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Molecular and Cellular Biology, June 1999, p. 4167-4181, Vol. 19, No. 6
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

GCD14p, a Repressor of GCN4 Translation, Cooperates with Gcd10p and Lhp1p in the Maturation of Initiator Methionyl-tRNA in Saccharomyces cerevisiae

Olga Calvo,¹ Rafael Cuesta,¹ James Anderson,² Noelia Gutiérrez,¹ Minerva Teresa García-Barrio,^1,2 Alan G. Hinnebusch,² and Mercedes Tamame^1,*

Instituto de Microbiología Bioquímica del CSIC/Universidad de Salamanca, 37007 Salamanca, Spain,¹ and Section on Molecular Genetics of Lower Eukaryotes, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, Maryland 20892²

Received 30 October 1998/Returned for modification 23 December 1998/Accepted 3 March 1999

Gcd10p and Gcd14p were first identified genetically as repressors of GCN4 mRNA translation in Saccharomyces cerevisiae. Recent findings indicate that Gcd10p and Gcd14p reside in a nuclear complex required for the presence of 1-methyladenosine in tRNAs. Here we show that Gcd14p is an essential protein with predicted binding motifs for S-adenosylmethionine, consistent with a direct function in tRNA methylation. Two different gcd14 mutants exhibit defects in cell growth and accumulate high levels of initiator methionyl-tRNA (tRNA_i^Met) precursors containing 5' and 3' extensions, suggesting a defect in processing of the primary transcript. Dosage suppressors of gcd10 mutations, encoding tRNA_i^Met (hcIMT1 to hcIMT4; hc indicates that the gene is carried on a high-copy-number plasmid) or a homologue of human La protein implicated in tRNA 3'-end formation (hcLHP1), also suppressed gcd14 mutations. In fact, the lethality of a GCD14 deletion was suppressed by hcIMT4, indicating that the essential function of Gcd14p is required for biogenesis of tRNA_i^Met. A mutation in GCD10 or deletion of LHP1 exacerbated the defects in cell growth and expression of mature tRNA_i^Met in gcd14 mutants, consistent with functional interactions between Gcd14p, Gcd10p, and Lhp1p in vivo. Surprisingly, the amounts of NME1 and RPR1, the RNA components of RNases P and MRP, were substantially lower in gcd14 lhp1::LEU2 double mutants than in the corresponding single mutants, whereas 5S rRNA was present at wild-type levels. Our findings suggest that Gcd14p and Lhp1p cooperate in the maturation of a subset of RNA polymerase III transcripts.

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^* Corresponding author. Mailing address: Instituto de Microbiología Bioquímica del CSIC/Universidad de Salamanca, Edificio Departamental de Biología, Campus Miguel de Unamuno, 37007 Salamanca, Spain. Phone: 34-923-121673. Fax: 34-923-224876. E-mail: tamame{at}gugu.usal.es.

M.T. dedicates this paper to the memory of Tomás Santos.

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Molecular and Cellular Biology, June 1999, p. 4167-4181, Vol. 19, No. 6
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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