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

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 tRNA_i^Met suppresses the Gcd⁻ phenotype of rpl33a-G76R cells, dissociation of tRNA_i^Met 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.

SUPPLEMENTAL MATERIAL

We thank Dionisio Martín-Zanca and Rosa Esteban for helpful suggestions regarding this work and Thomas Dever for critical reading of the manuscript. We also thank John L. Woolford for the nop7 and drs1 mutants and for the hcNOP7 and hcDRS1 plasmids, Alan Sachs for the hcPAB1 plasmid, and Javier de las Rivas for help with the 3D model of rpL33.

P.M.-M. has been supported by fellowship AP2000-4183 granted by the Spanish Ministerio de Educación y Ciencia (MEC). M.T. was supported by MEC grants BMC 2001-1676 and PTR-1995-1010.