Abstract
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.
Highlights
Studies of rRNA processing reported a split processing scheme for the independent maturation of the large and small subunit RNAs [5]
The deletion of the ITS2 sequence has been shown to be critical to the maturation of the large subunit and to have severe effects on the level of 18 S rRNA production [8], and as already indicated, a U3 snoRNA1⁄75Ј-ETS complex appears to be essential for the initiation of pre-rRNA processing overall
At least in part, these processes contribute to a quality control mechanism that helps ensure that only functional RNA is incorporated into mature ribosomal particles [8]
Summary
The deletion of the ITS2 sequence has been shown to be critical to the maturation of the large subunit and to have severe effects on the level of 18 S rRNA production [8], and as already indicated, a U3 snoRNA1⁄75Ј-ETS complex appears to be essential for the initiation of pre-rRNA processing overall. Using gel retardation studies we were able to demonstrate specific interactions between individual transcribed spacer sequences and cellular proteins [10], and using ITS1 RNA we were able to isolate a complex of proteins that interacted with this spacer sequence [12] This complex, a ribosome assembly chaperone (RAC), contained at least 20 proteins ranging in size from 20 to 200 kDa. Mass spectroscopy and computer analyses suggested that many of the proteins contained RNA binding motifs or were nuclear in localization. Subsequent comparative analyses indicate that a single protein complex has independent binding sites for all the transcribed spacers in the pre-rRNA
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