Ribosomal RNA (rRNA) is a critical catalytic component of the ribosome, a complex and massive ribonucleoprotein particle present in all living cells. Pre-rRNA is transcribed as a single long precursor molecule that is modified, folded, processed and assembled with proteins to yield a mature ribosome. Over the past year my laboratory has made significant progress in better understanding some of the cis- and trans-acting components involved in pre-rRNA processing. More specifically, we have identified a cis-acting element in pre-rRNA, the formation of which is critical for processing events that yield mature rRNA in yeast. In addition, we have begun to analyze the proteins comprising the U8snoRNP, a trans-acting small nucleolar ribonucleoprotein particle which I have previously demonstrated is critical for pre-rRNA processing in the Xenopus oocyte model system. In the absence of U8 RNA pre-rRNA processing is inhibited and no mature rRNA will accumulate. Last year I published a paper presenting data and a model describing the mechanisms by which U8 appeared to facilitate pre-rRNA processing in the Xenopus oocyte. This model predicted a specific intramolecular interaction, the formation of which should be critical for pre-rRNA processing. Because of the complexity of the Xenopus oocyte and the high likelihood of ambiguous results in that system, I first used the yeast system to test a smaller aspect of this model directly, using genetic methods. The experiments in yeast have been very fruitful, proving unequivocally that formation of this intramolecular interaction is critical for processing. Additional experiments in yeast experiment have implicated other cis-acting elements (either primary sequence or secondary structure) which also play an important roles in processing. These additional elements are currently being examined. One outcome of the work in yeast is that the results predict additional experiments which now need to be performed in Xenopus, the vertebrate model system. The paper published last year also presented evidence that the U8 RNA component of the U8 RNP was necessary but not sufficient to facilitate processing. The proteins associated with, or recruited by U8 RNA are also essential for processing, some of which appear to act in a species specific manner. To examine these proteins and their role in processing more closely, my lab has been using gel shift assays and crosslinking experiments to identify and enrich for U8-specific proteins via standard column chromatography methods. To this end, we have identified putative U8 RNA-specific binding proteins that we are in the process of characterizing in more detail. By taking advantage of the two different model systems, yeast and Xenopus, we hope to better understand the mechanisms of pre-rRNA processing and identify and the cis and trans-acting components involved. Identification of common components as well as species specific elements will help us understand the basic mechanisms at play in the universal process of pre-rRNA maturation.
