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Discovery of the Essential Roles of CRK9 in Gene Expression and Of University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 5-27-2015 Lessons from Trypanosome TFIIH: Discovery of the Essential Roles of CRK9 in Gene Expression and of the Divergent Helicase Paralog XPB-R in Nucleotide Excision Repair Nitika Badjatia University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Badjatia, Nitika, "Lessons from Trypanosome TFIIH: Discovery of the Essential Roles of CRK9 in Gene Expression and of the Divergent Helicase Paralog XPB-R in Nucleotide Excision Repair" (2015). Doctoral Dissertations. 866. https://opencommons.uconn.edu/dissertations/866 Lessons from Trypanosome TFIIH: Discovery of the Essential Roles of CRK9 in Gene Expression and of the Divergent Helicase Paralog XPB-R in Nucleotide Excision Repair Nitika Badjatia University of Connecticut, [2015] Eukaryotic TFIIH consists of a core of seven subunits, including the DNA helicase Xeroderma Pigmentosum B (XPB), and a cyclin-dependent kinase (CDK)-activating complex (CAK) that contains CDK7. XPB is crucial for DNA unwinding during transcription and nucleotide excision repair (NER) while the CAK complex phosphorylates RNA polymerase II (RNAPII) carboxy terminal domain (CTD), enabling the enzyme’s promoter clearance. Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are lethal human parasites, belonging to the early-diverged order Kinetoplastida. They transcribe genes polycistronically and require spliced leader (SL) trans splicing for the maturation of mRNAs from polycistronic precursors. SL RNA gene (SLRNA) transcription by RNAPII depends on trypanosome TFIIH complex which contains orthologs of all core subunits but lacks the CAK complex. Despite this, the trypanosome CTD is phosphorylated and essential for SLRNA transcription. Gene silencing of CDC2-related kinase 9 (CRK9) revealed its importance for parasite survival and CTD phosphorylation. Interestingly, this loss of phosphorylation did not cause a specific RNAPII transcription defect. Instead, CRK9 silencing blocked trans splicing and caused hypomethylation of SL RNA’s extensively modified cap. Sedimentation analysis of tandem affinity purified CRK9 revealed a putative tripartite complex including a novel L-type cyclin (CYC12), and a CRK9-associated protein (CRK9AP). Silencing CYC12 or CRK9AP recapitulated the defects observed upon CRK9 silencing, confirming that they functionally partner with CRK9 in vivo. CRK9AP depletion caused a rapid co-loss of CRK9 and CYC12, suggesting its role in complex assembly. This project identified CRK9 as crucial for parasite- i Nitika Badjatia – University of Connecticut, [2015] specific gene expression. As a CDK, CRK9 is a promising drug target against trypanosomes which was validated in a mouse model. Another feature related to trypanosome TFIIH is the presence of two divergent paralogs of XPB in kinetoplastid genomes while only the larger XPB paralog consistently co-purified with TFIIH. Gene knockout of the second paralog, termed XPB-R showed that XPB-R is specialized in NER but dispensable for transcription. While XPB-R does not assemble into a TFIIH complex, reciprocal co-immunoprecipitations revealed an interaction with the p52 ortholog, a TFIIH component and known regulator of XPB in other systems, indicating that trypanosomes possess a TFIIH whose function is restricted to transcription and a XPB-R/p52 repair complex. ii Lessons from Trypanosome TFIIH: Discovery of the Essential Roles of CRK9 in Gene Expression and of the Divergent Helicase Paralog XPB-R in Nucleotide Excision Repair Nitika Badjatia B.Sc., Devi Ahilya University, 2006 M.Sc., Devi Ahilya University, 2008 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut [2015] iii Copyright by Nitika Badjatia [2015] iv APPROVAL PAGE Doctor of Philosophy Dissertation Lessons from trypanosome TFIIH: Discovery of the Essential Roles of CRK9 in Gene Expression and of the Divergent Helicase Paralog XPB-R in Nucleotide Excision Repair Presented by Nitika Badjatia, B.Sc., M.Sc. Major Advisor ___________________________________________________________________ Arthur Günzl, Ph.D. Associate Advisor ___________________________________________________________________ Asis Das, Ph.D. Associate Advisor ___________________________________________________________________ Blanka Rogina, Ph.D. Associate Advisor ___________________________________________________________________ Bruce Mayer, Ph.D. Associate Advisor ___________________________________________________________________ Gordon Carmichael University of Connecticut [2015] v Table of Contents Table of Contents…………………………………………………………………………...…………...vi Publications and Contributions to the Thesis Projects…………………………...