Most Recent Presentations
(Students are italicizied.)
"To Splice or Not to Splice: an Ultraconserved Mode of Gene Regulation," and guest lectured in an Advanced Biochemistry Course on the Molecular Biology of Cancer titled "My very own lung cancer case study: targeted therapies" in the Chemistry and Biochemistry Department at Washington and Lee University. November 2014.
"Linking the C-Terminal Domain Code of RNA Polymerase II to Modulating Chromatin States in fission yeast Schizosaccharomyces pombe”. Cold Spring Harbor Laboratory Eukaryotic mRNA Processing Meeting. Cold Spring Harbor, NY. August 2013. Co-authored with Robert Nichols (Biochemistry ’14), Beate Schwer (Cornell Weill Medical College) and Jeff Pleiss (Cornell University).
Regulation of gene expression is essential for all living organisms. One critical step in modulating gene expression is altering the ability of the transcriptional enzyme, RNA Polymerase II (RNAPII), to access DNA by manipulating chromatin states. The carboxy-terminal domain (CTD) of RNAPII, is believed to play a critical role in chromatin remodeling through its recruitment of factors that modify histones. Conserved throughout evolution, the CTD contains a repeated Y1S2P3T4S5P6S7 heptapeptide sequence that undergoes dynamic posttranslational modifications. The capability of each serine in the sequence to undergo phosphorylation and dephosphorylation creates a readable ‘code’ for recruiting factors that can determine when processing events such as chromatin remodeling to occur. In order to characterize how specific phosphorylation marks in the CTD affect gene expression, mutants of fission yeast Schizosaccharomyces pombe were rendered defective for phosphorylation by substituting a nonphosphorylatable alanine in place of each serine in position 2 in the heptad sequence (S2A), each position 7 serine (S7A), or all serines in position 2 and position 7 in combination (S2A/S7A). In addition, a fourth mutant was created in which the position 7 serines were substituted for the phosphomimetic glutamic acid (S7E). We have performed microarray experiments with these mutants to study the genome-wide effects of eliminating and altering these phosphorylation events. In agreement with previous results, we have observed the expression of STE11, which is required for mating in S. pombe, to be low in the S2A mutant, whereas levels are restored in the double mutant S2A/S7A. However, while others have observed defects in snRNA levels with these mutants in human cells, we do not see a similar decrease with our S. pombe mutants. Interestingly, analyses of our microarray data reveals an upregulation of positionally related clusters of genes, specifically in some telomeric regions. Further quantitative PCR analysis of genes in these telomeric regions confirm a significant upregulation of gene expression in the telomeric regions spanning approximately 50kb. Our microarray analyses and subsequent qPCR validation suggest a role for the dynamic phosphorylation and dephosphorylation of serines within the CTD code in modulating the chromatin states in large telomeric regions of S. pombe.
"Investigating the Role of Phosphorylation Sites of the RNA Polymerase II CTD Code on Global Gene Expression in Schizosaccharomyces pombe." RNA 2012 conference. Ann Arbor, MI. May 2012. Co-authored with Robert Nichols, Jeffrey Pleiss, Beate Schwer.
Regulation of gene expression is essential for all living organisms. In eukaryotes, genetic information stored in the form of DNA must first be transcribed to a transient messenger RNA molecule that is highly processed prior to translation to functional protein. The structure of the transcriptional enzyme RNA polymerase II is believed to play a critical role in modulating gene expression. Specifically, the dynamic phosphorylation pattern on the carboxy terminal domain (CTD) of RNA polymerase II has been demonstrated to aid in the recruitment of numerous nuclear factors involved in RNA processing. Conserved throughout evolution, the Y1S2P3T4S5P6S7 heptapeptide repeat can be phosphorylated at any of the serines at positions 2, 5, and 7. The modification of residues of the CTD repeats is thought to create a readable ‘code’ for controlling transcription initiation, elongation, termination, changes in chromatin structure, and regulation of capping, splicing and polyadenylation. In order to characterize how the absence of specific phosphorylation marks in the CTD affect gene expression, mutants of fission yeast Schizosacchromyces pombe were rendered defective for phosphorylation by substituting alanines for serines at either positions 2 and 7 individually or in combination, S2A, S7A, and S2A/S7A respectively. Previous studies demonstrated mutants lacking some of these phosphorylation events were deficient in the expression of genes required for sexual differentiation. To examine the genome-wide effects these mutations have on gene expression as well as changes in pre-mRNA splicing, we conducted microarray analyses. We used custom designed splicing sensitive microarrays to allow us to detect upregulation or downregulation of specific introns, exons, or splice junctions for the S. pombe genome. In agreement with previous results, the expression of ste11, which is required for mating in S. pombe, is low in the S2A mutant, whereas levels are restored in the double mutant S2A/S7A. In addition, our microarray analyses reveal numerous changes in expression of both individual genes, as well as positionally related clusters of genes. These results and their consequences will be discussed.
"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.
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.
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.