Comparative Functional Study of the Viral Telomerase RNA Based on Natural Mutations

Laetitia Fragnet,Emmanuel Kut,Denis Rasschaert

doi:10.1074/jbc.m501163200

Abstract

Telomerase activity is present in most malignant tumors and provides a mechanism for the unlimited potential for division of neoplastic cells. We previously characterized the first identified viral telomerase RNA (vTR) encoded by the Marek's disease virus (MDV) (Fragnet, L., Blasco, M. A., Klapper, W., and Rasschaert, D. (2003) J. Virol. 77, 5985-5996). This avian herpesvirus induces T-lymphomas. We demonstrated that the vTR subunit of the oncogenic MDV-RB1B strain is functional and would be more efficient than its chicken counterpart, cTR, which is 88% homologous. We take advantage of the functionality of those natural mutant TRs to investigate the involvement of the mutations of vTR on its efficiency in a heterologous murine cell system and in a homologous in vitro system using the recombinant chicken telomerase reverse transcriptase. The P2 helix of the pseudoknot seems to be more stable in vTR than in cTR, and this may account for the higher activity of vTR than cTR. Moreover, the five adenines just upstream from the P3 helix of vTR may also play an important role in its efficiency. We also established that the substitution of a single nucleotide at the 3'-extremity of the H-box of the vaccine MDV-Rispens strain vTR resulted in a lack of its accumulation within the cell, especially in the nucleus, correlated with a decrease in telomerase activity.

Highlights

Telomeres are nucleoprotein structures found at the extremities of eukaryotic linear chromosomes [1]
We demonstrated that the viral telomerase RNA (vTR) subunit of the oncogenic Marek’s disease virus (MDV)-RB1B strain is functional and would be more efficient than its chicken counterpart, cTR, which is 88% homologous
We investigated the functional significance of the mutations in the vTR gene of the non-oncogenic MDV-Rispens strains by comparing this gene with the vTR of the oncogenic MDV-RB1B strain

Summary

Introduction

Telomeres are nucleoprotein structures found at the extremities of eukaryotic linear chromosomes [1]. A recent study [18] has shown that the telomerase complex would function as a multimer and that the pseudoknot domain is involved in TR dimerization and interaction with TERT. The third domain, the H/ACA CR7 domain, is dispensable for in vitro telomerase activity but essential for TR activity in vivo This domain is involved in the nuclear and nucleolar targeting of TR, resulting in 3Ј-end processing and their intracellular stability [20, 21]. Three forms of congenital dyskeratosis have been described [22, 23], of which the autosomal dominant form is the most severe This form of the disease is characterized by mutations in the hTR gene and, in particular, the deletion of the H/ACA CR7 domain of hTR, which is responsible for the lack of telomerase activity due to failure of accumulation of the TR

Methods

Results

Conclusion