…………….…...vii List of Tables…………………………………………………………………………………………...…ix List of Figures……………………………………………………………………………………………..x Chapter I. Introduction………………………………………………………………………..................1 Chapter II. Trypanosoma brucei harbors a divergent XPB helicase paralog that is specialized in nucleotide excision repair and conserved among kinetoplastid organism……………………………………………......................34 Chapter III. Trypanosome cdc2-related kinase 9 controls spliced leader RNA cap4 methylation and phosphorylation of the RNA polymerase II subunit RPB1……………………………68 Chapter IV. The cyclin-dependent kinase CRK9, a new L-type cyclin and a CRK9-associated protein cooperate in facilitating spliced leader trans splicing in Trypanosoma brucei……………………………..…100 Chapter V. The spliceosomal PRP19 complex of trypanosomes………………………………...131 Chapter VI. Discussion and future directions……………………………………………………….167 References……………………………………………………………………………………………..182 Attachment 1. Supporting information related to chapter II….…………………...……..………..211 2. Supporting information related to chapter III ………………….....……….………231 3. Supporting information related to chapter IV………………………………………236 4. Supporting information related to chapter V…………… …………………………249 5. Review: Günzl A, Kirkham JK, Nguyen TN, Badjatia N, Park SH. Mono-allelic VSG expression by RNA polymerase I in Trypanosoma brucei: expression site control from both ends? Gene. 2015; 556:68-73………………………….…257 vi Publications and Contributions to the Thesis Projects 1. Chapter II: In this study, I functionally characterized the smaller XPB (XPB-R) paralog in Trypanosoma brucei. I performed all the experiments described in this study except the generation of phylogenetic trees with XPB sequences from 33 species. Dr. Ju Huck Lee generated the clonal cell line that exclusively expressed C-terminally PTP- tagged XPB-R and assisted me with in vitro transcription analysis of either mock or XPB-R depleted extracts. This work was published in Badjatia N, Nguyen TN, Lee JH, Günzl A. Trypanosoma brucei harbours a divergent XPB helicase paralogue that is specialized in nucleotide excision repair and conserved among kinetoplastid organisms. Mol Microbiol 90 (2013), 1293-1308. 2. Chapter III: To identify trypanosome cyclin dependent kinase that phosphorylates the carboxy terminal domain of trypanosome RPB1, Dr. Lee generated clonal RNAi cell lines for expression silencing of cdc2-related kinase (CRK)1, CRK3, CRK7 and CRK9 and performed initial RNA analyses after RNAi experiments with each of these cell lines. Thereafter, I confirmed the phenotypes observed upon silencing of CRK9 in independent experiments and performed qRT-PCR analysis. I also analyzed nascent RNA, methylation of cap4 and m7G cap in CRK9 silenced cells. The loss of RPB1 phosphorylation upon CRK9 silencing was studied by Dr. Daniela Ambrósio. To ensure that the phenotypes observed upon CRK9 silencing are not due to an off-target effect, I generated new RNAi cell lines in which we targeted 3′-UTR of the CRK9 mRNA and rescue cell lines that express wild type or T533A mutated CRK9 which are resistant to the RNAi response. This work was published in Badjatia N, Ambrósio DL, Lee JH, Günzl A. Trypanosome cdc2- vii related kinase 9 controls spliced leader RNA cap4 methylation and phosphorylation of RNA polymerase II subunit RPB1.Mol Cell Biol 33 (2013), 1965-1975. 3. Chapter IV: To biochemically characterize CRK9 enzyme complex, I generated a cell line that exclusively expressed C-terminally PTP tagged CRK9. Thereafter, Dr. Sung Hee Park and I performed PTP purifications of CRK9 in two independent experiments. The final eluates of CRK9 PTP purification were independently analyzed by the Keck Facility at Yale University. Dr. Daniela Ambrósio performed sedimentation analysis and in vitro kinase assays with PTP purified CRK9 complex. Dr. Park did mice infection studies with bloodstream form CRK9 UTR RNAi and CRK9-HA rescue cell lines. Justin Kirkham helped me in raising polyclonal immune serum against CRK9 and CRK9AP. I generated all the cell lines used in this project. I also performed RNA and protein analysis subsequent to expression silencing of CYC12 and CRK9AP in procyclic and bloodstream form T. brucei. Lastly, I generated phylogenetic trees with the sequence alignment of cyclin sequences from model organisms and kinetoplastid CYC12 sequences. A manuscript is under preparation for this study. 4. Chapter V: In this study, Dr. Daniela Ambrósio characterized the trypanosome PRP19 complex. She performed all the experiments in this study with the exception of immunofluorescence assay and primer extension analysis of cap4 methylation
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