Understanding alternative splicing using deep mutagenesis

Alternative pre-mRNA splicing is a regulated step in eukaryotic gene expression in which introns are removed from the transcript and exons are joined together to form a mature mRNA. To study the effects of mutations on alternative splicing, we built a mutant library containing all combinations of 12...

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Autor: Baeza Centurión, Pablo
Formato: tesis doctoral
Estado:Versión publicada
Fecha de publicación:2020
País:España
Recursos:CBUC, CESCA
Repositorio:TDR. Tesis Doctorales en Red
OAI Identifier:oai:www.tdx.cat:10803/668749
Acesso em linha:http://hdl.handle.net/10803/668749
Access Level:acceso abierto
Palavra-chave:Alternative splicing
Mutation
Gene regulation
Regulatory sequence
RNA
Empalme alternativo
Mutación
Regulación genética
Secuencia reguladora
575
id ES_cd9972ae9ccc7b7359cc75ce0e20bf26
oai_identifier_str oai:www.tdx.cat:10803/668749
network_acronym_str ES
network_name_str España
repository_id_str
dc.title.none.fl_str_mv Understanding alternative splicing using deep mutagenesis
title Understanding alternative splicing using deep mutagenesis
spellingShingle Understanding alternative splicing using deep mutagenesis
Baeza Centurión, Pablo
Alternative splicing
Mutation
Gene regulation
Regulatory sequence
RNA
Empalme alternativo
Mutación
Regulación genética
Secuencia reguladora
575
title_short Understanding alternative splicing using deep mutagenesis
title_full Understanding alternative splicing using deep mutagenesis
title_fullStr Understanding alternative splicing using deep mutagenesis
title_full_unstemmed Understanding alternative splicing using deep mutagenesis
title_sort Understanding alternative splicing using deep mutagenesis
dc.creator.none.fl_str_mv Baeza Centurión, Pablo
author Baeza Centurión, Pablo
author_facet Baeza Centurión, Pablo
author_role author
dc.contributor.none.fl_str_mv Lehner, Ben
Universitat Pompeu Fabra. Departament de Ciències Experimentals i de la Salut
dc.subject.none.fl_str_mv Alternative splicing
Mutation
Gene regulation
Regulatory sequence
RNA
Empalme alternativo
Mutación
Regulación genética
Secuencia reguladora
575
topic Alternative splicing
Mutation
Gene regulation
Regulatory sequence
RNA
Empalme alternativo
Mutación
Regulación genética
Secuencia reguladora
575
description Alternative pre-mRNA splicing is a regulated step in eukaryotic gene expression in which introns are removed from the transcript and exons are joined together to form a mature mRNA. To study the effects of mutations on alternative splicing, we built a mutant library containing all combinations of 12 mutations that accumulated during the emergence of an alternatively-spliced human exon: FAS exon 6. This allowed us to study the effects of individual nucleotide substitutions in thousands of different closely-related genetic contexts. We show that the effect of the same mutation on exon inclusion depends non-monotonically on the inclusion levels of the exon before the mutation is made, with mutations having their strongest splice-altering effects in exons with intermediate levels of inclusion, and their smallest effects in exons that are always skipped or always included. After performing deep mutagenesis in two highly included (constitutive) exons, we find that, in agreement with our previous results, mutations have a very small effect on the inclusion of constitutive exons with the exception of mutations in exon-intron boundaries (splice sites). Since alternative splicing is frequently perturbed in human genetic diseases, we then put these results in a more practical context to address the question of the extent to which random mutations in the human genome are likely to have splice-altering effects. Since most human exons are highly included, we conclude that a random mutation is unlikely to have a splice altering effect, and that altered splicing should only be considered a likely proximal disease mechanism for mutations that affect alternatively spliced exons (included at intermediate levels) or disrupt splice sites.
publishDate 2020
dc.date.none.fl_str_mv 2020
2020
2022
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 http://hdl.handle.net/10803/668749
url http://hdl.handle.net/10803/668749
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv http://creativecommons.org/licenses/by-nc/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 217 p.
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Universitat Pompeu Fabra
publisher.none.fl_str_mv Universitat Pompeu Fabra
dc.source.none.fl_str_mv TDX (Tesis Doctorals en Xarxa)
