Short ITSs are a particular type of microsatellite and our group demonstrated that originated from a mechanism different from the canonical DNA polymerase slippage that leads to the progressive expansion or contraction of microsatellites. The comparative analysis of human ITS loci with the empty orthologs in primates showed that s-ITSs appear suddenly during evolution and allowed to propose that the introduction of telomeric-like repeats occurs during the repair of DNA double strand breaks (Nergadze et al. 2004) and that this insertion is mediated by telomerase. This model was supported by the observation, in the mouse and rat genomes, of 14 and 3 ITSs, respectively, flanked by sequences reverse transcribed from the 3’ end of the Telomerase RNA Component (TERC-ITSs). The recent identification of a horse TERC-ITS locus (Santagostino et al. 2020) prompted us to verify whether the insertion of TERC-derived sequences together with short telomeric repeat stretches can be found in the genomes of other species, thus suggesting that they are the result of a general mechanism typical of organisms in which telomeres are maintained by telomerase. To this purpose, I carried out an extensive search of TERC-ITSs in the genomic sequence of 21 vertebrates including 19 mammals, a bird and an amphibian. I also integrated all these observations with previous data from our laboratory regarding rodents (Nergadze et al. 2007) and horse (Santagostino et al. 2020). We found TERC-like insertions in 16 out of 21 genomes. I identified the presence of 21 TERC-ITSs. In 16 TERC-ITSs the length of the reverse transcribed TERC region is comprised between 20 and 33 nucleotides while the length of the ITSs is variable ranging from 6 to 43 nucleotides. Exceptions are represented by Chinese hamster (4 TERC-ITSs) and wallaby (a TERC-ITS), in which both the retrotranscribed TERC region and the ITSs are longer compared to other animals and comparable with those previously identified in mouse and rat (Nergadze et al. 2007). The sequence conservation of TERC-ITS loci was assessed using these loci as query for a BLAST search against the genome of evolutionary related species. Moreover, we found one locus in squirrel monkey and one locus in cow containing TERC-like sequences but missing the TTAGG repeat. For these loci, an empty orthologous locus was identified. Given their sequence organization, these loci can be considered pseudogenes. Finally, TERC pseudogenes were identified in pig, wallaby and in African clawed frog. Interestingly, the region of TERC retrotranscribed during the insertion of TERC-ITSs is different in different species. In rodents the 3’ end of TERC is the sequence retrotranscribed at interstitial sites, while, in mammals and amphibians these insertions mainly derive from the pseudoknot/template domain. The genomes of several species of Drosophila were also analyzed and no TERC-ITSs were identified. Since it is well known that telomeres in this species are maintained by a mechanism based on the reverse transcription of retrotransposons that integrate specifically at chromosome ends, they were utilized as outgroup and confirmed that TERC-ITSs and TERC-like insertions only occur in species where telomeres are maintained via telomerase-mediated mechanism. In conclusion, our work confirms our previous hypothesis that the Telomerase RNA Component can participate to the repair of DNA double strand breaks (Nergadze et al. 2007; Santagostino et al. 2020) via NHEJ, giving rise to TERC-ITSs or TERC-like insertions. These data, together with the previous findings of our laboratory (Nergadze et al. 2007; Santagostino et al. 2020), are the direct evidence that, telomerase can reverse transcribe portions of its RNA moiety at non-terminal sites in a transposon-like fashion.
L’analisi comparativa dei loci ITS umani con loci ortologhi in altri primati ha mostrato che le s-ITSs si sono formate, durante l’evoluzione, attraverso l’inserimento di sequenze simili a quelle telomeriche durante la riparazione del DNA in siti di rottura a doppio filamento (Nergadze et al. 2004) tramite il coinvolgimento della telomerasi. Questo modello è stato supportato dall’osservazione, nel genoma di topo e ratto, di 14 e 3 ITSs, rispettivamente, fiancheggiante da sequenze retrotranscritte derivanti dal 3’ della componente a RNA della telomerasi (TERC-ITSs). Questa scoperta suggerisce il forte coinvolgimento della telomerasi durante l’inserzione delle ITSs nel riparo dei siti di rottura a doppio filamento del DNA, attraverso un processo simile a quello della retrotransposizione (Nergadze et al. 2007). La recente identificazione di una TERC-ITS nel genoma di cavallo (Santagostino et al. 2020) ci ha spinto a verificare se l’inserzione di sequenze derivanti da TERC insieme a corte sequenze telomeriche può essere anche identificata nel genoma di altre specie. Per questo, ho svolto un’analisi esaustiva con il fine di identificare la presenza di TERC-ITSs nel genoma di 21 specie di vertebrati inclusi 19 mammiferi, un uccello e un anfibio. Sorprendentemente, abbiamo trovato inserzioni di sequenze derivanti da TERC in 16 genomi su un totale di 21 specie analizzate. Sono state identificate 21 TERC-ITSs. In 16 la lunghezza della regione retrotranscritta di TERC è compresa tra 20 e 33 nucleotidi mentre la lunghezza della ITSs è variabile tra 6 e 43 nucleotidi. Eccezioni sono rappresentate dal wallaby e dal criceto cinese, nei quali sia la regione retrotranscritta di TERC, sia le ITS sono più lunghe se paragonate a quelle di altri animali, ma risultano simili con quelle precedentemente identificate in topo e ratto (Nergadze et al. 2007). Per verificare la conservazione dei loci TERC-ITS abbiamo svolto un’analisi nei genomi di specie strettamente correlate da un punto di vista evolutivo. Inoltre, abbiamo identificato la presenza di un locus