Maki Inada

Maki Inada

Associate Professor, Department of Biology
Faculty, School of Humanities and Sciences
Faculty, Biochemistry

Student Presentations: recent

Ben Jung (Biochemistry '16). Poster presentation.” Investigating the regulation of splicing of RPS30 paralogs that arose form genome duplication in S. cerevisiae”. Northeast Regional Meeting of American Chemical Society. Ithaca College. June 2015. Ben also won a poster award.

Abstract:
Regulation of gene expression is a vital process in life, from controlling cell’s structure and function, to governing development, as well as responding to the environment. Gene expression refers to the flow of genetic information stored in DNA, which is transcribed into mRNA, then translated into functional proteins. Eukaryotic mRNAs have non-protein coding sequences (introns) that need to be removed. This is the process called RNA splicing, executed by the spliceosome. RNA splicing is an important regulatory step to ensure proper gene expression.

We use Saccharomyces cerevisiae (budding yeast) as our model organism to study how RNA splicing is regulated. It serves as a great model since yeast has similar splicing machineries with higher order eukaryotes. Interestingly in higher eukaryotes, there are families of proteins that regulate splicing. However, yeast is not thought to have these proteins, hence is thought to lack splicing regulation. Nonetheless, previous experiments1 have shown evidence of selective splicing behaviors in yeast with gene paralogs, or duplicated genes. These paralog genes in the S. cerevisiae genome have arisen from a genome duplication event 100 million years ago.  These paralog genes have nearly identical exon sequences, but it has been observed that some of these genes are spliced differently under different conditions.

In order to study how splicing could be differently regulated in yeast despite lacking known homologous regulatory proteins, my project characterizes how splicing is regulated through quantifying the difference of splicing under various conditions. Based on findings from previous experiments, my project focuses on splicing of a paralog gene pair Ribosomal Protein S30 (RPS30A&RPS30B) in a particular spliceosomal component mutant strain (prp16-302). In addition, I am examining the splicing efficiency of each of these paralog genes, and characterizing the splicing behaviors in time course experiments after exposure to stress conditions such as cold temperature. My results reveal that RPS30A and RPS30B are spliced differently both in standard growth conditions and in response to cold temperature.

This work will help expand our current understanding of how splicing is regulated in eukaryotes. Understanding how RNA splicing is regulated can elucidate how abnormal splicing could lead to genetic disorders and cancer.

Robert Nichols (Biochemistry '14)Poster presentation. "Linking the C-Terminal Domain Code of RNA Polymerase II to Modulating Chromatin States in Schizosaccharomyces pombe" , RNA Society Meeting.  Madison, WI, May 2015. Co-authored with Ruby Benn and Reyal Hoxie (Biochemistry ’15),  Jeffrey A. Pleiss (Cornell University),  Beate Schwer (Weill Cornell Medical College), Maki Inada.
 

ABSTRACT:

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 RNAPII 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 influence when processing events such as chromatin remodeling 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. 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, nor do we see any large changes in global splicing efficiency. Interestingly, analyses of our microarray data reveals an upregulation of positionally related clusters of genes, specifically at both ends of chromosomes one and two, but not chromosome three. Further quantitative PCR analysis of genes in these subtelomeric regions confirm a significant upregulation of gene expression in these regions spanning approximately 50-100kb. 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 subtelomeric regions of S. pombe.

 

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