (RPR1),
responsible for 5'-end processing of all tRNAs. Consistently,
overexpression of a mutant pre-tRNATyr that cannot
be processed by RNase P had a Gcd
phenotype.
Interestingly, the Gcd
phenotype of hcPUS4
also was reversed by overexpressing LOS1, required for
efficient nuclear export of tRNA, and los1
cells have a Gcd
phenotype. Overproduced PUS4 appears to impede
5'-end processing or export of certain tRNAs in the nucleus in a
manner remedied by increased expression of RNase P or LOS1,
respectively. The mutant tRNAVal* showed nuclear
accumulation in otherwise wild-type cells, suggesting a defect in
export to the cytoplasm. We propose that yeast contains a nuclear
surveillance system that perceives defects in processing or export of
tRNA and evokes a reduction in translation initiation at the step
of initiator tRNAMet binding to the ribosome.
*
Corresponding author. Mailing address: Laboratory of
Eukaryotic Gene Regulation, National Institute of Child Health and
Human Development, Bldg. 6A, Rm. B1A-13A, Bethesda, MD 20892. Phone: (301) 496-4480. Fax: (301) 496-6828. E-mail:
ahinnebusch{at}nih.gov.
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