Human telomeres are synthesized by the ribonucleoprotein telomerase. The abundance of hTR, the telomerase RNA component, is post-transcriptionally regulated by several enzymes and RNA-binding complexes that cooperate to convert longer hTR precursors to the mature form. A complex balance between 3¿ processing and degradation of hTR isoforms ultimately determines the steady-state levels of mature hTR that is incorporated into telomerase. We found that TGS1, the hypermethylase that converts the hTR 5' mono-methylguanosine cap to a tri-methylguanosine cap (TMG), favors nuclear retention of hTR and limits hTR abundance, thus tuning down telomerase activity. This project is aimed at characterizing the role of TGS1 in the regulation of telomerase biogenesis and activity in human cells, using TGS1 mutant cell lines generated by CRISPR-mediated genome editing. We have evidence that chemical inhibition of TGS1 activity affects telomere length in vivo and will explore the hypothesis that TGS1 inhibitors can be exploited as potential therapeutic tools for the treatment of telomerase insufficiency syndromes caused by reductions in the hTR levels.
In Drosophila, telomerase has been lost and telomeres are elongated by targeted transposition of retroelements at the DNA ends, while a specific capping complex, terminin, protects telomeres independently of sequence. Despite these differences, Drosophila melanogaster proved to be an excellent model system for the identification and characterization of proteins implicated in telomere maintenance, as fly telomeres are also protected by conserved non-terminin proteins, most of which have human counterparts involved in telomere maintenance. We will explore whether the fly homologue of TGS1, DTgs1, which physically interacts with terminin, is required for telomere homeostasis, by investigating its role in the biogenesis and expression of the telomeric retrotransposons Het-A and TART.
One of the hallmarks of cancer is unlimited proliferative capacity, which is naturally limited by progressive telomere shortening, which eventually leads to permanent growth arrest. To overcome this barrier, most cancer cells reactivate telomerase to maintain their telomeres. On the other hand, critically short telomeres are involved in age related diseases, or telomeropathies, a wide spectrum of conditions caused by the excessive shortening of telomeres. The most common short-telomere syndromes are Dyskeratosis congenita (DKC, characterized by leukoplakia, abnormal skin pigmentation and nail dystrophy), Idiopathic Pulmonary Fibrosis (IPF, characterized by progressive failure of lung function), familial liver cirrhosis, aplastic anemia and sporadic acute myelogenous leukemia (AML) (32). All these conditions are characterized by an elevated susceptibility to developing cancers (33). The common cause of these disorders is impaired telomere maintenance, and the consequent loss of stem cell populations, that is particularly critical in highly proliferative tissues. The underlying causative genetic defects are mutations in several genes involved in various aspects of telomere maintenance, such as the biogenesis, assembly, activity and recruitment of telomerase, telomere protection, telomere replication, stability of the T-loop (34). Interestingly, mutations in the genes encoding the RNA or the protein component of telomerase, hTR and TERT, respectively, are associated with a dominant mode of inheritance of the short telomere syndromes. The haploinsufficiency for hTR or TERT indicate that both telomerase components are limiting for telomerase activity. DKC1 and PARN, two proteins directly involved in the 3' end processing of hTR, ensure the stability of the hTR transcript and are both involved in the pathogenesis of recessive forms of DKC, indicating that proper regulation of hTR abundance is a key factor to prevent pathogenic telomere shortening (35). Our finding that TGS1 is a negative regulator of hTR abundance uncovers a novel mechanisms that contributes to the modulation of telomerase abundance and offers a novel perspective in the search for terapeuthic targets aimed at increasing hTR levels, in cells in which the hTR dosage is critically limiting. Recent work has shown that inactivation of the PAPD5 deadenylase in DKC1 human embryonic stem cells rescues telomerase and lengthens telomeres (36). Based on the same rationale, TGS1 inhibition could be effective in cells from patients carrying mutations in the Dyskerin (DKC1) or PARN genes. Our discovery that an analogue of the methyl donor S-adenosyl-methionine effectively inhibits TGS1 activity in vitro and in vivo and induces a net increase in hTR levels after a few days of treatment is a promising avenue to device potential treatments for both PARN and DKC1-dependent forms of Dyskeratosis congenital. Since the development of the anti-TMG cap specific antibody by the Luhrmann group in 1983, several RNA targets which undergo cap hypermethylation were identified, including snRNAs, snoRNAs and telomerase RNA, however, the functional significance of cap hypermethylation has remained elusive. Our work provided the first demonstration of the consequences of the loss of cap hypermethylation in the maturation of hTR, showing that the TMG cap does not contribute to the catalytic activity of telomerase, but it rather controls the intracellular trafficking of this RNA and increases its stability. The experiments proposed in this project will contribute to clarifying the mechanisms through which the TMG cap prevents hTR degradation and affects the dynamics of the 3' processing of hTR.
Finally, this project has the objective of understanding the functional significance of cap hypermethylation in an evolutionary perspective. We will investigate the roles of TGS1-mediated hypermethylation in Drosophila, an organism in which both the RNA and the protein components of telomerase have been lost and the task of elongating telomeres has been taken on by the RNAs of telomere-specific retrotransposons and their dedicated reverse transcriptase machinery. In light of our recent findings that human TGS1 can fully compensate for mutations in its fly orthologue, DTgs1, it will be interesting to learn to what extent the functions of the two proteins overlap and in particular whether the retrotransposon RNAs require cap hypermethylation and TGS1 for their biogenesis, similar to telomerase RNA.
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