An alternative telomerase RNA in Arabidopsis modulates enzyme activity in response to DNA damage

Abstract

Telomerase replenishes telomere tracts by reiteratively copying its RNA template, TER. Unlike other model organisms, Arabidopsis thaliana harbors two divergent TER genes. However, only TER1 is required for telomere maintenance. Here we examine the function of TER2. We show that TER2 is spliced and its 39 end is truncated in vivo to generate a third TER isoform, TER2 S . TERT preferentially associates with TER2 > TER1 > TER2 S . Moreover, TER2 and TER2 S assemble with Ku and POT1b (protection of telomeres), forming RNP (ribonucleoprotein) complexes distinct from TER1 RNP. Plants null for TER2 display increased telomerase enzyme activity, while TER2 overexpression inhibits telomere synthesis from TER1 and leads to telomere shortening. These findings argue that TER2 negatively regulates telomerase by sequestering TERT in a nonproductive RNP complex. Introduction of DNA double-strand breaks by zeocin leads to an immediate and specific spike in TER2 and a concomitant decrease in telomerase enzyme activity. This response is not triggered by replication stress or telomere dysfunction and is abrogated in ter2 mutants. We conclude that Arabidopsis telomerase is modulated by TER2, a novel DNA damageinduced noncoding RNA that works in concert with the canonical TER to promote genome integrity.

A Transposable Element within the Non-canonical Telomerase RNA of Arabidopsis thaliana Modulates Telomerase in Response to DNA Damage

“…Liquid Murashige and Skoog (MS) medium were used for germination and growing [16]. After transferring cold-treated seeds to MS, plants were grown at 22°C under long day light condition for ~7 days.…”

DDM1 guards against telomere truncation in Arabidopsis

“…Similarly, there was no change in telomerase subunits including the processivity factor POT1a (Surovtseva et al 2007; Renfrew et al 2014), the canonical telomerase RNA TER1 (Cifuentes-Rojas et al 2011) and the telomerase regulatory lncRNA TER2 (Cifuentes-Rojas et al 2012). …”

“…To further assess genome stability, we used RT-PCR analysis to test whether G6 ddm1-2 mutants mount a global DNA damage response by monitoring transcripts that are elevated in plants experiencing telomere dysfunction (Boltz et al 2012; Cifuentes-Rojas et al 2012; Hashimura and Ueguchi 2011). We found no significant induction of GR1 (Gamma response 1), BRCA1 (Breast cancer susceptibility 1) or RAD51 , although PARP1 (poly [ADP-ribose] polymerase 1) was somewhat increased (1.5-fold, p value > 0.05, t test) (Fig.…”

“…Reverse transcription was performed with 1 μg total RNA with the qScript cDNA SuperMix (Quanta Biosciences). mRNA levels were assessed by quantitative PCR with primers described previously to detect telomere-related transcripts (Cifuentes-Rojas et al 2011; Leehy et al 2013) or DDR transcripts (Boltz et al 2012; Cifuentes-Rojas et al 2012; Hashimura and Ueguchi 2011), using SsoAdvanced Universal Supermix (Bio-Rad). RNA from at least three individual plants was used for each genetic background and at least two technical replicates were run for each reaction.…”

RNA recognition by the DNA end-binding Ku heterodimer

“…Outside of the Saccharomycotina clade, KBSs have not been detected in the TERs of the fission yeast Schizosaccharomyces pombe or the filamentous ascomycete Neurospora crassa (Webb and Zakian 2008;Qi et al 2012). Although the precise Ku-binding sites have not been identified, Ku is reported to bind to both human TER and specific TER isoforms in Arabidopsis thaliana (Ting et al 2005(Ting et al , 2009Cifuentes-Rojas et al 2012) (see also Chai et al 2002).…”