The essential Gcd10p-Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA

Gcd10p and Gcd14p are essential proteins required for the initiation of protein synthesis and translational repression of GCN4 mRNA. The phenotypes of gcd10 mutants were suppressed by high-copy-number IMT genes, encoding initiator methionyl tRNA (tRNA(i)(Met)), or LHP1, encoding the yeast homolog of the human La autoantigen. The gcd10-504 mutation led to a reduction in steady-state levels of mature tRNA(i)(Met), attributable to increased turnover rather than decreased synthesis of pre-tRNA(i)(Met). Remarkably, the lethality of a GCD10 deletion was suppressed by high-copy-number IMT4, indicating that its role in expression of mature tRNA(i)(Met) is the essential function of Gcd10p. A gcd14-2 mutant also showed reduced amounts of mature tRNA(i)(Met), but in addition, displayed a defect in pre-tRNA(i)(Met) processing. Gcd10p and Gcd14p were found to be subunits of a protein complex with prominent nuclear localization, suggesting a direct role in tRNA(i)(Met) maturation. The chromatographic behavior of elongator and initiator tRNA(Met) on a RPC-5 column indicated that both species are altered structurally in gcd10 Delta cells, and analysis of base modifications revealed that 1-methyladenosine (m(1)A) is undetectable in gcd10 Delta tRNA. Interestingly, gcd10 and gcd14 mutations had no effect on processing or accumulation of elongator tRNA(Met), which also contains m(1)A at position 58, suggesting a unique requirement for this base modification in initiator maturation.