Wide mutational analysis to ascertain the functional roles of eL33 in ribosome biogenesis and translation initiation

Abstract

An extensive mutational analysis of RPL33A, encoding the yeast ribosomal protein L33A (eL33) allowed us to identify several novel rpl33a mutants with different translational phenotypes. Most of the rpl33a mutants are defective in the processing of 35S and 27S pre-rRNA precursors and the production of mature rRNAs, exhibiting reductions in the amounts of ribosomal subunits and altered polysome profiles. Some of the rpl33a mutants exhibit a Gcd− phenotype of constitutive derepression of GCN4 translation and strong slow growth phenotypes at several temperatures. Interestingly, some of the later mutants also show a detectable increase in the UUG/AUG translation initiation ratio that can be suppressed by eIF1 overexpression, suggesting a requirement for eL33 and a correct 60S/40S subunit ratio for the proper recognition of the AUG start codon. In addition to producing differential reductions in the rates of pre-rRNA maturation and perhaps in r-protein assembly, most of the point rpl33a mutations alter specific molecular interactions of eL33 with the rRNAs and other r-proteins in the 60S structure. Thus, rpl33a mutations cause distinctive effects on the abundance and/or functionality of 60S subunits, leading to more or less pronounced defects in the rates and fidelity of mRNA translation.