The ribosomal RNA (rRNA) in cells of all organisms is transcribed as a single long precursor RNA molecule which, through various RNA:RNA and RNA:protein interactions assembles into mature ribosomal subunits. In vertebrates there exist small nucleolar ribonucleolar protein (snoRNP) particles within nucleoli, some of which are essential for processingpre-rRNA molecules. Previously, I demonstrated that U8 snoRNA is essential in vivo for accumulation of mature 28S and 5.8S rRNAs. In the past year work was completed and published which demonstrated a region of direct interaction between U8 snoRNA and a segment within the pre-rRNA corresponding to the 5+ end of the mature 28S rRNA. This region of 28S rRNA is known to base pair with 5.8S rRNA to form an evolutionarily conserved stem present in rRNA from every organism where rRNA has been examined. This work showed that mutations, insertions or deletions within the region of U8 involved in the interaction will not function in pre-rRNA processing. In vivo competitions have confirmed that this region of U8 RNA must be available in the intact particle in vivo and in fact is involved in an RNA:RNA base pairing interaction. Cross-species hybrid molecules demonstrated that the interaction between the U8 snoRNA and pre-rRNA is necessary, but not sufficient to facilitate pre-rRNA processing; U8-specific proteins probably stabilize the interaction, modulate the timing of the cleavage or recruit the catalytic machinery. Thus, U8 snoRNA directly interacts with the pre-rRNA to achieve a folded conformation which is a substrate for cleavage. Since the work described above indicated essential base pairing interactions within pre-rRNA, rDNA clones are being generated in the yeast system to directly assay the necessity of some interactions for processing, with the hope of being able to uncouple the base pairing role of U8 RNA from the structural modifications induced in refolding pre-rRNA. Since it is not feasible to do these types of experiments in Xenopus, this work is being done in yeast, the only other well defined in vivo system available for examination of pre-rRNA processing. Another project in the lab involves examining the genomic organization of U8 snoRNA in Xenopus. A genomic clone encoding two tandem copies of theU8 gene in Xenopus was sequenced. The two genes are not identical to each other, nor is either identical to the previously reported RNA sequence of U8. Interestingly, despite the sequence differences, both genomic clones are functional in vivo, providing additional information regarding essential functional domains in U8 RNA. There appear to be seven or eight copies of the U8 gene present in the Xenopus genome, and additional U8 genes are being examined.
