Role of hnRNP A/B proteins in telomere maintenance and telomerase activity

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, A2and 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 hTR 57-72 mediated 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. 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. VI Publications included in this thesisNo publications included. Contributions by others to the t...