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"RNA POLYMERASE II-MEDIATED TRANSCRIPTION IN TRYPANOSOMA BRUCEI"

by
Mahrukh M. Banday
Microbiology and Molecular Genetics Program
M.V.Sc. 2005,Punjab Agricultural University, India
DVM. 2002 Kerala Agricultural University, India


Thesis Advisor: Vivian Bellofatto, Ph.D.
Professor
Department of Microbiology and Molecular Genetics

Thursday, March 21, 2013
10:00 A.M., ICPH-Auditorium


Abstract

RNA polymerase II carboxyl terminal domain (RNAP II CTD) is essential for growth and viability of all well studied eukaryotes. A typical RNAP II CTD in yeast and mammals contains tandem repeats of heptapeptide YSPTSPS, which orchestrates essential transcriptional and co-transcriptional functions associated with cellular mRNA production. During transcription the heptapeptide repeats undergo reversible phosphorylation which generates a phospho-CTD code that regulates diverse processes including transcription initiation, elongation, capping, splicing, and polyadenylation. However, Trypanosoma brucei, a primitive parasitic protozoan that causes human African trypanosomiasis, lacks these tandem repeats in its RNAP II CTD, swapping them for a non-repeated sequence termed as the pseudo-CTD. We have established that the pseudo-CTD is essential for parasite viability and contains a dynamic set of phospho-serine residues. Since trypanosomes diverged early during evolution, they maintain life processes that are unique. For example, protein coding genes are transcribed as polycistronic pre-mRNAs that are resolved into individual mature mRNAs by a trans-splicing reaction that adds a cap4 containing 39-nucleotide spliced leader RNA. Our hypothesis is that this unique gene expression strategy requires a parasite-specific pseudo-CTD code, and that by cracking this code, we will understand the involvement of the pseudo-CTD in basic transcriptional and co-transcriptional biology of the parasite. We have established a versatile and powerful experimental setup in which cells are depleted of endogenous RNAP II (by tetracycline-regulated RNAi mediated knockdown) and simultaneously supplied with an ectopic, tagged copy of RNAi-resistant RNAP II. We have confirmed that the ectopic-tagged RNAP II is incorporated into a transcription-competent RNAP II holoenzyme. Using our conditionally expressed RNAP II CTD system, we have tested the function of mutant RNAP II proteins with several different serine-to-alanine substitutions. Our study shows that specific serines are indeed essential for trypanosome viability. However, the set of mutant pseudo-CTDs tested did not alter the m7GpppN-cap formation on the splice leader RNA, as assayed by RNA immunoprecipitation and primer extension. Conversely, some mutants showed a distinct affect on cap4 modifications, a process specific to the generation of hypermethylated cap structure found on the splice leader RNAs, which are required for the translation of all trypanosome mRNAs. Similarly, nascent RNA transcription assays demonstrated that specific RNAP II CTD mutants were defective in transcription elongation. We are further exploring validating this global pre-mRNA transcription defect using RNA-seq and high throughput sequencing of trypanosome RNA


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