The ribosomal RNA 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, a few of which are essential for processing the precursor molecules. I previously demonstrated that U8 snoRNA is essential in vivo for accumulation of mature 28S and 5.8S rRNAs. Using evolutionary and indirect molecular approaches, I've identified 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 basepairs with 5.8S rRNA to form an evolutionarily conserved stem. 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 to basepair with pre-rRNA for processing to occur. Thus, it appears that the U8 snoRNA is acting like a molecular chaperone by directly interacting with the pre-rRNA to achieve an intermediate folded conformation which is cleaved; U8 snoRNA is released and the rRNA achieves a more stable, alternately folded conformation with 5.8S rRNA which is presumably that found in the mature ribosome. Using cross-species hybrid U8 RNAs in vivo, I've demonstrated that the snoRNA:pre-rRNA interaction is essential for processing, but not sufficient to direct efficient cleavage of the pre-rRNA substrate; the proteins comprising, or recruited by the U8 snoRNP modulate the efficiency of cleavage, perhaps by altering the U8 RNA structure. To examine this effect more closely we've begun studying the proteins associated with snoRNPs to learn more about the mechanisms by which the snoRNAs recognize, interact with, and facilitate processing of rRNA. We have used the yeast two-hybrid system to identify proteins that interact with fibrillarin, a 34-kDa protein which is associated with many, but not all of the nucleolar snoRNAs. Using a Xenopus cDNA library we have isolated a potential fibrillarin interactor via the yeast two hybrid system and identified the protein as the Xenopus homologue of the human SMN protein. We are currently examining the biological relevance of the fibrillarin:SMN interaction by looking for this interaction in vivo in Xenopus oocytes to insure the interaction is not specific to the yeast two-hybrid system.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Intramural Research (Z01)
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