Ribosome biogenesis is an essential and complex multistep pathway which exists in all living cells. The precursor rRNA (pre-rRNA) encodes three separate RNAs that are transcribed as a single precursor molecule that must be correctly modified, folded, processed and assembled with proteins to yield the two mature ribosomal subunits that comprise the functional ribosome. The focus of the research in my lab has been to identify and characterize cis-acting elements and trans-acting factors critical for the processing events of pre-rRNA processing, and thus essential for cell survival. Over the past year my lab has made progress on three fronts. First, we are using the genetics available in yeast, S.cerevisiae, to examine a predicted intramolecular interaction in pre-rRNA necessary for subsequent processing steps. Second, we are using biochemical methods to identify proteins that comprise the Xenopus U8 small nucleolar ribonucleoprotein particle (U8 snoRNP), an essential trans-acting factor required for accumulation of newly formed large ribosomal subunits. Third, we are examining the kinetics of pre-rRNA processing to learn more, at a mechanistic level, about the roles that snoRNPs, particularly U8, play in pre-rRNA processing. One focus in the lab involves a more detailed examination of our previously described a model for the mechanism by which U8 snoRNA may facilitate pre-rRNA processing in the Xenopus oocyte (1). This model predicted that formation of a specific intramolecular interaction in pre-rRNA should be critical for pre-rRNA processing. Because of the many different aspects of RNA processing addressed by this model and complexity of the Xenopus oocyte system, the yeast system was used to directly test this one aspect of the model. The feasibility of genetic and biochemical manipulations in yeast made it possible to directly test the effect of point mutations in this region upon the ability to process pre-rRNA. Our early experiments in yeast unequivocally demonstrated that formation of this intramolecular interaction is critical for pre-rRNA processing (2). Over the past year additional experiments in yeast have implicated other cis-acting elements that play important roles in processing; these appear to function as structural elements and sequence plays little role in recognition of these structures (3). The data obtained in these yeast studies will later be applied to parallel experiments in Xenopus, which to date is the only existing model system for examining rRNA processing in vertebrates. A second focus is a continuation of our characterization of trans-acting factors essential for pre-rRNA processing in vertebrates. I previously demonstrated that U8 snoRNP is essential for pre-rRNA processing in Xenopus oocytes. In the absence of U8 RNA, pre-rRNA processing is inhibited and no mature rRNA accumulates (1). Mutageneis of U8 RNA indicated that sequences at the 5’ end of U8 RNA were necessary, but not sufficient to direct pre-rRNA processing; presumably U8 RNP proteins affected the stability of the U8 RNA and the efficiency of processing (1). To better understand how the U8 RNP functions in vivo, we have been identifying proteins which specifically bind U8 RNA in vitro. We recently reported our identification of a 29 kDa protein from Xenopus ovary extracts which specifically binds U8 RNA (4). This protein binds U8 RNA with high affinity and can be crosslinked to U8 snoRNA. In vitro competition binding assays indicated this protein is unique to the U8 RNP and is not a common or shared protein present in other snoRNPs (4). We are continuing to characterize the X29 protein and identify other U8 RNA binding proteins to gain a better mechanistic understanding of how the U8 RNP facilitates processing. A third focus of the lab has been an examination of snoRNA localization in oocytes and the kinetics of pre-rRNA processing in Xenopus oocytes (5). The kinetics of pre-rRNA processing were examined in oocytes treated with transcriptional inhibitors to separate transcription-dependent events from those involved in processing. When performed in snoRNA-depleted oocytes, the ‘rescue-of-function’ assay demonstrated a failure of snoRNAs to mediated processing of previously accumulated rRNA precursors. This result is consistent with a scenario where these snoRNAs must be present co-transcriptionally to facilitate processing (5) and supports our theory that the snoRNAs act, in part, as molecular chaperones to facilitate pre-rRNA folding. Characterization of U8 snoRNA genes in Xenopus identified naturally occurring U8 sequence variants that are functional in vivo (6). This natural variation allowed us to identify a conserved octamer sequence in U8 snoRNA present in all vertebrate U8 snoRNAs known to date, including Xenopus, mouse, rat and human. Characterization of the octamer and identification of proteins that bind this sequence may give insight into conserved functional mechanisms and provide additional information about the unique role of U8 snoRNP in pre-rRNA processing. In using two model systems and taking advantage of their differences we hope to better understand the basic mechanisms of pre-rRNA processing and to identify conserved and unique cis- and trans-acting components involved in pre-rRNA maturation. Identification of common components as well as species specific elements will help us understand the basic mechanisms at play in the universal and complex process of ribosome biogenesis.
