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The Effect of Galactose on the Expression of Genes Regulated by Rrp6p

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The Effect of Galactose on the Expression of Genes Regulated by Rrp6p The Review: A Journal of Undergraduate Student Research Volume 18 Article 1 2017 The Effect of Galactose on the Expression of Genes Regulated by Rrp6p Mary Megan Pelkowski Saint John Fisher College, [email protected] Kevin Callahan Saint John Fisher College, [email protected] Follow this and additional works at: https://fisherpub.sjfc.edu/ur Part of the Biochemistry Commons, and the Molecular Genetics Commons How has open access to Fisher Digital Publications benefited ou?y Recommended Citation Pelkowski, Mary Megan and Callahan, Kevin. "The Effect of Galactose on the Expression of Genes Regulated by Rrp6p." The Review: A Journal of Undergraduate Student Research 18 (2017): -. Web. [date of access]. <https://fisherpub.sjfc.edu/ur/vol18/iss1/1>. This document is posted at https://fisherpub.sjfc.edu/ur/vol18/iss1/1 and is brought to you for free and open access by Fisher Digital Publications at St. John Fisher College. For more information, please contact [email protected]. The Effect of Galactose on the Expression of Genes Regulated by Rrp6p Abstract Gene expression is a multi-faceted phenomenon, governed not only by the sequence of nucleotides, but also by the extent to which a particular gene gets transcribed, how the transcript is processed, and whether or not the transcript ever makes it out of the nucleus. Rrp6p is a 5’-3’ exonuclease that can function independently and as part of the nuclear exosome in Saccharomyces cerevisiae (Portin, 2014). It degrades various types of aberrant RNA species including small nuclear RNAs, small nucleolar RNAs, telomerase RNA, unspliced RNAs, and RNAs that have not been properly packaged for export (Butler & Mitchell, 2010). This exosome mediated degradation is important as the accumulation of unprocessed mRNA transcripts can be harmful to the cell. These experiments sought to quantify changes in RNA levels in rrp6-Δ strains grown in glucose versus those grown in galactose compared to wild type strains grown in each of the carbon sources. The goal was to determine if there was an interplay between the genes regulated by Rrp6p and the genes involved in the switch from glucose metabolism to galactose metabolism. Based on the data, it appears that the absence of Rrp6p increases transcription in glucose while, in galactose, the absence of Rrp6p increases transcription to a significantly lesser degree than in glucose and in some cases decreases transcription. This article is available in The Review: A Journal of Undergraduate Student Research: https://fisherpub.sjfc.edu/ur/ vol18/iss1/1 Pelkowski and Callahan: Effect of Galactose on the Expression of Genes Regulated by Rrp6p The Effect of Galactose on the Expression of Genes Regulated by Rrp6p Mary Pelkowski and Kevin Callahan PhD ABSTRACT Gene expression is a multi-faceted phenomenon, governed not only by the sequence of nucleotides, but also by the extent to which a particular gene gets transcribed, how the transcript is processed, and whether or not the transcript ever makes it out of the nucleus. Rrp6p is a 5’-3’ exonuclease that can function independently and as part of the nuclear exosome in Saccharomyces cerevisiae (Portin, 2014). It degrades various types of aberrant RNA species including small nuclear RNAs, small nucleolar RNAs, telomerase RNA, unspliced RNAs, and RNAs that have not been properly packaged for export (Butler & Mitchell, 2010). This exosome mediated degradation is important as the accumulation of unprocessed mRNA transcripts can be harmful to the cell. These experiments sought to quantify changes in RNA levels in rrp6-∆ strains grown in glucose versus those grown in galactose compared to wild type strains grown in each of the carbon sources. The goal was to determine if there was an interplay between the genes regulated by Rrp6p and the genes involved in the switch from glucose metabolism to galactose metabolism. Based on the data, it appears that the absence of Rrp6p increases transcription in glucose while, in galactose, the absence of Rrp6p increases transcription to a significantly lesser degree than in glucose and in some cases decreases transcription. Introduction An area of genetics that has not been elucidated to quite as great of an extent yet is that of DNA was first discovered by Friedrich epigenetics, the change in gene expression Miescher in human leukocytes in 1869. without the change in DNA sequence. There However, the importance of DNA was not are many factors responsible for whether or not known until 1944 when the work of Collin a certain gene is expressed and to what extent a MacLeod, Oswald Avery, and Maclyn McCarty gene is expressed that go beyond the simplistic revealed that DNA was the transformative Central Dogma of biology, such as post- agent. This was accomplished by isolating and transcriptional and post-translational genetic purifying the cell-free substance capable of regulation. Rrp6p, found