nella scimmia scoiattolo e un locus in mucca contenenti sequenze derivanti da TERC ma mancanti della ripetizione telomerica. Per questi loci, un locus ortologo vuoto è stato identificato. Data la loro organizzazione, possono essere considerati come pseudogeni. In conclusione, pseudogeni di TERC sono stati identificati in maiale, wallaby e in Xenopus. Questi risultati dimostrano che differenti regioni di TERC possono essere retrotranscritte durante l’inserzione delle TERC-ITSs in specie differenti. L’analisi di sequenze di inserzione derivanti da TERC ha mostrato che, nei roditori il 3’ terminale di TERC è la sequenza retrotranscritta in siti interstiziali, mentre, in altri mammiferi e negli anfibi queste inserzioni derivano dal dominio chiamato pseudoknot/templato. Inoltre, è stato analizzato anche il genoma di diverse species di Drosophila e nessuna TERC-ITS è stata identificata. Siccome è ben risaputo che in questa specie i telomeri sono mantenuti da un meccanismo basato sulla retrotranscrizione di retrotransposoni che si integrano specificamente alle estremità dei cromosomi, sono state utilizzate come outgroup ed hanno confermato che le TERC-ITSs e le inserzioni derivanti da TERC avvengono solo in specie nelle quali i telomeri sono mantenuti attraverso un meccanismo mediato dalla telomerasi. In conclusione, il nostro lavoro conferma la precedente ipotesi che la componente a RNA della telomerasi può partecipare alla riparazione dei siti di rottura a doppio filamento del DNA attraverso NHEJ, originando TERC-ITSs o inserzioni derivanti da TERC. Questi dati, insieme alle precedenti scoperte del nostro laboratorio, sono la diretta evidenza che la telomerasi può retrotranscrivere porzioni del suo motivo a RNA a siti non-terminali con un meccanismo simile a quello dei retrotransposoni.
Retrotranscrizione della componente a RNA della telomerasi a siti di rottura a doppio filamento del DNA durante l’evoluzione del genoma dei vertebrati
SOLA, LORENZO
2019/2020
Abstract
Short ITSs are a particular type of microsatellite and our group demonstrated that originated from a mechanism different from the canonical DNA polymerase slippage that leads to the progressive expansion or contraction of microsatellites. The comparative analysis of human ITS loci with the empty orthologs in primates showed that s-ITSs appear suddenly during evolution and allowed to propose that the introduction of telomeric-like repeats occurs during the repair of DNA double strand breaks (Nergadze et al. 2004) and that this insertion is mediated by telomerase. This model was supported by the observation, in the mouse and rat genomes, of 14 and 3 ITSs, respectively, flanked by sequences reverse transcribed from the 3’ end of the Telomerase RNA Component (TERC-ITSs). The recent identification of a horse TERC-ITS locus (Santagostino et al. 2020) prompted us to verify whether the insertion of TERC-derived sequences together with short telomeric repeat stretches can be found in the genomes of other species, thus suggesting that they are the result of a general mechanism typical of organisms in which telomeres are maintained by telomerase. To this purpose, I carried out an extensive search of TERC-ITSs in the genomic sequence of 21 vertebrates including 19 mammals, a bird and an amphibian. I also integrated all these observations with previous data from our laboratory regarding rodents (Nergadze et al. 2007) and horse (Santagostino et al. 2020). We found TERC-like insertions in 16 out of 21 genomes. I identified the presence of 21 TERC-ITSs. In 16 TERC-ITSs the length of the reverse transcribed TERC region is comprised between 20 and 33 nucleotides while the length of the ITSs is variable ranging from 6 to 43 nucleotides. Exceptions are represented by Chinese hamster (4 TERC-ITSs) and wallaby (a TERC-ITS), in which both the retrotranscribed TERC region and the ITSs are longer compared to other animals and comparable with those previously identified in mouse and rat (Nergadze et al. 2007). The sequence conservation of TERC-ITS loci was assessed using these loci as query for a BLAST search against the genome of evolutionary related species. Moreover, we found one locus in squirrel monkey and one locus in cow containing TERC-like sequences but missing the TTAGG repeat. For these loci, an empty orthologous locus was identified. Given their sequence organization, these loci can be considered pseudogenes. Finally, TERC pseudogenes were identified in pig, wallaby and in African clawed frog. Interestingly, the region of TERC retrotranscribed during the insertion of TERC-ITSs is different in different species. In rodents the 3’ end of TERC is the sequence retrotranscribed at interstitial sites, while, in mammals and amphibians these insertions mainly derive from the pseudoknot/template domain. The genomes of several species of Drosophila were also analyzed and no TERC-ITSs were identified. Since it is well known that telomeres in this species are maintained by a mechanism based on the reverse transcription of retrotransposons that integrate specifically at chromosome ends, they were utilized as outgroup and confirmed that TERC-ITSs and TERC-like insertions only occur in species where telomeres are maintained via telomerase-mediated mechanism. In conclusion, our work confirms our previous hypothesis that the Telomerase RNA Component can participate to the repair of DNA double strand breaks (Nergadze et al. 2007; Santagostino et al. 2020) via NHEJ, giving rise to TERC-ITSs or TERC-like insertions. These data, together with the previous findings of our laboratory (Nergadze et al. 2007; Santagostino et al. 2020), are the direct evidence that, telomerase can reverse transcribe portions of its RNA moiety at non-terminal sites in a transposon-like fashion.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/20.500.14239/11893