<p>The unique C-terminal domain (CTD) of RNA polymerase II (RNAPII), composed of tandem heptad repeats of the consensus sequence YSPTSPS, is subject to differential phosphorylation throughout the transcription cycle. Several RNA processing factors have been shown to bind the appropriately phosphorylated CTD, and this facilitates their localization to nascent pre-mRNA during transcription. In <italic>Saccharomyces cerevisiae</italic>, the mRNA export protein Yra1 (ALY/REF in metazoa) has been shown to cotranscriptionally associate with mRNA and is thought to deliver it to the nuclear pore complex for export to the cytoplasm. Based on a previous proteomics screen, I hypothesized that Yra1 is a <italic>bona fide</italic> phosphoCTD associated protein (PCAP) and that this interaction is responsible for the pattern of Yra1 cotranscriptional association observed <italic>in vivo</italic>. Using <italic>in vitro</italic> binding assays, I show that Yra1 directly binds the hyperphosphorylated form of the CTD characteristic of elongating RNAPII. Using truncations of Yra1, I determined that its phosphoCTD-interacting domain (PCID) resides in the segment comprising amino acids 18-184, which, interestingly, also contains the RNA Recognition Motif (RRM) (residues 77-184). Using UV crosslinking, I found that the RRM alone can bind RNA, although a larger protein segment, extending to the C-terminus (aa 77-226), displays stronger RNA binding activity. Even though the RRM is implicated in both RNA and CTD binding, certain RRM point mutations separate these two functions: thus, mutations that produce defects in RNA binding do not affect CTD binding. Both functions are important <italic>in vivo</italic>, in that RNA binding-defective or CTD binding-defective versions of Yra1 engender growth and mRNA export defects. I also report the construction and characterization of a useful new temperature sensitive <italic>YRA1</italic> allele (<italic>R107AF126A</italic>). Finally, using chromatin immunoprecipitation, I demonstrate that removing the N-terminal 76 amino acids of Yra1 (all of the PCID up to the RRM) results in a 10-fold decrease in Yra1 recruitment to genes during elongation. These results indicate that the PCTD is likely involved directly in cotranscriptional recruitment of Yra1 to active genes.</p> / Dissertation

Magister Scientiae (Medical Bioscience) - MSc(MBS) / The blood brain barrier (BBB) is a signaling interface between the blood and the central nervous system (CNS), which prohibits the entry of harmful blood-borne substances into the brain micro-environment, thus maintaining brain homeostasis. The crucial role of the BBB is protecting the CNS, which may adversely be affected by alcohol. The central component of the BBB, endothelial cells (ECs), regulates BBB transport by regulating the permeability both transcellularly and through their paracellular junctions, by structures called tight junctions (TJs) that are composed of proteins. The aim of this study was to investigate the in vitro effects of ethanol (EtOH) and fermented rooibos (Rf) on a monolayer of bEnd5 mouse brain ECs, by determining the effects of EtOH and Rf on bEnd5 (i) cell viability (ii) cell proliferation (iii) rate of cell division (iv) cell toxicity (v) claudin-5 transcription (vi) permeability across a monolayer of bEnd5 ECs and (vii) morphology, for a selected experimental timeline of 24, 48, 72, and 96hrs. We then investigated if the simultaneous exposure of Rf and EtOH could reverse or alleviate the EtOHinduced effects on the bEnd5 ECs. EtOH metabolism induces oxidative stress and results in a range of adverse physiological effects. Aspathalus linearis (rooibos) contains many phenolic compounds, of which the main antioxidant activity is attributed to aspalathin. Our underlining hypothesis is that the antioxidants in an aqueous rooibos extract may therefore protect against the potential oxidant damaging effects of alcohol on the BBB. Cells were exposed for 24hrs to selected concentrations of EtOH (25mM and 100mM), a concentration of Rf containing equivalent of 1.9nM aspalathin, and the combinations of EtOH and Rf. Cell viability and cell toxicity was determined, while cell proliferation and rate of cell division was estimated using the trypan blue exclusion assay. Real time quantitative PCR was implemented to quantify claudin-5 transcription, normalized against housekeeping genes, GAPDH and HPRT. Transepithelial electrical resistance (TEER) was measured using the Ohm Millicell-electrical resistance system, while bEnd5 monolayer morphology was analysed using the Zeiss scanning electron microscope. Both concentrations of EtOH led to an overall decrease in cell viability, and a decreased number of live cells across 72hrs. Consistent with this, EtOH resulted in increased cell toxicity across the 96hr experimental timeframe and a diminished rate of cell division. The transcription of claudin-5 in bEnd5 ECs exposed to 25mM and 100mM EtOH varied dramatically across the 96hr timeframe. While 25mM EtOH resulted in an overall decrease in TEER, cells exposed to 100mM EtOH only decreased TEER between 48 and 96hrs. Morphologically, both concentrations of EtOH led to compromised paracellular spaces as endorsed by high definition SEM analysis. The administration of Rf on its own resulted in an initial decrease in viability, followed by recovery between 72 and 96hrs. Exposure to Rf diminished live cell numbers at 72 and 96hrs, accompanied by a compromised rate of cell division and an overall increase in cell toxicity. In addition, Rf down-regulated claudin-5 transcription across the course of the experiment, particularly between 24 and 48hrs. In alignment with this, Rf also led to an increase in BBB permeability from 24 to 96hrs. However, SEM studies were not able to discriminate any differences between control and Rf treated cells. Our study showed that the BBB could be protected against the adverse effects of EtOH, and this at the plasma concentration induced by 500ml’s of Rooibos tea. The simultaneous exposure of Rf and EtOH was able to negate the effects of EtOH on cell viability, cell proliferation, and cell toxicity but exacerbated the effects of EtOH on claudin-5 transcription and paracellular permeability. Morphologically, co-exposure with Rf only reversed the effects of 25mM EtOH while exacerbating the effects of 100mM EtOH at 96hrs. In conclusion, EtOH was shown to be detrimental to the integrity of bEnd5 ECs, and the addition of a minuscule quantity of t h e Rf extract was able to partially alleviate excess ROS-induced effects. / National Research Foundation (NRF)

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