Ribosomal Protein L33 Is Required for Ribosome Biogenesis, Subunit Joining, and Repression of GCN4 Translation

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Abstract
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 (ΔA). At 28°C, however, the 60S biogenesis defect is less severe in rpl33a-G76R than in ΔA 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 ΔA. We suggest that rpl33a-G76R alters the 60S subunit in a way that impedes ribosomal-subunit joining and thereby allows 48S rRNA complexes to abort initiation at uORF4, resume scanning, and initiate downstream at GCN4. Because overexpressing tRNAiMet suppresses the Gcd− phenotype of rpl33a-G76R cells, dissociation of tRNAiMet 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.