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

Abstract

Telomeres cap the ends of eukaryotic chromosomes, protecting them from degradation and erroneous recombination events which may lead to genome instability. Telomeres are transcribed giving rise to telomeric repeat-containing RNAs, called TERRA. The TERRA long noncoding RNAs have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. While TERRA RNAs are predominantly nuclear and localize at telomeres, little is known about the dynamics and function of TERRA molecules expressed from individual telomeres. Herein, we developed an assay to image endogenous TERRA molecules expressed from a single telomere in living human cancer cells. We show that single-telomere TERRA can be detected as TERRA RNA single particles which freely diffuse within the nucleus. Furthermore, TERRA molecules aggregate forming TERRA clusters. Three-dimensional size distribution and single particle tracking analyses revealed distinct sizes and dynamics for TERRA RNA single particles and clusters. Simultaneous time lapse confocal imaging of TERRA particles and telomeres showed that TERRA clusters transiently co-localize with telomeres. Finally, we used chemically modified antisense oligonucleotides to deplete TERRA molecules expressed from a single telomere. Single-telomere TERRA depletion resulted in increased DNA damage at telomeres and elsewhere in the genome. These results suggest that single-telomere TERRA transcripts participate in the maintenance of genomic integrity in human cancer cells.

The Emerging Roles of TERRA in Telomere Maintenance and Genome Stability

“…It remains to be determined whether MS2-tagged TERRA transcripts preferentially associate with their telomere of origin, as previously observed in yeast [91]. Importantly, depletion of MS2-tagged TERRA transcripts via ASO-LNA resulted in the activation of DNA damage responses predominantly at extratelomeric regions and at a single telomere signal, as detected by immunofluorescence experiments [90]. These findings suggest that TERRA transcribed from a single telomere can impact on DNA damage responses at telomeres and extratelomeric sites.…”

“…Thus TERRA/TRF proteins interaction may not be limited to chromosome ends and may be involved in the recruitment of TERRA transcripts to extratelomeric sites. Indeed, RNA FISH analyses have shown that only a subset of TERRA molecules accumulates at telomeres [89] and live cell imaging studies revealed that TERRA transcripts only transiently localize at chromosome ends, in humans as in yeast [90,91,92]. These findings suggested that TERRA molecules may also bind to extratelomeric regions of chromosomes.…”

“…Recently, TERRA transcripts expressed from subtelomere 15q were tagged using a short MS2 sequence integrated at the subtelomere 15q TERRA locus in the human stomach cancer cell line AGS [90]. The MS2-tagged TERRA transcripts were visualized by fluorescence microscopy by co-expressing a GFP-fused MS2 RNA binding protein (MS2-GFP).…”

“…The MS2-tagged TERRA transcripts were visualized by fluorescence microscopy by co-expressing a GFP-fused MS2 RNA binding protein (MS2-GFP). This approach enabled the following of single-telomere TERRA transcripts localization in living cells by fluorescence microscopy revealing that TERRA molecules transiently localize at chromosome ends [90]. It remains to be determined whether MS2-tagged TERRA transcripts preferentially associate with their telomere of origin, as previously observed in yeast [91].…”

Noncoding telomeric repeat‐containing RNA inhibits the progression of hepatocellular carcinoma by regulating telomerase‐mediated telomere length

“…*P < .05; **P < .01. shCtrl, control shRNA vector; shTRF1 or shTRF2, shRNA vector against TTAGGG repeat binding factor (TRF)1 or TRF2; siCtrl, control siRNA; siTERRA, siRNA against TERRA medulloblastoma, stomach adenocarcinoma, head and neck cancer, and cervical carcinoma. [12][13][14][15]27,28 However, the functional role of TERRA in hepatocarcinogenesis is largely unknown. In this study, the expression levels of TERRA and its regulators (TRF1 and TRF2) were investigated in HCC.…”