A chaperone for ribosome maturation.

The nascent pre-rRNA of eukaryotic ribosomes is fully transcribed and assembled into an 80-90 S nucleolar particle before being cleaved into mature ribosomal RNA. The interdependence of steps in the processing of this precursor RNA indicates that RNA processing, at least in part, acts as a quality control mechanism that helps ensure that only functional RNA is incorporated into mature ribosomes. In search of structural components that underlie this interdependence using the Schizosaccharomyces pombe internal transcribed spacer 1 (ITS) as a ligand for affinity chromatography of ITS1-specific proteins, we have isolated a large spliceosome-like protein complex, a ribosome assembly chaperone (RAC) of 20 or more polypeptides (Lalev, A. I., Abeyrathne, P. D., and Nazar, R. N. (2000) J. Mol. Biol. 302, 65-77). When the ITS2 spacer was used in the present study to isolate ITS2-specific proteins, the same proteins were identified consistent with a complex containing multiple specific binding sites. Subsequent competition binding studies indicated that the protein complex actually contains independent binding sites for all four of the transcribed spacers in the pre-rRNA. Because disruption of protein-binding sites in these spacer RNAs is known to severely affect rRNA processing, taken together these results suggest that the RAC complex is a chaperone for ribosome maturation acting as a "rack" on which critical structure is organized.

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.

Structural equivalence in the transcribed spacers of pre-rRNA transcripts in Schizosaccharomyces pombe.

The structure of the internal transcribed spacer 2 (ITS2) in Schizosaccharomyces pombe was re-evaluated with respect to phylogenetically conserved features in yeasts, features in other transcribed spacer regions as well as the binding of transacting factors which potentially play a role in ribosomal maturation. Computer analyses and probes for nuclease protection indicate a very simple core structure consisting of a single extended hairpin which includes the interacting termini of the mature 5.8S and 25S rRNAs. Comparisons with ITS2 sequences in greatly diverging organisms indicate that the same feature also can be recognized. This is especially clear in organisms that contain very short sequences in which the putative structures are much less ambiguous. Diversity between organisms is the result of changes in hairpin length as well as the addition of branched helices. Protein binding and gel retardation studies with the S.pombe ITS2 further indicate that, as observed in the 3" external transcribed spacer (ETS) and ITS1 regions, the extended hairpin is not only the site of intermediate RNA cleavage during rRNA processing but also a site for specific interactions with one or more soluble factors. Taken together with other analyses on transcribed spacer regions, the present data suggest that the spacer regions all may act in a similar fashion, not only to organize the maturing terminal sequences, but also serve to organize specific soluble factors possibly acting with snoRNAs or in a manner which is analogous with that of the free snoRNPs.

Conserved core structure in the internal transcribed spacer 1 of the Schizosaccharomyces pombe precursor ribosomal RNA.

The structure of the internal transcribed spacer 1 (ITS1) in Schizosaccharomyces pombe was examined with respect to phylogenetically conserved features in yeasts as well as the binding of transacting factors that potentially play a role in ribosomal maturation. Computer analyses and probes for nuclease protection indicate a compact, more highly organized structure than previously proposed in Saccharomyces cerevisiae, with distinct structural features which can be recognized in S. cerevisiae. These include a central extended hairpin structure as well as smaller hairpins immediately adjacent to the maturing termini. Comparisons with ITS sequences in more diverse organisms indicate that the same features also can be recognized. This is especially clear in organisms which contain very short sequences in which the putative structures are much less ambiguous. Again nuclease protection analyses in one of these, Verticillium albo-atrum, confirm a central hairpin with additional hairpins linked to the maturing termini. Protein binding and gel retardation studies with the S. pombe ITS1 further indicate that, as observed in the 3' external transcripted spacer (ETS) region, the extended hairpin is not only the site of intermediate RNA cleavage during rRNA processing, but also a site for specific interactions with one or more soluble factors. Taken together with other analyses on transcribed spacer regions, the present data provide evidence that the spacer regions act not only to organize the maturing terminal sequences but also may serve to organize specific soluble factors, possibly acting in a manner which is analogous with that of the free small nucleolar ribonucleo protein particles (snoRNPs).

Structural features in the 3' external transcribed spacer affecting intragenic processing of yeast rRNA.

A highly conserved extended hairpin structure in the 3' external transcribed spacer (3' ETS) region of nascent eukaryotic rRNA transcripts is essential for the maturation of the large ribosomal subunit RNAs (5.8 S and 25 to 28 S rRNAs). Systematic changes were introduced into this structure by PCR-mediated mutagenesis and the mutant rDNAs were expressed in vivo to determine the structural features that are essential for rRNA maturation. Changes in the lower half of the stem or the large loop at the end had little or no effect on the maturation of either the 5.8 S or 25 S rRNA, but changes that disrupted secondary structure in the upper half of this stem had equal and dramatic effects on both RNAs. When the RNA stem was incubated with a cellular protein extract, gel retardation studies indicated that the stem forms a ribonucleoprotein complex, and a comparison with mutant RNA indicated that protein binding could be compromised by changes that were critical for rRNA maturation. Sequence comparisons with other spacer regions as well as snRNAs reveal some structural analogy, which, when taken together with the mutational studies, raise the possibility that this hairpin functions during RNA processing in a manner that may be analogous with that of free snRNPs.