reponame:TDR. Tesis Doctorales en Red
instname:CBUC, CESCA
instname_str CBUC, CESCA
reponame_str TDR. Tesis Doctorales en Red
collection TDR. Tesis Doctorales en Red
repository.name.fl_str_mv
repository.mail.fl_str_mv
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spelling Understanding alternative splicing using deep mutagenesisBaeza Centurión, PabloAlternative splicingMutationGene regulationRegulatory sequenceRNAEmpalme alternativoMutaciónRegulación genéticaSecuencia reguladora575Alternative pre-mRNA splicing is a regulated step in eukaryotic gene expression in which introns are removed from the transcript and exons are joined together to form a mature mRNA. To study the effects of mutations on alternative splicing, we built a mutant library containing all combinations of 12 mutations that accumulated during the emergence of an alternatively-spliced human exon: FAS exon 6. This allowed us to study the effects of individual nucleotide substitutions in thousands of different closely-related genetic contexts. We show that the effect of the same mutation on exon inclusion depends non-monotonically on the inclusion levels of the exon before the mutation is made, with mutations having their strongest splice-altering effects in exons with intermediate levels of inclusion, and their smallest effects in exons that are always skipped or always included. After performing deep mutagenesis in two highly included (constitutive) exons, we find that, in agreement with our previous results, mutations have a very small effect on the inclusion of constitutive exons with the exception of mutations in exon-intron boundaries (splice sites). Since alternative splicing is frequently perturbed in human genetic diseases, we then put these results in a more practical context to address the question of the extent to which random mutations in the human genome are likely to have splice-altering effects. Since most human exons are highly included, we conclude that a random mutation is unlikely to have a splice altering effect, and that altered splicing should only be considered a likely proximal disease mechanism for mutations that affect alternatively spliced exons (included at intermediate levels) or disrupt splice sites.El empalme alternativo es un proceso de la expresión génica en eucariontes en el que los intrones del transcrito se eliminan, dejando únicamente exones para formar un RNAm maduro. Para estudiar los efectos de mutaciones en este proceso, diseñamos una librería de mutantes con las 12 mutaciones (y todas sus combinaciones) que surgieron a lo largo de la evolución enhumanos de un exón alternativo: el exón 6 de FAS. Esto nos permitió estudiar los efectos de cada mutación en miles de contextos genéticos distintos. Descubrimos que la misma mutación puede tener efectos muy diferentes en el empalme de un exón dependiendo de los niveles de inclusión del mismo. Los mayores efectos se observan en exones con niveles intermedios de inclusión, mientras que los menores efectos ocurren en exones con niveles de inclusión muy altos o muy bajos. Tras mutagenizar dos exones constitutivos, confirmamos que, con la excepción de mutaciones en los sitios de empalme, es poco probable que una mutación afecte la inclusión de dichos exones. Dado que el empalme alternativo es un proceso se encuentra alterado en muchas enfermedades genéticas humanas, pusimos nuestros resultados en un contexto más práctico al plantearnos qué probabilidad hay de que una mutación al azar sea capaz de alterar dicho proceso. Ya que la gran mayoría de exones humanos tienen altos niveles de inclusión, concluimos que es poco probable que una mutación escogida al azar sea capaz de alterar los niveles de inclusión de algún exón. De hecho, esto sólo es probable en el caso de mutaciones en los sitios de empalme o de aquellas que afecten la inclusión de un exón alternativo.Programa de doctorat en BiomedicinaUniversitat Pompeu FabraLehner, BenUniversitat Pompeu Fabra. Departament de Ciències Experimentals i de la Salut202020222020info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersion217 p.application/pdfapplication/pdfhttp://hdl.handle.net/10803/668749TDX (Tesis Doctorals en Xarxa)reponame:TDR. Tesis Doctorales en Redinstname:CBUC, CESCAInglésL'accés als continguts d'aquesta tesi queda condicionat a l'acceptació de les condicions d'ús establertes per la següent llicència Creative Commons: http://creativecommons.org/licenses/by-nc/4.0/http://creativecommons.org/licenses/by-nc/4.0/info:eu-repo/semantics/openAccessoai:www.tdx.cat:10803/6687492026-06-14T12:46:07Z
score 15,301629

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