The essential Gcd10p–Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA
- James Anderson1,
- Lon Phan1,
- Rafael Cuesta3,
- Bradley A. Carlson2,
- Marie Pak1,
- Katsura Asano1,
- Glenn R. Björk4,
- Mercedes Tamame3, and
- Alan G. Hinnebusch1,5
- 1Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892 USA; 2Basic Research Laboratory, Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892 USA; 3Instituto de Microbiología Bioquímica, Consejo Superior de Investigaciones Científicas, Universidad de Salamanca, Salamanca, Spain; 4Department of Microbiology, Umeå University, Umeå, Sweden
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 (tRNAi 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 tRNAi Met, attributable to increased turnover rather than decreased synthesis of pre-tRNAi Met. Remarkably, the lethality of a GCD10 deletion was suppressed by high-copy-number IMT4, indicating that its role in expression of mature tRNAi Met is the essential function of Gcd10p. A gcd14-2 mutant also showed reduced amounts of mature tRNAi Met, but in addition, displayed a defect in pre-tRNAi Met processing. Gcd10p and Gcd14p were found to be subunits of a protein complex with prominent nuclear localization, suggesting a direct role in tRNAi Metmaturation. The chromatographic behavior of elongator and initiator tRNAMet 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 tRNAMet, which also contains m1A at position 58, suggesting a unique requirement for this base modification in initiator maturation.
Keywords
Footnotes
-
↵5 Corresponding author.
-
E-MAIL ahinnebusch{at}nih.gov; FAX (301) 496-6828.
-
- Received August 2, 1998.
- Accepted October 9, 1998.
- Cold Spring Harbor Laboratory Press
