Gcd10p andMet maturation. The chromatographic behavior of elongator and initiator tRNA Met on a RPC-5 column indicated that both species are altered structurally in gcd10⌬ cells, and analysis of base modifications revealed that 1-methyladenosine (m 1 A) is undetectable in gcd10⌬ 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.

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 (tRNAiMet) precursors containing 5' and 3' extensions, suggesting a defect in processing of the primary transcript. Dosage suppressors of gcd10 mutations, encoding tRNAiMet (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 tRNAiMet. A mutation in GCD10 or deletion of LHP1 exacerbated the defects in cell growth and expression of mature tRNAiMet 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.

We identified a mutation in the 60S ribosomal protein L33A (rpl33a-G76R) that elicits derepression of GCN4 translation (Gcd ؊ phenotype) by allowing scanning preinitiation complexes to bypass inhibitory upstream open reading frame 4 (uORF4) independently of prior uORF1 translation and reinitiation. At 37°C, rpl33a-G76R confers defects in 60S biogenesis comparable to those produced by the deletion of RPL33A (⌬A). At 28°C, however, the 60S biogenesis defect is less severe in rpl33a-G76R than in ⌬A cells, yet rpl33a-G76R confers greater derepression of GCN4 and a larger reduction in general translation. Hence, it appears that rpl33a-G76R has a stronger effect on ribosomal-subunit joining than does a comparable reduction of wild-type 60S levels conferred by ⌬A. We suggest that rpl33a-G76R alters the 60S subunit in a way that impedes ribosomalsubunit joining and thereby allows 48S rRNA complexes to abort initiation at uORF4, resume scanning, and initiate downstream at GCN4. Because overexpressing tRNA i Met suppresses the Gcd ؊ phenotype of rpl33a-G76R cells, dissociation of tRNA iMet from the 40S subunit may be responsible for abortive initiation at uORF4 in this mutant. We further demonstrate that rpl33a-G76R impairs the efficient processing of 35S and 27S pre-rRNAs and reduces the accumulation of all four mature rRNAs, indicating an important role for L33 in the biogenesis of both ribosomal subunits.Cell growth and division are highly interconnected processes that require protein synthesis, which in turns requires the biogenesis of ribosomes, soluble translation factors, and charged tRNA species. In Saccharomyces cerevisiae, ribosome biogenesis consumes a great amount of energy and is tightly regulated according to nutrient availability and to signals depending on other macromolecular pathways (77). The production of 60S and 40S ribosomal subunits is a highly dynamic process that begins with the transcription of rRNA by RNA polymerase I (35S rRNA precursor to 25S, 18S, and 5.8S rRNAs) and RNA polymerase III (5S rRNA) in the nucleolus and ends with export of preribosomal 60S and 40S subunits to the cytoplasm, where final steps of maturation occur. The maturation pathway of the 35S pre-rRNA to yield 25S, 18S, and 5.8S rRNAs (see Fig. 4A) is closely coordinated with the assembly of 79 ribosomal proteins (r-proteins) and more than 150 trans-acting factors involved in the synthesis, maturation, and transport of the ribosomal subunits (reviewed in references 11, 41, 53, and 73). The association of 35S pre-rRNA with the U3-snoRNP complex, transiently associated trans-acting factors, and several r-proteins, mostly belonging to the 40S subunit, leads to the formation of the 90S nucleolar complexes, where cleavage at the A0, A1, and A2 processing sites occurs (16, 28; reviewed in reference 29). The pre-40S particle, which contains some newly associated maturation factors and some already present in the 90S particle, is released after A0-A1-A2 cleavage and transported to the cytoplasm, where cleavage of 20S pre-rRNA to matur...

GCN4 mRNA is translated by a reinitiation mechanism involving four short upstream open reading frames (uORFs) in its leader sequence. Decreasing the activity of eukaryotic initiation factor-2 (eIF-2) by phosphorylation inhibits general translation in yeast but stimulates GCN4 expression by allowing ribosomes to scan past the uORFs and reinitiate at GCN4 instead. GCDIO was first identified genetically as a translational repressor of GCN4. We show here that GCD10 is an essential protein of 54.6 kD that is required in vivo for the initiation of total protein synthesis. GCD10 binds RNA in vitro and we present strong biochemical evidence that it is identical to the RNA-binding subunit of yeast initiation factor-3 (eIF-3). eIF-3 is a multisubunit complex that stimulates translation initiation in vitro at several different steps. We suggest that gcdlO mutations decrease the ability of eIF-3 to stimulate binding of eIF-2/GTP/Met-tRNAi ~et ternary comPlexes to small ribosomal subunits in vivo. This would explain why mutations in eiF-3 mimic eIF-2a phosphorylation in allowing ribosomes to bypass the uORFs and reinitiate at GCN4. Our results indicate that GCN4 expression provides a sensitive in vivo assay for the function of eIF-3 in initiation complex formation.

Transient exposure of mycelia from Aspergillus niger and Aspergillus nidulans to the cytidine analog 5-azacytidine, leading to no more than 0.3 to 0.5% substitution for cytosine by 5-azacytosine in A. nidulans DNA, resulted in the conversion of a high fraction of the cell population (more than 20%) to a mitotically and meiotically stable "fluffy" developmental phenotype. The phenotypic variants are characterized by the developmentally timed production of a profuse fluffy network of undifferentiated aerial hyphae that seem to escape signals governing vegetative growth. Genetic analysis with six different fluffy clones reveals that this trait is not cytoplasmically coded, is recessive in heterozygous diploids but codominant in heterokaryons, and exhibits a 1:1 Mendelian segregation pattern upon sexual sporulation of heterozygous diploids. Complementation and mitotic haploidization studies indicated that all variants are affected in the same gene, which can be tentatively located on chromosome VIII of A. nidulans. Molecular analysis to search for modified bases showed that DNA methylation is negligible in in both A. niger and A. nidulans and that no differences could be detected among DNAs from wild-type cells, fluffy clones, or mycelia exposed to 5-azacytidine. It thus appears that high-frequency conversion of fungal mycelia to a stable, variant developmental phenotype by 5-azacytidine is the result of some kind of target action on a single nuclear gene and that this conversion can occur in organisms virtually devoid of DNA methylation.