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.
Researchers from the University of Trento have developed an assay to image endogenous TERRA molecules expressed from a single telomere in living human cancer cells. They 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, the researchers 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.
Imaging Tel15q TERRA-MS2 molecules in living AGS cells
(A) Images of AGS WT (control), clone 1 and clone 2 cells expressing MS2-GFP. Tel15q TERRA-MS2 foci are indicated by arrows (yellow for cluster and white for particles). Scale bar: 5 µm. (B) Two populations of Tel15q TERRA-MS2 foci are detected in AGS clones. (C) Expression of Tel15q TERRA-MS2 particles in AGS WT, clone 1 and clone 2 cells (N = 88 to 157 cells). (D) Expression of Tel15q TERRA-MS2 clusters in AGS WT, clone 1 and clone 2 cells (N = 95 to 154 cells). (E) Diffusion coefficients (in log D) of Tel15q TERRA-MS2 particles and clusters from clones 1 and 2. Diffusion coefficients of telomeres (TRF1-mCherry) and Cajal bodies (Coilin-mCherry) are included for comparison. (F) Overlay between the frequency distributions of Tel15q TERRA-MS2 clusters, particles and telomeres diffusion coefficients (log D).