THE ROLE OF THE SCHIZOSACCHAROMYCES-POMBE GAR2 PROTEIN IN NUCLEOLAR STRUCTURE AND FUNCTION DEPENDS ON THE CONCERTED ACTION OF ITS HIGHLY-CHARGED N-TERMINUS AND ITS RNA-BINDING DOMAINS

Nonribosomal nucleolar protein gar2 is required for 18S rRNA and 40S r ibosomal subunit production in Schizosaccharomyces pombe. We have inve stigated the consequences of the absence of each structural domain of gar2 on cell growth, 18S rRNA production, and nucleolar structure. Del etion of gar2 RNA-binding domains (RBDs) causes stronger inhibition of growth and 18S rRNA accumulation than the absence of the whole protei n, suggesting that other factors may be titrated by its remaining N-te rminal basic/acidic serine-rich domain. These drastic functional defec ts correlate with striking nucleolar hypertrophy. Point mutations in t he conserved RNP1 motifs of gar2 RBDs supposed to inhibit RNA-protein interactions are sufficient to induce severe nucleolar modifications b ut only in the presence of the N-terminal domain of the protein. Gar2 and its mutants also distribute differently in glycerol gradients: gar 2 lacking its RBDs is found either free or assembled into significantl y larger complexes than the wild-type protein. We propose that gar2 he lps the assembly on rRNA of factors necessary for 40S subunit synthesi s by providing a physical link between them. These factors may be recr uited by the N-terminal domain of gar2 and may not be released if inte raction of gar2 with rRNA is impaired.