Unravelling TERRA regulation by the interplay of two mechanisms: RALY interaction and telomere-specific polyadenylation

Telomeric repeat-containing RNA (TERRA) is a family of long non-coding RNAs transcribed at chromosome ends which play key roles in telomere maintenance. TERRA transcripts are of variable length and sequence that is characterized by a subtelomere-specific portion at the 5’ end and a common portion with telomeric repeats at the 3’ end. TERRA is implicated in different aspects of telomere biology, including the maintenance of their epigenetic landscape, the regulation of telomere elongation activity by telomerase or alternative recombination-based lengthening mechanisms and the conservation of genomic integrity at chromosome ends. However, several molecular mechanisms of TERRA biology remain elusive, as its stability and post-transcriptional regulation in human cells. This thesis unravels a novel regulatory mechanism of TERRA stability, that depends on two factors: the interaction with the hnRNP RALY and telomere-specific polyadenylation. RALY is an RNA binding protein that preferentially binds to RNAs containing poly(U) stretches and it plays key roles in RNA maturation and stabilization processes. An interactome study identified RALY as a TERRA interacting factor in mouse embryonic stem cells, but this interaction had not yet been characterized in humans. Our study unveils that in HeLa cells RALY interacts with TERRA transcribed by several telomeres through the binding of the repeated 3’ end and that this interaction has an impact on TERRA biology: RALY knockout or transient depletion results in lower TERRA global levels, impaired localization of TERRA at telomeres and telomere dysfunction induced foci. The downregulation of TERRA caused by RALY depletion affects certain TERRA subpopulations and, interestingly, the absence of RALY produces a drop in the half-life of these TERRA transcripts. Thus, RALY regulates TERRA stability in a telomere-specific manner. This study also provides evidence that TERRA polyadenylation is telomere-specific in diverse human cancer cell lines and that PABPN1 is enriched especially at polyadenylated TERRA transcripts. We then investigated polyadenylation as a factor for TERRA stability. The role of the TERRA poly(A) tail is more complex than representing only a stabilizer, as polyadenylation may also direct human TERRA transcripts to degradation instead. Notably, our results suggest that TERRA stability and decay are regulated by PABPN1 with the contribution of RALY, that preferentially stabilizes non-polyadenylated TERRA transcripts. In contrast, PABPN1 promotes TERRA decay. Given the distinct nuclear localization and features of poly(A)+ versus poly(A)- TERRA, we propose that different telomeres trigger distinct TERRA-mediated biological functions in cells.