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

Abstract

ABSTRACT 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 (hc IMT1 to hc IMT4 ; 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 (hc LHP1 ), also suppressed gcd14 mutations. In fact, the lethality of a GCD14 deletion was suppressed by hc IMT4 , 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.