Inada: Research in Biology
Investigating a mode of gene regulation that couples splicing and mRNA decay using experimental and computational approaches
The major goal of my research is to understand one of the most fundamental problems in molecular biology today: how do organisms regulate the expression of their genetic material or how are genes turned on and off? Whereas many mechanisms of transcriptional control are well characterized, we are focusing on the role of pre-mRNA splicing in regulating this process. Since most eukaryotic genes are interrupted by noncoding sequences called introns, proper gene expression requires the removal of these introns in a process called pre-mRNA splicing. More recently, pre-mRNA splicing has been suggested to play a role in regulation of the gene expression pathway. By changing the order in which the coding regions of genes are spliced together, a process termed alternative splicing, a significant amount of genomic and therefore proteomic diversity can be generated.
We are examining an unusual mode of alternative splicing, whereby the mRNA products are not predicted to encode a stable protein, but rather the resulting mRNAs are thought to be targeted for degradation via a cellular discard pathway. Importantly, this finding expanded the role of alternative splicing from generating protein diversity to include potent regulation of the levels of that transcript. By inhibiting this decay pathway we are able to detect transcripts that are subject to this form of regulation. Using an RNA interference approach in mammalian cells to inhibit the cellular decay pathway and coupling this with a sensitive quantitative PCR assay we can detect changes in cellular levels of alternative transcripts. Strikingly, when we examined the behavior of a family of proteins called SR proteins, which are themselves important modulators of alternative splicing, we found that the entire family was subject to this mode of regulation.
We are currently examining this mode of regulation in the model organism fission yeast, Schizosaccharomyces pombe. Importantly, splicing in S. pombe is very similar to splicing in mammalian systems and a single homolog of the human SR protein family has been identified in S. pombe, Srp2. My goal is to leverage the highly tractable experimental system provided by S. pombe to identify the molecular mechanisms by which this gene is regulated. We are undertaking experiments designed to identify important regulatory sequences within the SRP2 gene that are both necessary and sufficient for regulation. We are also using genome-wide approaches to further characterize the regulation of Srp2 splicing. By employing a high-throughput reverse genetic screen we hope to identify the full complement of trans-acting factors required for this regulation. Because of the similarities between splicing in S. pombe and higher eukaryotes, we expect that the lessons we learn about the mechanisms of splicing of Srp2 will be immediately applicable to our understanding of the mechanisms of regulated splicing in higher eukaryotes. Furthermore, because the regulation of splicing is essential for normal cellular function and its mis-regulation is often associated with disease, we expect the results of this work will have broad implications for the fields of biology, biochemistry and human health.
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