The telomeric repeat containing RNA TERRA represents a class of long non-coding RNAs transcribed from telomeres. TERRA has been shown to play key roles in telomere maintenance. During past years it has been reported that TERRA interacts with telomerase, suggesting it may act as regulator of telomerase. However, the role of TERRA in telomerase activity in human cells is still unknown and it needs to be further investigated. Herein, we investigate the role of TERRA in telomerase activity in human cancer cells by exploiting different experimental conditions. By using single molecule RNA FISH (smiFISH) combined with confocal microscopy, we demonstrated that TERRA and the telomerase RNA subunit hTR colocalize in cancer cells. We observed that these events mainly occur in the nucleoplasm, while a fraction of TERRA transcripts colocalize with hTR molecules also at telomeres. This result suggested us the possibility of a localization-dependent role of TERRA in the regulation of telomerase activity. Surprisingly, by studying TERRA and hTR interactions during telomere length homeostasis, we observed that fewer TERRA molecules localize at chromosome ends when telomeres are elongated by telomerase. Furthermore, the fraction of telomeric TERRA-hTR colocalizing foci markedly decreased during telomere elongation. We observed that telomere shortening correlated with increased TERRA levels, in line with previously reported findings. Intriguingly, by quantifying the integrated density of the telomeric foci in smiFISH experiments combined with immunofluorescence (IF), we observed that TERRA transcripts preferentially localize to telomeres showing a brighter signal intensity as compared to the average telomeric signal of the cell, suggesting a preferential recruitment of TERRA to longer chromosome ends. These results suggest that TERRA transcripts may localize to telomeres in trans and that a displacement of TERRA from chromosome ends may be required for telomere elongation by telomerase. To gain insight into the role of TERRA in the regulation of telomerase, we used antisense oligonucleotides to deplete TERRA in cells. Interestingly, TERRA depletion resulted in increased telomerase localization to telomeres. Altogether, our findings support a model in which telomeric TERRA transcripts act as negative regulators of telomerase activity at telomeres.
| Identifer | oai:union.ndltd.org:unitn.it/oai:iris.unitn.it:11572/382929 |
| Date | 20 July 2023 |
| Creators | Bettin, Nicole |
| Contributors | Bettin, Nicole, Cusanelli, Emilio |
| Publisher | Università degli studi di Trento, place:TRENTO |
| Source Sets | Università di Trento |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | firstpage:1, lastpage:113, numberofpages:113 |
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