Co-transcriptional quality control of mRNA biogenesis : impact for human genetic diseases

Detalhes bibliográficos
Autor(a) principal: Drago, Rita Catarina Vaz
Data de Publicação: 2018
Idioma: eng
Título da fonte: Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
Texto Completo: http://hdl.handle.net/10451/37217
Resumo: Tese de doutoramento, Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2018
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spelling Co-transcriptional quality control of mRNA biogenesis : impact for human genetic diseasesTranscriçãoSplicing3' end processingControlo de qualidade co-transcricionalDoença genéticaTeses de doutoramento - 2018Domínio/Área Científica::Ciências Médicas::Medicina BásicaTese de doutoramento, Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2018Protein coding genes are transcribed in the nucleus by RNA polymerase II (RNAPII) forming a precursor messenger RNA (pre-mRNA) that undergoes extensive processing including 5' capping, splicing, 3' end cleavage and polyadenylation to form a mature mRNA. Pre-mRNA processing takes place cotranscriptionally, potentiated by the carboxyl-terminal domain (CTD) of the largest subunit of RNAPII, in a way that transcription and processing machineries communicate with each other to coordinate mRNA biogenesis. After being released from the chromatin template, mRNAs diffuse through the nucleoplasm until they encounter a nuclear pore to be translocated to the cytoplasm where they are translated into proteins, the final outcome of gene expression. Mutations that alter the coding sequence or affect splicing often result in the introduction of premature termination codons (PTCs). If translated, the resulting mRNAs would give rise to truncated proteins with potential deleterious effect for the cell. However, this rarely occurs because eukaryotic cells are able to recognize and degrade mRNAs containing PTCs by a cytoplasmic pathway referred to as nonsense-mediated mRNA decay (NMD). NMD was the first reported example of a quality control mechanism of gene expression. The advantages of mRNA quality control started to be appreciated in the case of beta-thalassemia, as it was found that in most cases only homozygotes suffered from severe anemia. Heterozygotes tend to be phenotypically healthy because NMD prevents production of truncated forms of beta-globin. In addition, thalassemia-like beta-globin mutations resulting in mRNA processing defects induce a nuclear RNA surveillance mechanism that lead to the retention of RNAs near the transcription site. To study the quality control mechanisms that operate during mRNA biogenesis it is essential to fully understand the process of gene expression in health and disease. One pertinent question that was addressed in my PhD work was how general the co-transcriptional mRNA quality control mechanism is and what is its impact in human genetic diseases. To address this question, I used as model system lymphoblastoid cell lines from patients with genetic diseases caused by splicing mutations and mutations in the coding region that introduce a PTC. Quantification of nascent transcripts revealed that a subset of genes containing splicing mutations have reduced transcriptional activity. Inhibition of NMD did not alter the levels of chromatin-associated transcripts, suggesting that a transcription-coupled surveillance mechanism operates independently from NMD to reduce cellular levels of abnormal RNAs in the context of human genetic diseases. Disease-causing mutations that disrupt splicing are mostly localized in splice sites, however next-generation sequencing has revealed that mutations localized deep within introns (more than 100 base pairs away from exonintron junctions) can be the cause of human genetic diseases. Aiming to highlight the importance of studying variation in deep intronic sequences, I reviewed evidence from mRNA analysis and entire genomic sequencing indicating that deep-intronic pathogenic mutations are the cause of over 75 monogenic disorders as well as hereditary cancer syndromes. Interestingly, deep-intronic mutations most commonly create/activate non-canonical splice sites in the pre-mRNA molecule that subsequently lead to pseudo-exon inclusion in the mature mRNA. Since disruption of splicing causes approximately 30% of human genetic diseases, measurement of splicing efficiency is essential to understanding gene regulation in wild-type and splicing-mutated genes. A variety of approaches have been used to purify nascent transcripts and determine the efficiency of splicing. Specifically, purification of newly transcribed molecules using 4sU-tagging has been widely used. Classically, this approach relies on treatment with a thio-reactive reagent HPDP to biotinylate the tagged RNA, which is then affinity-purified with streptavidin. Taking advantage of an efficient biotinylation strategy that uses MTS reagent, I showed that nascent RNA purified with biotin-HPDP