Telomeric Noncoding RNA TERRA Is Induced by Telomere Shortening to Nucleate Telomerase Molecules at Short Telomeres

Abstract

Elongation of a short telomere depends on the action of multiple telomerase molecules, which are visible as telomerase RNA foci or clusters associated with telomeres in yeast and mammalian cells. How several telomerase molecules act on a single short telomere is unknown. Herein, we report that the telomeric noncoding RNA TERRA is involved in the nucleation of telomerase molecules into clusters prior to their recruitment at a short telomere. We find that telomere shortening induces TERRA expression, leading to the accumulation of TERRA molecules into a nuclear focus. Simultaneous time-lapse imaging of telomerase RNA and TERRA reveals spontaneous events of telomerase nucleation on TERRA foci in early S phase, generating TERRA-telomerase clusters. This cluster is subsequently recruited to the short telomere from which TERRA transcripts originate during S phase. We propose that telomere shortening induces noncoding RNA expression to coordinate the recruitment and activity of telomerase molecules at short telomeres.

Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells

“…However, single-telomere TERRA transcripts only transiently localize with telomeres, which is consistent with previous studies showing that only a small fraction of total TERRA transcripts co-localize with telomeres in fixed or living cells [5,17]. A transient localization of single-telomere TERRA molecules at chromosome ends was also previously observed in yeast by live-cell imaging, indicating that this is a conserved feature of TERRA transcripts [13]. By performing single particle tracking analyses, we observed that the diffusion coefficients of TERRA clusters overlap with the diffusion coefficients of telomeres.…”

“…Considering their sizes and kinetics, larger TERRA foci could represent clusters of TERRA transcripts which may be bound to chromatin or assembled in large ribonucleoprotein particles (RBPs). Interestingly, similar TERRA clusters have been observed in budding yeast [13], suggesting that the capacity of this lncRNA to form high-ordered structures may be conserved among eukaryotes.…”

“…We previously used the MS2-GFP system to tag and image endogenous TERRA transcripts expressed from a single telomere in living yeast cells [13]. The MS2 system relies on the high affinity binding between the bacteriophage MS2 stem-loop RNA and the bacteriophage MS2 RNA binding protein and it has been widely used to study endogenous RNA molecules in living cells of various organisms, including human [26,27].…”

“…Expression of TERRA is tightly regulated by the activity of several transcription regulators, including the chromatin organizing factor CTCF [9] and the transcription factors heat shock factor 1 (HSF1) [10], Snail [11], as well as the nuclear respiratory factor NRF1 [12]. Furthermore, telomere shortening is associated with increased TERRA levels in yeast as well as human cells [13-16]. RNA fluorescence in-situ hybridization (FISH) and live-cell imaging analyses have shown that a subset of TERRA transcripts localizes with human telomeres [5,6,17].…”

“…RNA fluorescence in-situ hybridization (FISH) and live-cell imaging analyses have shown that a subset of TERRA transcripts localizes with human telomeres [5,6,17]. At telomeres, TERRA molecules have been proposed to mediate several important functions, including regulation of heterochromatin formation [16], recruitment of chromosome end-processing and chromatin remodelling factors to dysfunctional telomeres [18,19], sustaining telomeric DNA replication [20], participating in telomere length homeostasis by regulating telomerase activity [13,14] or promoting homologous recombination among telomeres through formation of RNA-DNA heteroduplex (R-loops) at chromosome ends [21-24]. In addition to their preferential association with telomeres, recent evidence indicates that TERRA transcripts interact with numerous internal chromosomal regions to regulate widespread gene expression [25].…”