Understanding the biogenesis of telomerase RNA (Tlc1) in Saccharomyces cerevisiae

Abstract

Telomeres are conserved nucleoprotein structures present at the eukaryotic chromosome extremities that facilitates genome integrity and cellular proliferative capacity. Most eukaryotes employ telomerase, a specialized ribonucleoprotein complex, to counteract the progressive loss of terminal chromosomal DNA with every round of cell division. Telomerase, a reverse transcriptase, uses a specific segment of its RNA component as a template to reiteratively add the telomeric repeats at the ends of the chromosome. Abnormality in telomere regulation causes genomic instability with deleterious end results such as premature cell death or transformation into immortal cancer cells. These detrimental implications indicate the pressing need to decipher the biogenesis and regulation of telomerase. The RNA subunit of Saccharomyces cerevisiae telomerase (Tlc1) is initially transcribed as a longer precursor and is subsequently trimmed at its 3’ end to the mature form found in functional telomerase. Tlc1 harbors two unique transcriptional termination signals: Nrd1-Nab3-Sen1 (NNS) mediated termination signal and polyadenylation termination signal. The relevance of two transcriptional termination pathways and the 3’ processing mechanism that give rise to stable and functional mature Tlc1 has remained unclear. In this study, we demonstrate that NNS transcriptional termination pathway is not essential to maintain steady-state levels of mature Tlc1. In the absence of NNS termination signals, the poly(A+) precursor produced via the alternative polyadenylation termination pathway is successfully processed at the 3’ end and thereby contributes to the formation of stable mature form. In addition to the two known precursor forms resulting from NNS and polyadenylation pathways, we identified a third stable form. Furthermore, we made use of a ribozyme to bypass normal 3’ processing events by rapid cleavage of the Tlc1 at preselected sites. We observed that Tlc1 molecules processed in such a manner are highly unstable. Here, we discuss the role of processing factors, the loading of telomerase components and 3’ processing kinetics on determining the stability of Tlc1.