The abundance of hTR, the telomerase RNA, is negatively regulated by the TGS1 hypermethylase, which converts the hTR 5' mono-methylguanosine cap to a trimethylguanosine cap. TGS1 loss results in an increase of hTR dosage, upregulation of telomerase activity and telomere elongation. We have also found that chemical inhibition of TGS1 by the SAM analog sinefungin, elicits phenotypes similar to those observed upon genetic loss of TGS1. We are currently testing if chemical inhibition of TGS1 may be beneficial for diseases caused by excessive telomere shortening, due to mutations in genes controlling telomerase homeostasis, such as dyskeratosis congenita (DC) and pulmonary fibrosis. In these disorders, the primary defect is shortened telomeres, which in turn cause exhaustion of stem cells renewal, resulting in tissue homeostasis failure at organism level. We will assay sinefungin and its analogs for their ability to restore telomere length in different cell types, carrying mutations in the PARN or DKC1 genes (found in patients affected by DC), with the objective of correcting their telomere shortening phenotype, thus improving their proliferation potential.
We will also explore the role of TGS1 in the DNA damage response. Our recent work suggests the hypothesis that TGS1 might cooperate with the Survival Motor Neuron protein (SMN) in preventing the accumulation of transcriptional stress in the genome and in particular at transcription termination regions. Both TGS1 and Smn physically interact with Topoisomerase 2, which prevents R-loops accumulation. To address the functional relationships between these protein in the maintenance of genome stability, we will perform studies on Drosophila mutants in which Tgs1, Smn and Top2 functions are specifically depleted in the eye imaginal discs by RNAi, and in parallel, experiments in CRISPR-cas9-derived HeLA cells, deficient for TGS1 and SMN.
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) [38]. All these conditions are characterized by an elevated susceptibility to developing cancers [39]. 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 [6]. 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 [40]. Our finding that TGS1 is a negative regulator of hTR abundance uncovers a novel mechanism that contributes to the modulation of telomerase abundance and offers a novel perspective in the search for therapeutic 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 [24]. 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 congenita.
Another important aspect that we will address in this proposal, stems from our recent findings that SMN and TGS1-deficient cells experience spontaneous DNA damage. SMN has been implicated in preventing transcriptional stress and R-loops accumulation, especially at transcription termination regions [35]. Unpublished experiments performed in our lab, based on deep-sequencing transcriptome analyses on SMN and TGS1-deficient cells, revealed an accumulation of extended mRNA transcripts, likely arising from defective transcriptional termination. Here we will address if transcriptional stress at termination region could be a problem common to both SMN and TGS1 mutant backgrounds and if Topoisomerase 2 contributes to the maintenance of genome integrity in these mutants. These investigations are of key importance for unveiling which is the source of cellular stress that culminates in cell death of retinal precursors in fly mutants. These studies will have relevant implications for untangling the causes driving degeneration in neurons of patients affected by SMA. Given the close relationships between TGS1 and SMN[30], we believe our investigations will tackle unexplored aspects of RNA homeostasis, controlled by these proteins. The combination of the fly and human models to study TGS1 role in the DNA damage response, will help defining conserved pathways that may be essential to preserve genome stability also in neurons.
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