Understanding, predicting and preventing the impact of nonsense mutations on gene expression by deep mutational scanning

[eng] Premature termination codons (PTCs) are responsible for ~10–20% of inherited diseases and represent a major mechanism of tumor suppressor gene inactivation in cancer. Traditionally, PTCs are considered to induce transcript degradation via nonsense-mediated mRNA decay (NMD) and lead to the prod...

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Detalhes bibliográficos
Autor: Toledano Martin, Ignasi
Tipo de documento: tese
Estado:Versão publicada
Data de publicação:2025
País:España
Recursos:Universidad de Barcelona
Repositório:Dipòsit Digital de la UB
OAI Identifier:oai:diposit.ub.edu:2445/219877
Acesso em linha:https://hdl.handle.net/2445/219877
http://hdl.handle.net/10803/694048
Access Level:Acceso aberto
Palavra-chave:Mutació (Biologia)
RNA
Transcripció genètica
Mutation (Biology)
Genetic transcription
id ES_f3eb2190ed02bc4da32015cfa096f195
oai_identifier_str oai:diposit.ub.edu:2445/219877
network_acronym_str ES
network_name_str España
repository_id_str
dc.title.none.fl_str_mv Understanding, predicting and preventing the impact of nonsense mutations on gene expression by deep mutational scanning
title Understanding, predicting and preventing the impact of nonsense mutations on gene expression by deep mutational scanning
spellingShingle Understanding, predicting and preventing the impact of nonsense mutations on gene expression by deep mutational scanning
Toledano Martin, Ignasi
Mutació (Biologia)
RNA
Transcripció genètica
Mutation (Biology)
Genetic transcription
title_short Understanding, predicting and preventing the impact of nonsense mutations on gene expression by deep mutational scanning
title_full Understanding, predicting and preventing the impact of nonsense mutations on gene expression by deep mutational scanning
title_fullStr Understanding, predicting and preventing the impact of nonsense mutations on gene expression by deep mutational scanning
title_full_unstemmed Understanding, predicting and preventing the impact of nonsense mutations on gene expression by deep mutational scanning
title_sort Understanding, predicting and preventing the impact of nonsense mutations on gene expression by deep mutational scanning
dc.creator.none.fl_str_mv Toledano Martin, Ignasi
author Toledano Martin, Ignasi
author_facet Toledano Martin, Ignasi
author_role author
dc.contributor.none.fl_str_mv Supek, Fran
Lehner, Ben, 1978-
Universitat de Barcelona. Facultat de Biologia
dc.subject.none.fl_str_mv Mutació (Biologia)
RNA
Transcripció genètica
Mutation (Biology)
Genetic transcription
topic Mutació (Biologia)
RNA
Transcripció genètica
Mutation (Biology)
Genetic transcription
description [eng] Premature termination codons (PTCs) are responsible for ~10–20% of inherited diseases and represent a major mechanism of tumor suppressor gene inactivation in cancer. Traditionally, PTCs are considered to induce transcript degradation via nonsense-mediated mRNA decay (NMD) and lead to the production of truncated non-functional proteins. Nonsense suppression therapies aim to promote translational readthrough over PTCs, enabling the synthesis of full-length proteins. Both NMD and readthrough modulate the severity of disease phenotypes by regulating the abundance of the mRNA and the full-length protein; respectively. However, their efficiencies vary across PTCs. In this thesis, we employed deep mutagenesis methods to systematically quantify how sequence context and other genetic factors influence the mRNA levels and the full-length protein abundance of PTC-containing transcripts. First, we developed a methodological improvement for deep mutagenesis libraries generation. Second, a comprehensive assessment of drug-induced readthrough was performed, encompassing over 140,000 measurements and generating readthrough scores for 6,000 PTCs that cause genetic diseases and cancer. This massive dataset was subsequently leveraged to elucidate the effect of sequence context on readthrough and to train accurate predictive models to estimate drug-specific PTC readthrough genome-wide. We envisage these datasets will become a valuable resource to improve clinical trial design and the development of personalized nonsense suppression therapies. Third, we combined different libraries to test and extend hypotheses for how PTC position, exon length, sequence context and translation reinitiation interplay to determine NMD efficiency. Overall, this thesis provides a comprehensive view of how the sequence landscape influences the fate of PTC-containing transcripts. More broadly, it demonstrates the effectiveness of deep mutagenesis in uncovering sequence-to- activity relationships, highlighting the potential of this approach for investigating other mRNA-related processes.
publishDate 2025
dc.date.none.fl_str_mv 2025
dc.type.none.fl_str_mv info:eu-repo/semantics/doctoralThesis
info:eu-repo/semantics/publishedVersion
format doctoralThesis
status_str publishedVersion
dc.identifier.none.fl_str_mv https://hdl.handle.net/2445/219877
http://hdl.handle.net/10803/694048
url https://hdl.handle.net/2445/219877
http://hdl.handle.net/10803/694048
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv cc by-nd (c) Toledano Martin, Ignasi, 2025
http://creativecommons.org/licenses/by-nd/3.0/es/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv cc by-nd (c) Toledano Martin, Ignasi, 2025
http://creativecommons.org/licenses/by-nd/3.0/es/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Universitat de Barcelona
publisher.none.fl_str_mv Universitat de Barcelona
