Telomere maintenance is a key molecular process required to prevent cell senescence. Impairment of telomere maintenance results into severe diseases such as Dyskeratosis Congenita (DC), on the other hand aberrant activation of the telomere elongation allows cancer cells to escape senscence.
Telomerase is the key enzyme involved in telomere maintenance. This enzyme consists of a protein component (TERT) and a non-coding RNA moiety (TERC). TERC maturation is a key step in the assembly of a functional telomerase enzyme and requires DKC1 (a gene whose loss is causative of DC) and PARN (a polyA ribonuclease previously involved in small RNA biogenesis). Our preliminary data show that not only AGO2 interacts with DKC1, TERC and a small RNA derived from the 3' end of TERC (sm-TERC-RNA), but also that AGO2 depletion reduces telomere length, telomerase activity and clonogenicity of human cells. We therefore hypothesize that AGO2 might take part in TERC processing.
The proposed project aims at elucidating the molecular mechanisms through which AGO2, DKC1, PARN and a sm-TERC-RNA contribute to TERC maturation to ensure proper telomerase function. We will check by means of biochemical purifications the interactions between AGO2, TERC, PARN and DCK1, we will investigate which of these factors are involved in sm-TERC-RNA biogenesis by gene silencing/overexpression approaches in cell lines. We expect to clarify the role played by AGO2 and sm-TERC RNA in TERC maturation and identify molecular strategies to modulate telomerase activity which might be exploited to target cancer cells as well as to ameliorate life quality of DC patients.
The proposed research is highly innovative. Indeed, the involvement of AGO2 and a specific small RNA (sm-TERC-RNA) in the maturation of TERC are reported by us for the first time. Our research will validate a novel component of the complex mechanism of telomere maintenance, contributing to clarify the molecular mechanisms underlying this process.
Our project is supported by very strong preliminary data suggesting direct interactions between AGO2 and key components of the telomerase as well as functional data linking genetic loss of AGO2 and telomere shortening.
If confirmed, our findings will pave the way to the use of RNAi related strategies targeting TERC maturation and in turn telomerase activity with the development of RNAi drugs tailored to the treatment of cancer (to reduce telomerase activity) or DC (to rescue telomerase activity) with relevant repercussion on health. In fact, our hypothesis may provide a rationale to the long-standing observation that AGO2 is frequently over-expressed in tumor cells, by proving that AGO2 overexpression is able to enhance telomerase activity. If these findings will be supported a possible strategy to trigger cancer cell senescence could be direct targeting of AGO2. An alternative strategy might be targeting sm-TERC-RNA molecule through the delivery of antisense oligonucleotides able to prevent its association with endogenous target RNAs and therefore inhibiting its function. Targeting AGO2 or sm-TERC-RNA might prove promising approaches which might contribute to trigger senescence in tumor cells and in combination with other therapies help tumor eradication.
On the other hand, sm-TERC-RNA over-expression in DC patients could be beneficial to the treatment of the disease by compensating decreased amounts of mature TERC due to impairment of DKC1, PARN or heterozygous mutations of TERC gene itself. In fact, if our research will confirm that sm-TERC-RNA can promote TERC RNA maturation, it will be conceivable that its over-expression might restore mature TERC expression levels in DC patients. Furthermore, sm-TERC-RNA would only increase telomerase activity in cells which express both TERT and TERC, thus minimizing the risk of boosting telomerase activity in somatic cells. Furthermore, the use of an endogenous small RNA (sm-TERC-RNA) whose expression might be modulated in the future through targeted delivery will likely overcome many of the restrictions that currently apply to RNAi approaches.