Human telomerase regulation by associated proteins and alternatively spliced mRNA variants

Human telomerase is a ribonucleoprotein complex, minimally composed of a catalytic subunit, hTERT and an RNA component, hTR that contains the template used to synthesize telomere DNA. Telomerase is present in over 80% of all cancer cells. Therefore, an understanding of telomerase regulation is important for cancer research. This thesis work was focused upon the study of telomerase regulation by telomerase associated proteins and alternatively spliced hTERT mRNA variants. Tandem affinity purification (TAP) and immunopurifications with anti-hTERT antibody were used to visualize potential telomerase associated proteins. No specific proteins were identified in TAP-hTERT transfected cells. hTERT was purified from 293 telomerase positive cells, and proteins specifically associated with hTERT were visualized. Insertion 3 and 4 alternatively spliced hTERT mRNA variants were detected in telomerase positive cell lines by RT-PCR and Southern blot analysis. The gamma-deletion alternatively spliced hTERT mRNA variant was detected in Huh-7 liver cells. These results provide information and tools important for a greater understanding of telomerase