The ends of eukaryotic chromosomes are characterized by specific nucleoproteic structures called telomeres. Telomeres protect chromosome from recombination, exonuclease degradation and end-to-end fusion and guarantee their stability. Mammalian telomeric DNA is composed by the tandem repetition of the hexamer TTAGGG and it is tightly associated to the multiproteic complex called shelterin. In normal somatic cells, telomere length progressively shortens with age. When telomeres reach a critical length, cells become senescent and stop to divide. Traditionally, telomeres have always been considered transcriptionally silent, but this longstanding dogma was overturned in 2007, when it was discovered that they are transcribed into non-coding RNAs named “Telomeric Repeat-containing RNAs” (TERRA). The function of TERRA is still controversial, but it is hypothesized it could involve telomere maintenance through various mechanisms, still to be elucidated. Telomeric repeats are not exclusively located at chromosome ends; indeed, our group demonstrated that stretches of telomeric repeats called Interstitial Telomeric Sequences (ITSs) are also found at interstitial regions. During my internship we carried out an in silico search of ITS in the human reference genome sequence. We identified a total of 501 loci, 236 are contained in non genic sequences. The remaining 265 loci are comprised inside genic sequences (introns or UTRs), with possible major implications in gene expression regulation. In this work, we dug more into the transcription of ITSs. We searched the databases of human transcripts for the presence of cDNAs containing sequences homologous to ITS, located both in genic and non-genic sequences. Intronic loci could be transcribed together with the primary transcript of the gene or independently as non coding RNAs, while non genic loci could be transcribed per sé or together with unannotated genes. In both cases, since these RNA contain telomeric repeats, they can be considered a particular class of TERRA molecules. In particular, the EST analysis revealed a family of ITS loci contained inside a segmental duplication present at 28 loci in the genome. Surprisingly, we found that 27 of these loci were flanked by a TAR1 region (Telomere-Associated Repeat 1), typically found at subtelomeric promoters. This region displays a substantial homology with the functional parts of the TERRA promoter driving the transcription of telomeres, which our group described in a previous work, suggesting that also these interstitial promoters could be functional and drive the transcription of this group of ITSs. The analysis of the ENCODE project data showed that RNA polymerase II is found at these promoters and that they are classified as transcriptionally active. Finally, we experimentally demonstrated through Real Time qPCR that these ITSs are transcriptionally active. In conclusion, our work demonstrated for the first time that a fraction of human TERRA "reservoir" is composed by transcripts synthesized at ITS loci. Moreover, we described for the first time a group of ITS-loci flanked by a specific promoter sequence. We hypothesize that the transcription at the loci containing a promoter homologous to those found at subtelomeres is finely regulated. Additional experiments are required to further characterize these loci, to define their regulation, the function of the RNA molecules transcribed at ITS-loci and what is the connection with telomeric metabolism.
Le estremità dei cromosomi eucariotici sono caratterizzate da specifiche strutture nucleoproteiche chiamate telomeri. I telomeri proteggono i cromosomi dalla ricombinazione, dalla degradazione enzimatica e dalle fusioni e ne garantiscono la stabilità. Il DNA telomerico dei mammiferi è composto dalla ripetizione in tandem dell’esamero TTAGGG ed è strettamente associato al complesso multiproteico chiamato shelterina. Nelle cellule somatiche normali i telomeri si accorciano progressivamente con l'età. Quando i telomeri raggiungono una lunghezza critica, le cellule entrano in senescenza e smettono di dividersi. Tradizionalmente i telomeri sono sempre stati considerati trascrizionalmente silenti, ma questo dogma è stato smentito nel 2007 quando si scoprì che essi sono trascritti in RNA non codificanti chiamati “Telomeric Repeat-containing RNAs” (TERRA). La funzione di TERRA è ancora oggetto di controversie, ma si ipotizza che riguardi il mantenimento dei telomeri attraverso numerosi meccanismi ancora da delucidare. Le ripetizioni telomeriche non sono esclusivamente collocate alle estremità dei cromosomi, infatti il nostro gruppo ha dimostrato che tratti costituiti da ripetizioni telomeriche, chiamati Sequenze Telomeriche Interstiziali (ITS), sono collocati anche presso loci interni. Durante il mio internato di tesi abbiamo effettuato una ricerca in silico di ITS nella sequenza del genoma umano di riferimento. Abbiamo identificato un totale di 501 loci, 236 dei quali sono contenuti in sequenze non geniche. I rimanenti 265 loci sono compresi all’interno di sequenze geniche (introni o UTR), con possibili importanti implicazioni nella regolazione della trascrizione dei geni che li contengono. In questo lavoro, ci siamo soffermati in particolare sulla trascrizione delle ITS. Tramite ricerche in silico all’interno dei database dei trascritti umani, abbiamo trovato alcuni cDNA con sequenza omologa a ITS localizzate sia in sequenze geniche che non geniche. I