A novel protein subunit of telomerase from the ciliate Euplotes

The ends of eukaryotic chromosomes are bound by specialized proteins and thereby form nucleoprotein complexes that are called telomeres. Telomeric DNA is synthesized by an unusual reverse transcriptase, telomerase. The conserved core of this enzyme consists of a catalytic subunit and an intrinsic RNA component that provides the template for DNA polymerization. However, telomerase complexes from all organisms contain additional protein subunits and accessory factors that function in telomerase biogenesis and catalysis.; This thesis describes the characterization of a novel protein factor, p43, that was identified because it copurifies with active telomerase from the ciliate Euplotes aediculatus. Sequence analysis revealed the presence of a La motif, relating this factor to the La autoantigen functioning in the maturation of ribonucleoproteins by conferring nuclear retention and stabilizing labile regions of RNA structure. Biogenesis of p43 appears to require a programmed +l ribosomal frameshift, the first report of this extremely rare translational mechanism in ciliates.; Immunoprecipitation studies demonstrated that p43 is a true telomerase subunit present in most or all active telomerase complexes in the cell. In support of a conserved function in ciliates, the protein was found to associate with telomerase by binding to a conserved structural element of telomerase RNA. Several lines of evidence obtained from in vitro and in vivo experiments indicated that the vast majority of the cellular p43 pool is bound by telomerase, suggesting that the protein is, rather unexpectedly, not the general La protein of Euplotes but a bona fide telomerase-specific subunit.; Because of the structural similarities of their RNAs, Euplotes p43 also binds to the telomerase RNA subunit of Tetrahymena . Telomerase from this ciliate can be reconstituted in active form in vitro and p43 was found to modestly enhance its activity. Structural probing of the RNA bound by p43 implies that the stimulation observed in vitro may be caused by p43-induced conformational changes in the RNA. These in vitro findings suggest a telomerase-activating function for p43 without precluding additional roles for the protein in telomerase biogenesis in vivo.