contains a significantly higher proportion of unspliced long introns compared to RNAs purified with MTS-biotin. This argues that the splicing kinetics of long introns may be selectively underestimated in studies using biotin-HPDP, which may lead to mis-calculation of processing efficiency in different biological contexts. Disruption of 3' end processing can also be the cause of many human disorders. However, compared to splicing, this step of mRNA biogenesis has been less studied. To further study 3' end processing and transcription xxvi termination, I used a live-cell and single-molecule approach, in which time of release of two different reporter transcripts from the transcription site (TS) was measured. By using two different RNA labelling methods, MS2 and PP7, I showed that β-globin and IgM transcripts are released within 15-25 seconds after transcription of the 3' end of the gene. Furthermore, I showed that downregulating the cleavage factor CPSF3 by RNAi increases time of permanence at TS of both transcripts. Using a different RNA labelling method inserted past the poly(A) site (λN22), I determined that the time of transcription termination ranges between 20-80 seconds, with an average of 30 seconds. These results have important implications for a mechanistic understanding of mRNA biogenesis, particularly at 3' end. Altogether, the original data that resulted in this dissertation detailed the processes involved in mRNA synthesis and decay in the contexts of health and disease.Fonseca, M. Carmo, 1959-Custódio, Noélia Maria Fernandes, 1971-Repositório da Universidade de LisboaDrago, Rita Catarina Vaz2021-06-25T00:30:16Z201820182018-01-01T00:00:00Zdoctoral thesisinfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://hdl.handle.net/10451/37217TID:101459076enginfo:eu-repo/semantics/openAccessreponame:Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)instname:FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiainstacron:RCAAP2025-03-17T14:03:36Zoai:repositorio.ulisboa.pt:10451/37217Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireinfo@rcaap.ptopendoar:https://opendoar.ac.uk/repository/71602025-05-29T03:01:34.634208Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) - FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiafalse
dc.title.none.fl_str_mv Co-transcriptional quality control of mRNA biogenesis : impact for human genetic diseases
title Co-transcriptional quality control of mRNA biogenesis : impact for human genetic diseases
spellingShingle Co-transcriptional quality control of mRNA biogenesis : impact for human genetic diseases
Drago, Rita Catarina Vaz
Transcrição
Splicing
3' end processing
Controlo de qualidade co-transcricional
Doença genética
Teses de doutoramento - 2018
Domínio/Área Científica::Ciências Médicas::Medicina Básica
title_short Co-transcriptional quality control of mRNA biogenesis : impact for human genetic diseases
title_full Co-transcriptional quality control of mRNA biogenesis : impact for human genetic diseases
title_fullStr Co-transcriptional quality control of mRNA biogenesis : impact for human genetic diseases
title_full_unstemmed Co-transcriptional quality control of mRNA biogenesis : impact for human genetic diseases
title_sort Co-transcriptional quality control of mRNA biogenesis : impact for human genetic diseases
author Drago, Rita Catarina Vaz
author_facet Drago, Rita Catarina Vaz
author_role author
dc.contributor.none.fl_str_mv Fonseca, M. Carmo, 1959-
Custódio, Noélia Maria Fernandes, 1971-
Repositório da Universidade de Lisboa
dc.contributor.author.fl_str_mv Drago, Rita Catarina Vaz
dc.subject.por.fl_str_mv Transcrição
Splicing
3' end processing
Controlo de qualidade co-transcricional
Doença genética
Teses de doutoramento - 2018
Domínio/Área Científica::Ciências Médicas::Medicina Básica
topic Transcrição
Splicing
3' end processing
Controlo de qualidade co-transcricional
Doença genética
Teses de doutoramento - 2018
Domínio/Área Científica::Ciências Médicas::Medicina Básica
description Tese de doutoramento, Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2018
publishDate 2018
dc.date.none.fl_str_mv 2018
2018
2018-01-01T00:00:00Z
2021-06-25T00:30:16Z
dc.type.driver.fl_str_mv doctoral thesis
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10451/37217
TID:101459076
url http://hdl.handle.net/10451/37217
identifier_str_mv TID:101459076
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
instname:FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
instacron:RCAAP
instname_str FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
instacron_str RCAAP
institution RCAAP
reponame_str Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
collection Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
repository.name.fl_str_mv Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) - FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
repository.mail.fl_str_mv info@rcaap.pt
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