loci intronici potrebbero essere trascritti insieme al trascritto primario del gene o indipendentemente come RNA non codificanti, mentre i loci non genici potrebbero essere trascritti per sé o insieme a geni non annotati. In entrambi i casi, poiché questi RNA contengono ripetizioni telomeriche possono essere considerati una classe particolare di molecole di TERRA. In particolare, l’analisi delle EST ha rivelato la presenza di una famiglia di ITS contenuta in una duplicazione segmentale presente 28 volte nel genoma. Sorprendentemente, abbiamo osservato che 27 di questi loci sono fiancheggiati da una sequenza TAR1 (Telomere-Associated Repeat 1), tipica dei promotori subtelomerici. Questa regione presenta una sostanziale omologia con le parti funzionali del promotore di TERRA che guida la trascrizione dei telomeri e che il nostro gruppo ha in precedenza descritto, suggerendo che anche questa sequenza potrebbe essere attiva e guidare la trascrizione di questo gruppo di ITS. L’analisi del progetto ENCODE ha rivelato che questi loci sono arricchiti in sequenze legate dall’RNA polimerasi II e che vengono classificati come trascrizionalmente attivi. Infine, tramite Real Time qPCR abbiamo dimostrato sperimentalmente che questi loci sono cattivi. In conclusione, il nostro lavoro dimostra per la prima volta che parte della “riserva” di TERRA nell’uomo è costituita da trascritti provenienti da ITS. Inoltre, abbiamo descritto per la prima volta delle ITS fiancheggiate da uno specifico promotore. Ipotizziamo che la trascrizione ai loci contenenti promotori omologhi a quelli subtelomerici sia finemente regolata. Ulteriori esperimenti sono richiesti per caratterizzare la regolazione di questi loci, definire la funzione delle molecole di RNA trascritte da queste ITS e quale relazione li leghi al metabolismo dei telomeri.
Trascrizione di sequenze telomeriche interstiziali umane
BOERO, ELENA
2015/2016
Abstract
The ends of eukaryotic chromosomes are characterized by specific nucleoproteic structures called telomeres. Telomeres protect chromosome from recombination, exonuclease degradation and end-to-end fusion and guarantee their stability. Mammalian telomeric DNA is composed by the tandem repetition of the hexamer TTAGGG and it is tightly associated to the multiproteic complex called shelterin. In normal somatic cells, telomere length progressively shortens with age. When telomeres reach a critical length, cells become senescent and stop to divide. Traditionally, telomeres have always been considered transcriptionally silent, but this longstanding dogma was overturned in 2007, when it was discovered that they are transcribed into non-coding RNAs named “Telomeric Repeat-containing RNAs” (TERRA). The function of TERRA is still controversial, but it is hypothesized it could involve telomere maintenance through various mechanisms, still to be elucidated. Telomeric repeats are not exclusively located at chromosome ends; indeed, our group demonstrated that stretches of telomeric repeats called Interstitial Telomeric Sequences (ITSs) are also found at interstitial regions. During my internship we carried out an in silico search of ITS in the human reference genome sequence. We identified a total of 501 loci, 236 are contained in non genic sequences. The remaining 265 loci are comprised inside genic sequences (introns or UTRs), with possible major implications in gene expression regulation. In this work, we dug more into the transcription of ITSs. We searched the databases of human transcripts for the presence of cDNAs containing sequences homologous to ITS, located both in genic and non-genic sequences. Intronic loci could be transcribed together with the primary transcript of the gene or independently as non coding RNAs, while non genic loci could be transcribed per sé or together with unannotated genes. In both cases, since these RNA contain telomeric repeats, they can be considered a particular class of TERRA molecules. In particular, the EST analysis revealed a family of ITS loci contained inside a segmental duplication present at 28 loci in the genome. Surprisingly, we found that 27 of these loci were flanked by a TAR1 region (Telomere-Associated Repeat 1), typically found at subtelomeric promoters. This region displays a substantial homology with the functional parts of the TERRA promoter driving the transcription of telomeres, which our group described in a previous work, suggesting that also these interstitial promoters could be functional and drive the transcription of this group of ITSs. The analysis of the ENCODE project data showed that RNA polymerase II is found at these promoters and that they are classified as transcriptionally active. Finally, we experimentally demonstrated through Real Time qPCR that these ITSs are transcriptionally active. In conclusion, our work demonstrated for the first time that a fraction of human TERRA "reservoir" is composed by transcripts synthesized at ITS loci. Moreover, we described for the first time a group of ITS-loci flanked by a specific promoter sequence. We hypothesize that the transcription at the loci containing a promoter homologous to those found at subtelomeres is finely regulated. Additional experiments are required to further characterize these loci, to define their regulation, the function of the RNA molecules transcribed at ITS-loci and what is the connection with telomeric metabolism.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/24429