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Unveiling co-transcriptional splicing kinetics using novel nascent transcriptomic techniques
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Orientador(es)
Resumo(s)
Most RNA splicing – the excision of non-coding regions within precursor messenger RNA (mRNA)
molecules – happens at the nascent moment, when the multiprotein complex RNA Polymerase II (Pol
II) is synthesizing the new RNA molecules. Post-translational phosphorylations on the C-terminal
domain (CTD) of the biggest Pol II subunit have been indicated as an inducer of different transcriptional
and processivity moments. However, how a temporary inhibition of transcription influences these
dynamics is still largely unexplored.
In this study, a temporary transcription inhibition was promoted in Hela cells with the reversible drug
5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). To analyse co-transcriptional splicing, it
was employed the mammalian native elongation transcript sequencing (mNET-seq) technique, that
isolates the latest transcribed nucleotides from nascent pre-mRNAs, in a Pol II CTD-specific manner.
The results of this work showed that there was a decrease of co-transcriptional splicing after treating
cells with DRB. 47% of exons before DRB had splicing intermediates peaks – indicative of splicing first
reaction – but only ~10% had these peaks 30 minutes after washing DRB. Splicing ratio distribution
also shifted towards 0 after DRB treatment.
It was also detected an increase of CTD Tyrosine 1 phosphorylation (Y1P) during and after the
treatment. The absence of spliceosome components in Y1P elongation complex and the low levels of
splicing signals in nascent RNA from Pol II Y1P suggest that this phosphorylation might not be associate
with co-transcriptional splicing. This could explain the decrease observed after transcription restart.
The association of CTD Serine 5 phosphorylation with splicing, discovered in previous studies, was also
identified in these analyses, and barely affected by the DRB treatment.
To conclude, these results suggest that higher Y1P levels inhibit co-transcription splicing, although
further experiments are necessary to discriminate its effect from the influence of S5P.
Descrição
Tese de mestrado, Bioinformática e Biologia Computacional, 2023, Universidade de Lisboa, Faculdade de Ciências
Palavras-chave
Splicing co-transcricional mammalian native elongation transcript sequencing (mNET-seq) carboxyl terminal domain da polymerase II (Pol II CTD) inibição da transcrição fosforilação da Tirosina 1 Teses de mestrado - 2023