U3 snoRNA in a fission yeast
U3 snoRNA is an essential trans-acting factor, which is required for rRNA processing in eukaryotic cells. This study examined the U3 snoRNA in ' S. pombe' in terms of its expression, which included the identification of the promoter elements and the processing/termination pathway, and its functional structure. To initiate the study, a "tagged" U3 snoRNA expression system was developed in which the products could be readily distinguished from endogenous transcripts. Despite greatly elevated levels of precursor in cells transformed with the "tagged" U3 snoRNA construct, the level of mature RNA remained constant, an observation which demonstrates tight regulation at the post-transcriptional level. Subsequent study on the promoter sequences indicated that transcription is dependent on two upstream elements, an important but not critical TATA-box found in other 'S. pombe' snRNA and rRNA gene promoters and an essential Homol D-box, often considered a TATA-box analogue in promoters of genes that encode ribosomal proteins. These results suggest that the U3 snoRNA promoter may reflect the special role of this RNA in ribosome biogenesis. Study on U3 snoRNA processing and transcript termination revealed an intimate relationship between these two pathways. Both processes do not rely on any specific sequence or structure, but are mediated by a Pac1p endonuclease cleavage downstream of the coding region. Although the cleavage site is near the 3' end coding region, it can be moved thousands of nucleotides downstream with no apparent effect on maturation or transcript termination. The results support a "reverse torpedoes" model in which a single cleavage allows exonuclease access to the transcript leading to transcription termination in one direction and RNA maturation in the other direction. Finally a search for the elements within the coding region, which might be important for RNA stability or function, was initiated. The results suggest that helical structures near the ends of the RNA are important to RNA stability, while other regions are less essential. While preliminary, these mutational analyses raised a number of questions regarding putatively identified protein binding sites and contributions to RNA function. The expression system remains a new tool for future analyses.