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Characterization of the physical and functional interactions between human TRF proteins and the telomeric long noncoding RNA TERRA

2023-03Tese de doutoramento Acesso restrito
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datacite.subject.fosCiências Médicas::Ciências da Saúdept_PT
dc.contributor.advisorAzzalin, Claus Maria
dc.contributor.authorLona Abreu, Patricia
dc.date.accessioned2024-03-22T17:57:41Z
dc.date.available2024-03-22T17:57:41Z
dc.date.issued2023-03
dc.date.submitted2022-11
dc.description.abstractTelomeres, the physical ends of linear eukaryotic chromosomes, are nucleoprotein structures comprising telomeric DNA, the multiprotein complex shelterin and the long noncoding (lnc) telomeric repeat-containing RNA (TERRA). These structures assemble at the end of chromosomes to avoid unwanted activation of DNA repair machineries and telomeric fusions, thus having an essential role in maintaining genome stability and cellular homeostasis. The protein complex shelterin is considered the guardian of telomere integrity. Within this complex, the telomeric double-stranded (ds) DNA binding proteins TRF1 and TRF2 are well known for their functions in supporting faithful telomeric DNA replication and chromosome end protection, respectively. Conversely, it has been shown that increased levels of TRF1 and TRF2 disturb telomere stability, indicating that association of TRF proteins with telomeric DNA has to be tightly controlled. However, the mechanisms controlling TRF protein assembly on telomeric chromatin remain to be fully elucidated. The lnc RNA TERRA also plays several functions at telomeres: it recruits several proteins and enzymatic functions to chromosome ends by acting as a scaffold molecule; it facilitates telomeric heterochromatin formation and telomere replication; and it is involved in telomere-length regulation. It is also known that TERRA binds to TRF1 and TRF2, both in vitro and in vivo, but the mechanisms regulating these interactions and their physiological relevance are fairly unexplored. Our laboratory previously showed that TRF2, through its N-terminal basic (B) domain, binds to TERRA and promotes its invasion into telomeric dsDNA. TRF2-mediated TERRA invasion leads to formation of telomeric RNA:DNA hybrids or R-loops (telR loops). We also demonstrated that the N-terminal acidic (A) domain of TRF1, when fused to TRF2 B domain, suppresses the binding of the latter to TERRA and that TRF1 inhibits TRF2-stimulated telR-loop formation. This suggests that TRF proteins communicate in order to regulate telR-loop formation in cells. Here, we further characterized the molecular mechanisms regulating the binding of TRF proteins to TERRA. We showed that the binding of both TRF1 and TRF2 to TERRA depends on the ability of this RNA to form G-quadruplex (G4s) structures. While both TRFs also bind to non-telomeric RNAs forming G4s, the binding to TERRA was more efficient. Additionally, we demonstrated a critical role of charged amino acids within the N-terminal domains of TRFs in TRF1-mediated regulation of TERRA-TRF2 B domain interactions. We also started to characterize TERRA binding sites outside the N-terminal domains of TRFs and showed that multiple domains within both TRF1 and TRF2 can bind to TERRA. We then demonstrated a role of TERRA in the regulation of TRFs association with telomeric dsDNA. TERRA overexpression in human cancer cells reduced the density of TRF proteins at telomeres and affected telomere integrity, as indicated by an increase in telomere fragility and loss of telomeric tracts. These phenotypes seemed to be directly associated to the reduction of TRF1 density at telomeres elicited by TERRA as they were no longer observed in cells with ectopic overexpression of TRF1. Based on these data, we propose a model where TERRA-mediated regulation of TRFs density at telomeres is crucial for telomeric DNA repair and elongation. This model challenges the current understanding of telomere structure and organization, which considers shelterin and TERRA as separate entities, and depicts a novel view of telomeres as dynamic DNA/RNA/protein particles where TERRA is a key regulator of shelterin association with telomeric DNA. Overall, this work highlights the importance of TERRA-TRF interactions in supporting regulated telR-loop formation and telomere stability, and, in turn cellular and tissue homeostasis, thereby avoiding disease conditions.pt_PT
dc.identifier.tid101560567pt_PT
dc.identifier.urihttp://hdl.handle.net/10451/63706
dc.language.isoengpt_PT
dc.relationKeeping telomeric R-loops in check: unravelling new functions of human TRF1 in chromosome end protection
dc.relationKeeping telomeric R-loops in check: unravelling new functions of human TRF1 in chromosome end protection
dc.subjectTelómerospt_PT
dc.subjectTRFspt_PT
dc.subjectTERRApt_PT
dc.subjectInterações RNA-proteínapt_PT
dc.subjectTelomerespt_PT
dc.subjectRNA-protein interactionspt_PT
dc.titleCharacterization of the physical and functional interactions between human TRF proteins and the telomeric long noncoding RNA TERRApt_PT
dc.typedoctoral thesis
dspace.entity.typePublication
oaire.awardNumberPD/BD/128284/2017
oaire.awardNumberCOVID/BD/151621/2021
oaire.awardTitleKeeping telomeric R-loops in check: unravelling new functions of human TRF1 in chromosome end protection
oaire.awardTitleKeeping telomeric R-loops in check: unravelling new functions of human TRF1 in chromosome end protection
oaire.awardURIinfo:eu-repo/grantAgreement/FCT//PD%2FBD%2F128284%2F2017/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/OE/COVID%2FBD%2F151621%2F2021/PT
oaire.fundingStreamOE
person.familyNameLona Abreu
person.givenNamePatricia
person.identifier.ciencia-id2914-7E3B-499B
person.identifier.orcid0000-0002-6387-8537
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.rightsrestrictedAccesspt_PT
rcaap.typedoctoralThesispt_PT
relation.isAuthorOfPublication22a39ac0-860f-474a-97db-cc460851f34c
relation.isAuthorOfPublication.latestForDiscovery22a39ac0-860f-474a-97db-cc460851f34c
relation.isProjectOfPublication9d7e7626-a434-4b0d-a901-b934b0796803
relation.isProjectOfPublicationf2303a2c-7aeb-4ae2-a212-b0a815059287
relation.isProjectOfPublication.latestForDiscoveryf2303a2c-7aeb-4ae2-a212-b0a815059287
thesis.degree.nameTese de doutoramento, Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2023pt_PT

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