Presentations
Most Recent Presentations
(Students are italicizied.)
"Investigating a mode of regulating gene expression via alternative splicing coupled with mRNA decay in the fission yeast Schizosaccharomyces pombe." Co-authored with Philip A Feinberg, Bushra Amreen, Jeffrey A Pleiss, Maki Inada. The Eukaryotic mRNA Meeting. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. August 2011.
Abstract:
Regulation of gene expression is required to maintain cellular function. Since most eukaryotic genes are interrupted by noncoding sequences called introns, proper gene expression requires the removal of these introns by the process of pre-mRNA splicing. Moreover, by changing the order in which the coding regions or exons of genes are spliced together, or alternative splicing, it has been observed that a significant amount of proteomic diversity can be generated. As such, alternative splicing can play a role in modulating the gene expression pathway.
We are examining a different mode of alternative splicing, whereby the mRNA products are not predicted to encode stable protein, but rather the resulting mRNAs are thought to be targeted for degradation via the cellular discard pathway of nonsense-mediated decay (NMD). This finding expanded the role of alternative splicing from generating protein diversity to include potent regulation of the levels of that transcript. By inhibiting NMD we are able to detect transcripts that are subject to this form of regulation. In mammals, 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 investigating this mode of regulation in the model organism fission yeast, S. pombe. Importantly, the core splicing machinery is highly conserved and the splicing architecture is similar between S. pombe and mammalian systems. Over half of genes in S. pombe contain introns and many are multi-intron genes. Also, a homolog of the human SR protein family has been identified in S. pombe, Srp2, suggestive of the presence of regulated alternative splicing. We are undertaking experiments designed to analyze whether Srp2 is similarly regulated by alternative splicing coupled to mRNA decay using RT-QPCR to detect the levels of different isoforms in an NMD-inhibited strain, upf1Δ. Our long term goal is to identify important regulatory sequences within the SRP2 gene that are required for this regulation. We are also using a genome-wide approach utilizing splicing-sensitive microarrays to identify other spliced isoforms that are targets of NMD and hence candidates for regulatory control. Because of the similarities between splicing in S. pombe and higher eukaryotes, we expect that the lessons we learn about the mechanisms of splicing in S. pombe will have broad implications in understanding global regulatory control.
"Genome-Wide Analyses of prp8 Alleles Implicated in the Two-State Model for Spliceosome Activitiy". RNA Society Meeting. Seattle, WA. June 2010. Co-authored with Jeffrey Pleiss.
Abstract:
Removal of noncoding introns from pre-rnRNA is catalyzed by the spliceosome, a large multi-component complex I that must be assembled anew for every splicing reaction. Splicing chemistry consists of two separate and sequential transesterification reactions: in the first step the branch site adenosine attacks the 5' splice site to produce the 5' exon and branched lariat intermediate; in the second step the 5' exon attacks the 3' splice site to produce the lariat intron and 1 spliced product. A two-state model for the spliceosome has been previously proposed, in which the conformations of the active site required for the first and second steps are in competition with each other (Query and Konarska 2004). Factors that modulate / and stabilize the first step result in inhibition of the second step and vice versa. A number of such opposiogprp8 alleles that affect the transition between the first and second step have been isolated and characterized to support this model (Query and Konarska 2004, Liu et al. 2007). To further investigate the role that prp8 plays in splicing activation, we have chosen to take two genome-wide approaches in Saccharomyces cerevisiae. First, we have conducted splicing-sensitive microarray analyses to determine the genes that are affected by each of these prp8 alleles. Second, we have taken a high through-put reverse genetic approach known as Synthetic Genetic h a y analysis to identify those factors that are involved in modulating the activity of these prp8 alleles. By determining both the transcripts affected and the complement of factors that genetically interact with each of these prp8 alleles, we will be better able to define how prp8 functions in the splicing pathway. Lastly, strategies for developing a research course for undergraduates utilizing these methods will be discussed.