451 Biotech Building
Overview: Research in our lab focuses broadly on the area of RNA biology. We are taking microarray-based approaches to examine questions both about the mechanism of pre-mRNA splicing, as well as the role of splicing as a control point for regulating gene expression. We are studying aspects of RNA processing both in the budding yeast Saccharomyces cerevisiae as well as the fission yeast Schizosaccharomyces pombe.
Background: A major focus of the work in my lab is to understand one of the most fundamental problems in molecular biology today: how do organisms regulate the expression of their genetic material? In particular, our work examines the role of pre-mRNA splicing in this process. Because the coding regions of most eukaryotic genes are interrupted by non-coding introns, appropriate expression of these genes requires the precise removal of their introns in a process catalyzed by the spliceosome. While the presence of introns in eukaryotic genes has been known for over three decades, the importance of pre-mRNA splicing in the gene expression pathway has been reinforced by the sequencing of the human genome. While many were initially surprised by the relatively small number of genes encoded in our genome, it soon became clear that a significant amount of genomic diversity could be generated by changing the order in which the coding regions of many genes are spliced together – a process termed alternative splicing (reviewed in Black Cell 2000; Blencowe Cell 2006). Indeed, the average human gene is interrupted by eight introns (Lander, et al. Nature 2001), and it is now clear that the splicing patterns for many genes change in different cell types and under different developmental conditions. The importance of pre-mRNA splicing and its appropriate regulation has been further augmented over the past few years by the number of disease states that have been associated with its mis-regulation (reviewed in Faustino and Cooper, Genes Dev 2003). While these examples make clear the importance of the process of alternative splicing, remarkably little is currently known about the capacity of the spliceosome to function as a regulatory control point in the gene expression pathway.
Recent Work: In my lab we are using splicing-specific microarrays that I designed during my post-doctoral work to examine splicing from a genome-wide perspective.
These microarrays allow us to simultaneously examine the behavior of all intron-containing genes in the budding yeast S. cerevisiae. By comparison with higher eukaryotes, splicing is relatively simple in S. cerevisiae. Whereas more than 95% of human genes are interrupted by at least one intron, only about 300 genes in S. cerevisiae (less than 5% of all genes) contain an intron, the vast majority of which contain only a single intron. As seen in the figure above, S. cerevisiae introns tend to conform to very strong consensus sequences at both the branch point (BP) and 5’ and 3’ splice sites (5’SS and 3’SS). Perhaps accordingly, S. cerevisiae appears to lack the large families of regulatory (SR and hnRNP) proteins that are found in higher eukaryotes. In the absence of such regulatory factors, and because of the relative conformity of introns in S. cerevisiae, it had been widely believed that S. cerevisiae lacked the capacity to specifically regulate pre-mRNA splicing. By combining the microarrays with a set of high-throughput methods that I also developed, we were able to make two important and previously unanticipated findings. First, by examining a panel of conditional mutants in core spliceosomal components, I showed that the spliceosome can efficiently discriminate among all transcripts (see Pleiss, et al. PLoS Biology 2007; reviewed in Skipper, Nat Rev Genetics 2007). This finding suggests that even in the absence of functional SR proteins the S. cerevisiae spliceosome has the potential to regulate transcript-specific events. Second, by examining the molecular responses to either of two environmental stresses (amino acid starvation or exposure to toxic levels of ethanol), I demonstrated that the splicing of distinct subsets of transcripts is rapidly and specifically regulated (see Pleiss, et al. Mol Cell 2007). The splicing responses to these stresses involve both up- and down-regulation, suggesting that pre-mRNA splicing can be an important control point for regulating gene expression. Because the core spliceosomal machinery present in S. cerevisiae is so highly conserved throughout eukaryotes, it is likely that the mechanisms controlling this regulation will also be widely conserved.
Future Projects: We are now in a position to use these techniques to examine the regulation of pre-mRNA splicing in the fission yeast S. pombe. While the simple, yet powerful, genetics of budding yeast made it an ideal model organism for elucidating the basic mechanism of pre-mRNA splicing, S. pombe is the ideal organism for elucidating mechanisms of splicing regulation from a genome-wide perspective. First, while the fission yeast genome is nearly identical in size to the budding yeast genome, nearly half of all S. pombe genes contain at least one intron, and many are interrupted by multiple introns – the number of introns varies from a minimum of zero to a maximum of 15 – greatly increasing the potential opportunities for alternative splicing. Furthermore, as seen in the figure on the previous page, the sequences found at the 5’ SS and BP of S. pombe introns are significantly more degenerate than those in S. cerevisiae, more closely resembling those found in higher eukaryotes. From a statistical perspective, this increased diversity provides a powerful opportunity to identify correlations between sequence identity and splicing behavior. And finally, a single protein has been identified in the S. pombe genome called Srp2 which functions as a bona fide homolog of the human SR proteins, allowing us for the first time to directly determine the primary consequences of inactivating an SR protein. Importantly, while splicing in S. pombe more closely resembles that seen in higher eukaryotes, it nevertheless remains a highly tractable genetic system, and one that is readily adaptable to all of the high-throughput methodologies that I developed in my previous work.
Things to know about the lab: We use a wide variety of techniques to approach the biological questions of interest, including genetics, genomics, biochemistry, molecular biology, and computational biology. As such, rotation projects are available for students who have diverse scientific interests. We will be accepting rotation student during all three periods, and will plan to accept two students into the lab.