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ROLE of Hnrnp A/B PROTEINS in TELOMERE MAINTENANCE and TELOMERASE ACTIVITY

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ROLE of Hnrnp A/B PROTEINS in TELOMERE MAINTENANCE and TELOMERASE ACTIVITY ROLE OF hnRNP A/B PROTEINS IN TELOMERE MAINTENANCE AND TELOMERASE ACTIVITY by EMAD AHMED SAIG (BSc and MSc) A thesis submitted for the degree of Master of Philosophy at The University of Queensland (2018) School of Chemistry and Molecular Bioscience Abstract Telomere shortening leads to eukaryotic cell senescence, whereas enhanced telomerase activity is associated with telomere expansion and growth of cancer cells. Several studies have suggested hnRNPs are important for telomere maintenance including their ability to bind telomeres, and telomerase. The hnRNPs A/B family are highly abundant multifunctional proteins that bind to single-stranded DNA and RNA and perform many roles in cellular regulation. In this study, affinity pull-down assays, biosensor and UV-cross-linking studies were used to confirm that recombinant hnRNPs A2 and A3, compared to recombinant hnRNP A1, specifically interact with single-stranded telomeric DNA repeat TTAGGG and hexamer repeat. Tandem RRMs are required for strong binding, which is little changed by exclusion of the glycine-rich domain (hnRNPs Δ GRD). Additionally, TRAP assays, that indicate telomerase activity, showed that hnRNPs A1, A2 and A3 bind to telomerase in cell extracts. It is not clear whether this association is mediated by binding a specific nucleotide region within telomerase RNA, or a protein component such as dyskerin, or both. In this study I have confirmed and refined the interaction site between the hnRNP A/B proteins and telomerase RNA (hTR). Serial UV-cross-linking RNA electrophoretic mobility shifting assays (REMSA) have shown that hnRNP A2 binds specifically with to a 17nt region within the RNA telomerase template (hTR). Deletion analysis of the hnRNP A/Bs revealed that these proteins bind preferentially to the hTR segment encompassing nucleotides hTR57- 72mediated mainly through the RRM1 sequence. Importantly, I found that hnRNP A2 can interact simultaneously with telomeric ssDNA repeats. Both hnRNP A1 and hnRNP A3 showed no binding to the short segments of hTR in this assay, yet they could form a complex with full-length hTR. This difference can be explained by the increased insolubility of A1 and A3. Nevertheless, as the template region itself appears not to associate in vitro with hnRNPs A1, A2 and A3, protein binding may be to the single-stranded nucleotides 56-61, which border the template. My results show that hnRNP A2 and the other hnRNP A/Bs bind to a specific region of hTR that lies immediately 3ʹ of the hTR57-72 template sequence. The binding of both II telomeric ssDNA and telomerase RNA by the hnRNP A/Bs could appropriately juxtapose these molecules for telomerase-mediated telomere extension. Moreover, hTR57-72 binds to hnRNP A2 in the presence of excess non-labeled Telo1, which indicates that the RNA-protein interaction is not displaced or diminished by high concentrations of Telo1 and points to independent binding sites on A2. Furthermore, I show that the second RNA-Recognition- Motif (RRM2) of hnRNP A2 binds preferentially to the ssDNA telomeric repeats, whereas the first RNA-Recognition-Motif (RRM1) specifically interacts with nucleotides hTR57-72 of telomerase RNA. This result supports a model where there is an intimate association between hnRNP A2, hTR57-72 and telomeric DNA and predicts an important role for the hnRNP A/Bs in facilitating telomer extension. Additionally, this study has indicated that both RRMs of hnRNP A2 are essential for either DNA-protein, or RNA-protein interactions, or both for simultaneous binding. In the chromatography experiments, RRM1 and RRM2 on their own were not able to mediate a simultaneous binding with both telomeric DNA and telomerase RNA, while short variant UP2 was successful to maintain this kind of binding feature. The results suggest that hnRNP A2 may help recruit telomerase to the ends of chromosomes. These hnRNP A/B family members are highly abundant in the cell nucleus, perform many roles in the cell and potentially influence telomere biogenesis. From these studies a model was proposed where the hnRNP A2 binding site is adjacent, or close to the template segment