in the nucleus of causing the genetic transformation demonstrated Saccharomyces cerevisiae, is an example of a in Frederick Griffith’s well-known work with protein that mediates post-transcriptional pneumococcus and showing that it was in fact genetic regulation. There is evidence that this DNA (Portin, 2014). Much progress has been protein may also be present in the cytoplasm of made since then. It is well understood that T. brucei, A. thaliana and humans (Butler & DNA is replicated during cell division, that Mitchell, 2010). Rrp6p functions in association RNA is transcribed from DNA, and that mRNA with the nuclear exosome and plays a key role is translated into amino acids specified by the in the regulation of RNA levels in two ways: it triplet codons of the mRNA strand. This is involved in transcript elongation and proper translation of amino acids yields the proteins packaging of RNA transcripts for export from that are ultimately responsible for the expression the nucleus, and it degrades aberrant RNA of information encoded by the DNA. The transcripts (Butler & Mitchell, 2010; Kilchert & sequencing of the entire human genome in 2004 Vailjeva, 2013). This study focuses on the latter marked a turning point in the attempt to of these two functions. Rrp6p, together with the understand the genetic code and generated many exosome core, Exo9, and the ribonuclease Dis3, more questions regarding the role of DNA (Portin, 2014). Published by Fisher Digital Publications, 2017 1 The Review: A Journal of Undergraduate Student Research, Vol. 18 [2017], Art. 1 forms the nuclear exosome, Exo11 (Butler & deletion of the RRP6 gene would result in Mitchell, 2010). increased levels of mRNA corresponding to the genes normally regulated by Rrp6p. This study Types of aberrant RNA targeted by the exosome investigated the effects on mRNA levels of include small nuclear RNAs (snRNAs), small deletion of the RRP6 gene in S. cerevisiae as nucleolar RNAs (snoRNAs), telomerase RNA, well as the effects of an alternate carbon source unspliced RNAs, and RNAs that have not been on mRNA levels in an rrp6- strain properly packaged for export. This exosome compared to wild type. Glucose is the preferred mediated degradation is important as the carbon source of S. cerevisiae and is accumulation of unprocessed mRNA transcripts metabolized to pyruvate and ATP via the can be harmful to the cell (Figure 1) (Kilchert & process of glycolysis. However, in the absence Vailjeva, 2013). Rrp6p can also act of glucose, other carbon sources may be utilized independently of the exosome complex and (Demir & Kurnaz, 2006). This experiment used serves to regulate concentrations of specific galactose as an alternative carbon source. mRNAs, thus contributing to feedback control Galactose undergoes a series of enzymatic and regulation of cell cycle events. For reactions that eventually convert it to glucose-6- instance, Rrp6p works with the polyadenylating phospate (G6P) which can then enter glycolysis TRAMP complex to degrade histone mRNAs at to yield ATP. Both galactose-induced the end of the S-phase of the cell cycle to ensure upregulation and repression induced by the that histone synthesis does not continue beyond absence of glucose are necessary for the S-phase and is involved in a pathway that expression of galactose-metabolizing enzymes degrades mRNA transcripts that are exported (Demire & Kurnaz, 2006). This study sought to out of the nucleus at slow rates (Butler & determine if the absence of Rrp6p combined Mitchell, 2010). with the introduction of galactose as the carbon As Rrp6p is involved in the degradation of source elicits novel changes in mRNA levels mRNA transcripts, it would be expected that that are not elicited by either factor individually. pre-mRNA pre snRNA/snoRNA pre rRNA 5.8S ITS2 25S 7mGppp 7mGppp Splicing Cleavage in ITS2 7mGppp 7mGppp Cleavage 7S 25S Rrp43 7mGppp MET Rrp46 Csl4 Hypomodified tRNAi pre-sn/snoRNAs Polyadenylation Rrp40 Mtr3 Rrp42 pre-rRNAs 7m Gppp A -8050 Rrp45 Rrp4 Rrp41 Trf4 Dis3 Air2 Mtr4 Rrp6 Transport nucleotides AAAAAAAAAAAn 7mGppp 5.8S Figure 1. Functions of the Nuclear Exosome. For pre-mRNAs the diagram implies that defects occurring at the indicated steps result in degradation of the transcript by the nuclear exosome. For pre-sn/snoRNA and 7S pre- rRNA, the core exosome and Rrp6p catalyzes 3’ end formation in a two-step reaction. https://fisherpub.sjfc.edu/ur/vol18/iss1/1 2 Pelkowski and Callahan: Effect of Galactose on the Expression of Genes Regulated by Rrp6p Materials and Methods Real time quantitative PCR Growth of yeast cultures Real time quantitative PCR was carried out using SYBR Green mix (Thermo Fisher Two different strains of Saccharomyces Scientific), forward and reverse primers for cerevisiae, wild type (WT) and an rrp6-∆ the genes of interest, and 4.4 µL cDNA for a strain, were each cultured in YPD at 37 °C total reaction volume of 10 µL. Primers and YP-Gal at 37 °C. Cells were collected included sequences for Actin, Nrd1, Nab2, at an optical density at 600 nm between 0.6 SnR45, and Cut652.
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