Essential structural features in the Schizosaccharomyces pombe pre-rRNA 5′ external transcribed spacer

Abstract

The proximal region in the 5′ external transcribed spacer (5′ETS) of the genes encoding ribosomal RNAs in Schizosaccharomyces pombe was examined with respect to structural features which underlie rRNA maturation. Computer analyses and partial digestion with nuclease probes indicate a crucifix-like structure composed primarily of three extended hairpins which are more highly ordered than previously proposed in Saccharomyces cerevisiae. A re-evaluation of the same region in S. cerevisiae indicates a conserved core structure, including the U3 snoRNA binding site within this higher-order structure. The sequences encoding the individual hairpins were deleted by PCR-mediated mutagenesis and the mutant rDNAs were expressed in vivo to determine the effect of these features on rRNA maturation. Quantitative hybridization analyses indicate that the first hairpin only has modest effects on 18 S rRNA maturation, but the other two regions are critical and no mature 18 S rRNA was observed. When smaller changes were systematically introduced into the critical regions, strong correlations were observed with known or putative events in rRNA maturation. Changes associated with an intermediate cleavage site in helix II and with the putative U3 snoRNA binding site were again critical to 18 S rRNA production. In each case, the effects were sequence dependent and not simply the result of disrupted structure. Further analyses of the 5.8 S rRNA indicate that the large ribosomal subunit RNA can be properly processed in each case but the efficiency is reduced by as much as 60 %, an observation which provides new evidence of interdependency in the maturation process. The results illustrate that rRNA processing is more critically dependent on the 5′ETS than previously believed.