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The role of damage-induced non coding RNA in the control of DNA damage response activation at telomeres in aging

Periodic Reporting for period 1 - TeloRNAging (The role of damage-induced non coding RNA in the control of DNA damage response activation at telomeres in aging)

Reporting period: 2019-10-01 to 2021-03-31

This project aims to elucidate the role of telomeric non-coding RNA (tncRNA), which are transcribed following telomere dysfunction, in the activation of the DNA damage response (DDR). By exploiting our unprecedented ability to inhibit DDR selectively at telomeres by inhibit tncRNA with antisense oligonucleotides (ASO), we will determine the specific contribution of telomeric DDR activation to the detrimental phenotypes associated with aging-related disorders.
The project is important for the society, because we will provide evidence for a novel target to treat age-related conditions, supporting the use of ASO, a class of drugs already used in the clinics, to treat different diseases.
The objectives of the projects are: to dissect the pathways controlling the transcription of dysfunctional telomeres; to investigate the molecular mechanisms through which tncRNA control the DDR activation at telomeres; and to study the impact of telomeric DDR inhibition on accelerated aging conditions and age-related diseases.
tncRNA have been detected and studied so far by RT-qPCR or hybridization-based methods only. In order to directly sequence long intact tncRNAs molecules, we exploited the Oxford Nanopore Technology (ONT) on a GridION device. This is the only technology which allows the sequencing of long RNA molecules, avoiding reverse transcription and PCR amplification steps. The specificity for the telomeric RNA transcripts is achieved by the ligation of sequence-specific adapters, to detect both the G- and the C-rich RNA species.
Unexpectedly, the tncRNAs seemed so far resistant to standard Nanopore sequencing protocols. Upon intense scrutiny, this seems likely due to an impaired ligation of the sequence-specific adapters. This may hint to post-transcriptional modifications that prohibit efficient ligation and robustr sequencing. We are therefore pursuing different and creative ways to achieve an effective direct sequencing protocol.

To study the dynamics of tncRNA transcription, we planned to generate a cell system in which we can specifically induce telomeric dysfunction and monitor telomere transcription in real time.
In collaboration with the laboratory of Emilio Cusanelli at CIBIO (Trento, Italy), we are generating a construct containing a cassette with 24 MS2 repeats flanked by telomeric repeats on both sides. We generated the recipient plasmid containing the telomeric repeats flanking a restriction site, and the donor plasmid with the 24 MS2 repeats and the hygromycin resistance gene flanked by FRT sequences. We are currently extracting the FRT-Hygro-FRT-24-MS2 cassette for the final cloning step in the receiving plasmid.
Due to the repetitive nature of telomeric and MS2 sequences, each step of this cloning strategy had major problems of recombination, which we overcome by using bacteria with low recombinogenic potential, grown in small volumes and at low temperature.
The complete cassette will be then integrated in the telomeres of MEFs, so that the endogenous C-rich tdilncRNA will be tagged by the MS2 element. We will use this tool to visualize in situ the transcription of C-rich tdilncRNAs and to identify dilncRNA-associated RBP, in the presence of telomere deprotection by Cre-mediated TRF2 KO.

Late generation mice genetically lacking Terc, the RNA component of telomerase, recapitulate several of the features of ageing humans and have been used as an accelerated aging model.
At the molecular and histological level, these animals are characterized by increased markers of DDR activation, accumulation of senescent and apoptotic cells in several organs including the lungs. In order to inhibit telomeric DDR, we systemically treated Terc-/- G3 mice at 2 months of age with ASOs targeting the tncRNA (tASO), or an ASO with a control sequence. Mice were sacrificed at 12 months of age, and several organs were collected for following analyses. We collected samples for RNA extraction, for immunohistochemical and for immunofluorescence stainings.

First, we extended our preliminary observations on the lungs of these mice. In addition to an improvement of the overall tissue morphology, we observed a partial restoration of the number of air spaces, suggesting a reduction in the fibrotic phenotype, and a reduction of the DDR in the animals treated with tASO, compared with control ASO-treated mice (Figure 1).

Next, we focused on the hematopoietic system, studying bone marrow and spleen morphology and architecture of the different experimental groups by hematoxylin-eosin staining. As independently evaluated by our pathologist, compared with wild type animals, spleens from control ASO-treated G3 Terc-/- mice were characterized by a multifocal-to-diffuse expansion of the red pulp associated with variable degree of white pulp regressive changes. The expansion of the red pulp was associated with increased signs of extramedullary hematopoiesis with increased density of myelopoietic and erythropoietic foci, and variable degree of megakaryocytic hyperplasia and clustering. The white pulp regression varied from focal and restricted to peri-vascular T-cell areas, to diffuse with effacement of the B-cell follicular architecture and marginal zone disruption. Differently, treatments with tASOs resulted in a lower degree of red pulp hyperplasia and by preserved white pulp architecture with maintenance of neat white pulp/red pulp interfaces.
In the bone marrow from control ASO-treated G3 Terc-/- mice, the hematopoietic parenchyma was characterized by a moderate to severe expansion of the granulopoietic lineage with increase of morphologically immature myeloid precursors associated with marked contraction of erythroid colonies and signs of dysmegakaryopoiesis and increased hemocatheresis/eythrophagocytosis. Consistently, the fraction of interstitial bone marrow lymphoid B cells was decreased and paralleled by the emergence of B cells inside dilated sinuses. Also in this organ, tASO treatments variably restored the normal myeloid-to-erythroid ratio predominantly limiting the expansion of morphologically immature granulocytic cells and reverted alterations in megakaryopoiesis and B-cell lymphopoiesis.
To obtain a semi-quantitative measure of the differences observed in the spleen and bone marrow morphology, we defined a scoring system, evaluating several parameters. This evaluation allowed us to conclude that tASO treatments are effective in reducing the pathological impact of telomere dysfunction (Figure 2).
With the results obtained so far, we have demonstrated that the inhibition of tncRNA in vivo, by systemic delivery of tASO, is sufficient to reduce the DDR activation at telomeres and to ameliorate the accelerated aging phenotypes in a mouse model, in three different organs (lung, bone marrow and spleen).
We plan to extend these observations to other organs, evaluating the effect of tASO treatment on the overall healthspan of these mice.

We will also apply the same approach on late generation telomerase KO mice treated with bleomycin, a well characterized model for idiopathic pulmonary fibrosis (IPF).

In addition, we are setting up a method to directly sequence the tncRNA, which will allow the identification of potential RNA modifications.

Finally, we are generating a cell system that can be used to monitor the tncRNA generated upon telomere dysfunction in real time in living cells.
Figure 2
Figure 1