dc.source.none.fl_str_mv Tesis Doctorals - Facultat - Biologia
reponame:Dipòsit Digital de la UB
instname:Universidad de Barcelona
instname_str Universidad de Barcelona
reponame_str Dipòsit Digital de la UB
collection Dipòsit Digital de la UB
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869424422288359425
spelling Understanding, predicting and preventing the impact of nonsense mutations on gene expression by deep mutational scanningToledano Martin, IgnasiMutació (Biologia)RNATranscripció genèticaMutation (Biology)Genetic transcription[eng] Premature termination codons (PTCs) are responsible for ~10–20% of inherited diseases and represent a major mechanism of tumor suppressor gene inactivation in cancer. Traditionally, PTCs are considered to induce transcript degradation via nonsense-mediated mRNA decay (NMD) and lead to the production of truncated non-functional proteins. Nonsense suppression therapies aim to promote translational readthrough over PTCs, enabling the synthesis of full-length proteins. Both NMD and readthrough modulate the severity of disease phenotypes by regulating the abundance of the mRNA and the full-length protein; respectively. However, their efficiencies vary across PTCs. In this thesis, we employed deep mutagenesis methods to systematically quantify how sequence context and other genetic factors influence the mRNA levels and the full-length protein abundance of PTC-containing transcripts. First, we developed a methodological improvement for deep mutagenesis libraries generation. Second, a comprehensive assessment of drug-induced readthrough was performed, encompassing over 140,000 measurements and generating readthrough scores for 6,000 PTCs that cause genetic diseases and cancer. This massive dataset was subsequently leveraged to elucidate the effect of sequence context on readthrough and to train accurate predictive models to estimate drug-specific PTC readthrough genome-wide. We envisage these datasets will become a valuable resource to improve clinical trial design and the development of personalized nonsense suppression therapies. Third, we combined different libraries to test and extend hypotheses for how PTC position, exon length, sequence context and translation reinitiation interplay to determine NMD efficiency. Overall, this thesis provides a comprehensive view of how the sequence landscape influences the fate of PTC-containing transcripts. More broadly, it demonstrates the effectiveness of deep mutagenesis in uncovering sequence-to- activity relationships, highlighting the potential of this approach for investigating other mRNA-related processes.[cat] Els codons de terminació prematura (PTCs) són responsables del ~10-20% de les malalties hereditàries i representen un mecanisme important d'inactivació dels gens supressors de tumors en el càncer. Tradicionalment, es considera que els PTCs indueixen la degradació del trànscrit mitjançant la degradació de l'ARNm mediada per codons stop (NMD) i causen la producció de proteïnes truncades no funcionals. Les teràpies de supressió de codons stop busquen promoure el ‘llegir a través’ dels PTCs (procés conegut com a readthrough), el que permet la síntesi de proteïnes completes. Tant la NMD com la ‘lectura a través’ modulen la gravetat de les malalties al regular l'abundància de l'ARNm i de la proteïna completa, respectivament. Tanmateix, les seves eficiències varien entre els PTCs. En aquesta tesi, hem utilitzat mètodes de mutagènesis massiva per quantificar sistemàticament com el context de la seqüència i altres factors genètics influeixen en l'abundància de l'ARNm i de la proteïna completa dels trànscrits que tenen PTCs. En primer lloc, hem desenvolupat una millora metodològica per a la generació de llibreries pels experiments de mutagènesis massiva. En segon lloc, hem fet una avaluació exhaustiva de com diferents fàrmacs estimulen el readthrough, on hem dut a terme més de 140,000 mesures per als 6,000 PTCs més comuns en malalties genètiques i càncer. Posteriorment, hem utilizat aquesta base de dades per estudiar com la seqüència afecta l’eficiència del readthrough i per entrenar models predictius precisos per estimar l’eficiència dels fàrmacs en tots els possibles PTCs que poden haver-hi en el genoma humà. Creiem que aquest estudi esdevindrà un recurs valuós per millorar el disseny d'assajos clínics i el desenvolupament de teràpies personalitzades de supressió de codons sense sentit. Finalment, en el tercer projecte, hem combinat diferents llibreries per investigar en més profunditat com la posició del PTC, la longitud de l'exó, el context de la seqüència i la reiniciació de la traducció interactuen per determinar l'eficiència de la NMD. En general, aquesta tesi ofereix una visió completa de com la seqüència influeix en el regulació dels trànscrits mutats per PTCs. Més enllà, demostra l'eficàcia del la mutagènesis massiva per estudiar les relacions entre seqüència i activitat, destacant el potencial d'aquesta estratègia per investigar altres processos regulats a nivell de l'ARNm.Universitat de BarcelonaSupek, FranLehner, Ben, 1978-Universitat de Barcelona. Facultat de Biologia2025info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://hdl.handle.net/2445/219877http://hdl.handle.net/10803/694048Tesis Doctorals - Facultat - Biologiareponame:Dipòsit Digital de la UBinstname:Universidad de BarcelonaIngléscc by-nd (c) Toledano Martin, Ignasi, 2025http://creativecommons.org/licenses/by-nd/3.0/es/info:eu-repo/semantics/openAccessoai:diposit.ub.edu:2445/2198772026-05-27T06:46:51Z
score 15.811543

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