EVOLUTIONARILY CONSERVED STRUCTURAL ELEMENTS ARE CRITICAL FOR PROCESSING OF INTERNAL TRANSCRIBED SPACER-2 FROM SACCHAROMYCES-CEREVISIAE PRECURSOR RIBOSOMAL-RNA

Structural features of Internal Transcribed Spacer 2 (ITS2) important for the correct and efficient removal of this spacer from Saccharomyce s cerevisiae pre-rRNA were identified by in vivo mutational analysis b ased upon phylogenetic comparison with its counterparts from four diff erent yeast species. Compatibility between ITS2 structure and the S. c erevisiae processing machinery was found to have been maintained over only a short evolutionary distance, in contrast to the situation for I TS1. Nevertheless, cis-acting elements required for correct and effici ent processing are confined predominantly to those regions of the spac er that show the highest degree of evolutionary conservation. Mutation or deletion of each of these regions severely reduced production of m ature 26 S, but not 17 S rRNA, mainly by impeding processing of the 29 S-B precursor. In some cases, however, conversion of 29 S-A into 29 S -B pre-rRNA also appeared to be affected. Deletion of non-conserved se gments, on the other hand, caused little or no disturbance in processi ng. Surprisingly some combinations of such individually neutral deleti ons had a severe negative effect on the removal of ITS2, suggesting a requirement for a higher-order structure of ITS2. Finally, even struct ural alterations of ITS2 that did not noticeably affect processing, si gnificantly reduced the growth rate of cells that exclusively express the mutant rDNA units. We take this as further evidence for a direct r ole of ITS2 in the formation of fully functional 60 S ribosomal subuni ts.