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The regulation of the transcription of the IMD2 gene in Saccharomyces cerevisiae is unusual because of the dependence on the concentration of intracellular guanine nucleotide pools and the choice between two alternative transcription initiation sites. Start site selection results in either a non-coding transcript terminating upstream of the start codon or a full-length IMD2 mRNA. Our working model is that a specific phosphorylation event on RNA polymerase II mediated by the Ctk1 kinase during elongation has a regulatory role on the transcription of IMD2. We will also consider the effect on regulated IMD2 start site switching of Sub1 as an anti-termination factor, which is present on yeast promoters and along the length of transcribed genes. Here we construct a plasmid with a NAT1 dominant drug resistance marker and a disruption-deletion cassette with flanking 5’RRP6 and 3’RRP6 regions in order to knock out RRP6 in ∆ctk1 and ∆sub1 strains and detect the short non-coding RNA. The absence of RRP6 function enables the detection of the short upstream RNA involved in IMD2 regulation. Northern analyses on the mutant strains, after inducing IMD2 transcription with mycophenolic acid or repressing it with guanine, will allow us to evaluate the effect of Ctk1 and Sub1 mutations on the transcription of IMD2 from the two start sites. This information will be important in understanding the regulation of the enzyme IMP dehydrogenase and may ultimately translate into clinical medicine.
The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) consists of a repeated heptameric sequence (Y1S2P3T4S5P6S7). Serine 2 is phosphorylated during elongation by the Ctk1 kinase and is required for the recruitment of polyadenylation factors needed for cotranscriptional 3’ end processing. 1 Ctk1 phosphorylation of Ser2 is thought to involve surpassing an elongation block and converting RNAP II into an elongating form. 2
Sub1 is an anti-termination factor found on yeast promoters and along the length of transcribed genes. It has been shown to interact with a polyadenylation factor and assist in elongation by regulating enzymes, kinases and phosphatases, which modify the CTD of RNAP II. 3
The IMD genes in yeast encode IMP dehydrogenase (IMPDH), which is a rate limiting enzyme involved in the de novo synthesis of guanine nucleotides and in the biosynthetic pathway of GTP. Transcription of IMD2 is regulated in yeast in response to the concentration of intracellular guanine nucleotides (see Fig. 1). 4 The Reines lab and others have characterized the regulation of the transcription of IMD2 under two intracellular conditions:
In guanine-replete conditions, an upstream transcription start site is used to create only short, non-coding transcripts. This transcription regulation mechanism leads to a non-coding transcript encoded upstream of the productive transcription initiation site and terminated at the intergenic-IMD2 terminator (IT).
Under guanine-depleted conditions, such as during exposure to the IMPDH-inhibitor mycophenolic acid (MPA), a distinct downstream adenine start site is used to create IMD2 mRNA.
To evaluate the effect of Ctk1 and Sub1 mutations on IMD2 start site selection and transcription levels, we will conduct northern analyses on the mutant strains after inducing IMD2 transcription with mycophenolic acid or repressing it with guanine.
To detect the short, non-coding RNA transcript of the region upstream of IMD2, we knocked out RRP6 in ∆ctk1 and ∆sub1 strains. The strains already included kanamycin (kanMX) drug resistance, a commonly used marker for generating knockout strains, so we transformed the strains with a newly constructed plasmid containing a NAT1 dominant drug resistance marker and a disruption-deletion cassette with flanking 5’RRP6 and 3’RRP6 regions. After inactivating RRP6, the RNA degradation system known as the nuclear exosome is no longer functional. 5, 6
Drug resistance genes from bacteria and fungi have been used in Saccharomyces cerevisiae in place of auxotrophic mutations as selectable markers in disruption-deletion cassettes. 7
Hypothesis
Loss of Ctk1 and Sub1 from yeast will result in dysregulation of the initiation site shift that regulates IMD2.
Successful construction of a plamid, pBS-NAT-RRP6, with a 5’RRP6-NAT1-3’RRP6 linear cassette
Transformation of ∆ctk1 and ∆sub1 yeast strains with pBS-NAT-RRP6 allowed for the creation of multiple deletions, ∆ctk1 rrp6::NAT and ∆sub1 rrp6::NAT, and the cognate isogenic wild-type for use as a control strain
Treat three new yeast strains with mycophenolic acid and guanine nucleotides
Isolate RNA
Run RNA on an agarose gel, blot to a filter, and probe with 32P-labeled IMD2 DNA
Conduct primer extension analyses to map 5’ ends of the short upstream RNA and full length IMD2 mRNA
Acknowledgments: We thank M. Harley Jenks and Thomas W. O’Rourke for expert technical assistance. This material is based upon work supported by the Howard Hughes Medical Institute under Grant No. 52005873 and by the National Institutes of Health under Grant No. GM46331.
1 Ahn, S. H., M. Kim, and S. Buratowski. Phosphorylation of Serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3' end processing. Mol. Cell 13 (2004): 67-76.
2 Egloff, S., and S. Murphy. Cracking the RNA polymerase II CTD code. Trends in Genetics 24, no. 6 (2008): 280-88.
3 Calvo, O., and J. L. Manley. The transcriptional coactivator PC4/Sub1 has multiple functions in RNA polymerase II transcription. EMBO 24 (2005): 1009-1020.
4 Kopcewicz, K. A., T. W. O' Rourke, and D. Reines. Metabolic regulation of Imd2 transcription and an unusual DNA element that generates short transcripts. Mol. Cell. Biol. 27, no. 8 (2007): 2821-29.
5 Davis, C. A., and M. Ares, Jr. Accumulation of unstable promoter-associated transcripts upon loss of the nuclear exosome subunit Rrp6p in Saccharomyces cerevisiae. PNAS 103, no. 9 (2006): 3262-67.
6 Jenks, M. H., T. W. O' Rourke, and D. Reines. Properties of an intergenic terminator and start site switching that regulates Imd2 transcription in yeast. Mol. Cell. Biol. 28, no. 12 (2008).
7 Goldstein, A. L., and J. H. McCusker. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces Cerevisiae. Yeast 15 (1999): 1541-53.
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