of hTR. The close proximity of the hnRNP A2 binding site to the template region on hTR and necessarily, to the end of the ssDNA 3’ overhang, supports the proposal that these proteins play an active role in the addition of new telomeric repeats for cellular maintenance. III Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. IV Publications during candidature Non Conference Abstracts: Oral Presentations Saig E. A., Rothnagel J., Smith R. Role of hnRNP A/B proteins in telomere maintenance and telomerase activity. Presented at the School of Chemistry and Molecular Biosciences 9th annual research student symposium, The University of Queensland, Australia. November 2013. Saig E. A., Rothnagel J., Smith R. Role of hnRNP A/B proteins in telomere maintenance and telomerase activity. Presented at the School of Medicine and Medical Science, Um Alqura University, KSA. January 2014. Saig E. A., Rothnagel J., Smith R. Role of hnRNP A/B proteins in telomere maintenance and telomerase activity. Presented at structural biology and biochemistry theme seminar, The University of Queensland, Australia, Brisbane. June 2014. Conference Abstracts: Poster Presentations Saig E. A., Rothnagel J., Smith R. Role of hnRNP A/B proteins in telomere maintenance and telomerase activity. Presented at School of Chemistry and Molecular Biosciences 8th annual research student symposium, The University of Queensland, Australia. November 2012 Saig E. A., Rothnagel J., Smith R. Role of hnRNP A/B proteins in telomere maintenance and telomerase activity. Presented at International postgraduate symposium in biomedical science, The University of Queensland, Australia. September 2012 Saig E. A., Rothnagel J., Smith R. Role of hnRNP A/B proteins in telomere maintenance and telomerase activity. Presented at the Telomere and DNA repair workshop, Sydney. October 2012 Saig E. A., Rothnagel J., Smith R. Role of hnRNP A/B proteins in telomere maintenance and telomerase activity. Presented at NanoBio Australia, The University of Queensland, Australia. July 2014 Saig E. A., Rothnagel J., Smith R. Role of hnRNP A/B proteins in telomere maintenance and telomerase activity. Presented at ComBio2014 conference, Canberra, Australia. September- October 2014. V Publications included in this thesis No publications included. Contributions by others to the thesis Supervisors, research design advice, critical review of thesis: A/Prof. Joe Rothnagel Prof. Ross Smith A/Prof. Peter Dodd Laboratory experiment advice: Dr. Amanda Nouwens Dr. Sergio Sara Statement of parts of the thesis submitted to qualify for the award of another degree None. VI Acknowledgements I have done this project not only for marks but to also to increase my knowledge. The studies in this thesis were performed in the School of Chemistry and Molecular Biosciences (SCMB) at the University of Queensland under the supervision of Assoc. Prof. Joe Rothnagel and Prof. Ross Smith, with financial assistance provided by SMMS and the UQ Graduate School. I would like to express my special thanks and gratitude to my supervisor, Prof. Ross Smith, as well as to my principal supervisor, Assoc.Prof. Joe Rothnagel, who gave me the golden opportunity to do this wonderful project on the topic (Role of hnRNP A/B on telomere maintenance and telomerase activity) not only in their laboratory, but also for mentoring me through those many turbulent years, which also helped me in doing a lot of research and I came to know about so many new things. I am really grateful to them. Many thanks are extended to the numerous people like Melissa Brown and lab members, who in some way have contributed to this thesis, either by directly sharing reagents or providing expert advice during many troubleshooting sessions that were had. Thanks to Dr. Amanda Nouwens who was responsible for not only technical advice, but also assistance on the thesis structure. Thanks to Assoc. Prof. Peter Dodd and his group members for helping me by providing reagents and technical advice on the molecular biology techniques employed. To all members of the Smith, Rothnagel, Dodd and Brown laboratories, past and present, for the daily scenes of humor that provided the much-needed escape from the daily routine of the bench. Nevertheless, I am also eager to express my special recognition and gratitude to my parents, my wife Dr.
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