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Single-molecule imaging of transcription dynamics
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Resumo(s)
Transcription is the molecular process that synthesizes an RNA molecule that is complementary to the DNA template. In eukaryotes, transcription is catalyzed by one of three RNA polymerases that share structural features and subunits but transcribe different types of genes. RNA polymerase II (RNA Pol II) transcribes genes that code for proteins and several long non-coding genes. RNA Pol II transcription involves three main stages: initiation; transition into productive elongation; and termination. This thesis focuses on transcription termination and aims at studying its functional links with RNA processing and the formation of non-canonical nucleic acids structures, such as R-loops. R-loops are by-products of transcription that also have been found to play a role in transcription regulation, however, it is not well understood how they affect transcription termination and mRNA processing. To decipher how R-loop formation affect the process of transcription and RNA processing, we directly examined with single-molecule sensitivity the synthesis of transcripts upstream and downstream the cleavage and polyadenylation site (CPAS) in the nucleus of living human cells and calculated the transcription rate of RNA Pol II for that regions. By using two different RNA labeling methods, PP7 and λN22, we show that R-loops suppression impairs transcription termination. Further, we show that R-loop formation after the CPAS is not essential for an immediate transcription termination. A model for transcription termination, the torpedo model, suggests that exoribonuclease 2 (XRN2) digests the unprotected 5’ end of the RNA transcript until it collides with the RNA Pol II, leading to its dissociation from the template DNA and favoring transcription termination. Here, we provide evidence for the torpedo model by observing a non-immediate termination when XRN2 is depleted. Moreover, we show that splicing inhibition impairs transcript cleavage and transcription termination. Our data provide new insights in the role of R-loops in transcription termination and mRNA processing.
Descrição
Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2019
Palavras-chave
R-loops Terminação da transcrição Splicing Microscopia confocal de células vivas com sensibilidade de para detetar moléculas individuais Cinética da RNA polimerase II Modelo do torpedo Teses de mestrado - 2019