Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 1

The interdependency of steps in the processing of pre-rRNA in Schizosacchar omyces pombe suggests that RNA processing, at least in part, acts as a qual ity control mechanism which helps assure that only functional RNA is incorp orated into mature ribosomes. To determine further the role of the transcri bed spacer regions in rRNA processing and to detect interactions which unde rlie the interdependencies, the ITS1 sequence was examined for its ability to form ribonucleoprotein complexes with cellular proteins. When incubated with protein extract, the spacer formed a specific large RNP. This complex was stable to fractionation by agarose or polyacrylamide gel electrophoresi s. Modification exclusion analyses indicated that the proteins interact wit h a helical domain which is conserved in the internal transcribed spacers. Mutagenic analyses confirmed an interaction with this sequence and indicate d that this domain is critical to the efficient maturation of the precursor RNA. The protein constituents, purified by affinity chromatography using t he ITS1 sequence, retained an ability to form stable RNP. Protein analyses of gel purified complex, prepared with affinity-purified proteins, indicate d at least 20 protein components ranging in size from 20-200 kDa. Peptide m apping by Maldi-Toff mass spectroscopy identified eight hypothetical RNA bi nding proteins which included four different RNA-binding motifs. Another pr otein was putatively identified as a pseudouridylate synthase. Additional R NA constituents were not detected. The significance of this complex with re spect to rRNA maturation and interdependence in rRNA processing is discusse d. (C) 2000 Academic Press.