The essential Gcd10p-Gcd14p nuclear complex 15 required for 1- methyladenosine modification and maturation of initiator methionyl-tRNA

Abstract

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 gcd10Δ cells, and analysis of base modifications revealed that 1- methyladenosine (m1A) is undetectable in gcd10Δ tRNA. Interestingly, gcd10 and gcd14 mutations had no effect on processing or accumulation of elongator tRNA(Met), which also contains m1A at position 58, suggesting a unique requirement for this base modification in initiator maturation.