Maki Inada: Summer
- Using a genome-wide microarray-based approach, identify and characterize novel genes regulated by splicing coupled to mRNA decay
- Identify conserved cis-regulatory sequences important for changes in splicing
- Examine the role of kinases in gene regulation
- Study the effects of co-transcriptional recruitment of regulatory factors on gene control
Investigating an unusual mode of gene regulation that couples splicing and mRNA decay
My research focuses on one of the most fundamental questions in molecular biology today: how do organisms regulate expression of their genetic material by turning genes on or off. Previously, I and others have identified a novel and unusual mode of gene regulation. Whereas mechanisms of transcriptional control have been widely studied, I am focusing on the role of splicing in regulating this process. Since most eukaryotic genes are interrupted by noncoding sequences, proper gene expression requires the removal of these sequences via the process known as splicing. Splicing has already been known to play an important role in generating genomic diversity. By changing the order in which the coding regions of genes are spliced together, different proteins can be produced from a single transcript via a process termed alternative splicing. I am examining a different mode of alternative splicing in which splicing events lead not to production of different proteins, but rather to degradation of specific transcripts. This would lead to subsequent down regulation as a mechanism of gene control.
We are examining this mode of gene regulation in the fission yeast, Schizosaccharomyces pombe. While my previous work was done in mammalian cells, S. pombe is in many ways an ideal system in which to undertake these studies. While splicing in S. pombe is highly similar to splicing in mammalian systems, S. pombe is a much more tractable genetic system for dissecting the molecular mechanisms of this regulatory pathway. S. pombe is also easy to work with and therefore highly amenable for research with undergraduates. Our goal is to leverage the experimental system provided by S. pombe to dissect the molecular details that govern the decision to splice or not to splice in regulating gene expression.
Recent experiments conducted by students in my laboratory and students in the Experimental Biochemistry laboratory course have identified a number of novel candidates genes that we hypothesize may be regulated by this unusual mode of gene regulation. Because the products of such a pathway would be normally rapidly degraded, much of this type of alternative splicing has previously remained undetected. We postulated we would be able to detect transcripts that are subject to this form of regulation by inhibiting the cellular decay pathway. We therefore examined yeast cells that are defective for this decay pathway and used a microarray-based approach to identify the genome-wide array of candidate transcripts. Our genome-wide approach is very powerful in that it allows us to identify all possible candidate transcripts. With a list of exciting candidate genes in hand, careful validation and characterization are now necessary. We will be conducting quantitative PCR experiments to measure regulatory effects on transcript levels. Because of the similarities between splicing in S. pombe and higher eukaryotes, we expect that the lessons we learn about the mechanisms of splicing will be immediately applicable to our understanding of the mechanisms of regulated splicing in higher eukaryotes, such as humans. Furthermore, because the regulation of splicing is essential for normal cellular function and its mis-regulation is often associated with cancer, we expect the results of this work will have broad implications for human health and disease.
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