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 Schizosaccharomyces pombe suggests that RNA processing, at least in part, acts as a quality control mechanism which helps assure that only functional RNA is incorporated into mature ribosomes. To determine further the role of the transcribed spacer regions in rRNA processing and to detect interactions which underlie 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 electrophoresis. Modification exclusion analyses indicated that the proteins interact with a helical domain which is conserved in the internal transcribed spacers. Mutagenic analyses confirmed an interaction with this sequence and indicated that this domain is critical to the efficient maturation of the precursor RNA. The protein constituents, purified by affinity chromatography using the ITS1 sequence, retained an ability to form stable RNP. Protein analyses of gel purified complex, prepared with affinity-purified proteins, indicated at least 20 protein components ranging in size from 20-200 kDa. Peptide mapping by Maldi-Toff mass spectroscopy identified eight hypothetical RNA binding proteins which included four different RNA-binding motifs. Another protein was putatively identified as a pseudouridylate synthase. Additional RNA constituents were not detected. The significance of this complex with respect to rRNA maturation and interdependence in rRNA processing is discussed.
Lalev
A I
AI
Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
Abeyrathne
P D
PD
Nazar
R N
RN
D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D005800:Genes, Fungal / Q000235:genetics; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D010449:Peptide Mapping; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D012322:RNA Precursors; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins