THE 18S RIBOSOMAL-RNA DIMETHYLASE DIM1P IS REQUIRED FOR PRE-RIBOSOMAL-RNA PROCESSING IN YEAST

The mg(2)(6)A(1779)m(2)(6)A(1780) dimethylation at the 3' end of the s mall subunit rRNA has been conserved in evolution from bacteria to euk aryotes. The yeast 188 rRNA dimethylase gene DIM1 was cloned previousl y by complementation in Escherichia coli and shown to be essential for viability in yeast. A conditional GAL10::dim1 strain was constructed to allow the depletion of Dim1p from the cell. During depletion, dimet hylation of the pre-rRNA is progressively inhibited and pre-rRNA proce ssing at cleavage sites A1 and A2 is concomitantly lost. In consequenc e, the mature 188 rRNA and its 20S precursor drastically underaccumula te. This has the effect of preventing the synthesis of nonmethylated r RNA. To test whether the processing defect is a consequence of the abs ence of the dimethylated nucleotides or of the Dim1p dimethylase itsel f, a cis-acting mutation was created in which both dimethylated adenos ines are replaced by guanosine residues. Methylation cannot occur on t his mutant pre-rRNA, but no clear pre-rRNA processing defect is seen. Moreover, methylation of the wild-type pre-rRNA predominantly occurs a fter cleavage at sites A1 and A2. This shows that formation of the m(2 )(6)A(1779)m(2)(6)A(1780) dimethylation is not required for pre-rRNA p rocessing. We propose that the binding of Dim1p to the pre-ribosomal p article is monitored to ensure that only dimethylated pre-rRNA molecul es are processed to 188 rRNA.