Interconnections between transcription and pre-mRNA splicing
thesisposted on 2016-10-31, 11:37 authored by Oksana Gonchar
Eukaryotic gene expression involves many processes some of which are transcription and pre-mRNA splicing. It has been shown that the majority of splicing events are functionally or physically coupled to RNA polymerase II transcription machinery. This suggests that transcription and splicing processes might influence one another. For example, a number of studies have implicated snRNPs, in particular U1 snRNP, in transcription initiation and elongation. In this work, interconnections between transcription and splicing were tested using an in vitro RNA polymerase II transcription/splicing assays (Pol II-TSRs). The results obtained in this study showed that inhibition of U1 but not U2 or U6 snRNPs led to a major reduction in transcript levels using different DNA templates. However, interference with initiation was excluded because it was found that this effect was the result of reduced RNA stability. Moreover, similar results were observed both with transcription by T7 RNA polymerase and with purified transcripts added to the extract. These results allow to conclude that the U1 snRNP has a novel function in protecting RNA from degradation. Further investigations showed that the RNA is protected by the U1 snRNP against 5’ exonucleases and 3’ exonucleases and possibly endonucleases. It was found that the presence of 5’ splice site (5’SS) is necessary for RNA stability. These data suggest that the U1 snRNP through a direct interaction with the 5’SS of the pre-mRNA protects it from degradation and only U1 snRNP but not the active spliceosome is required for RNA stability. It was observed that under splicing conditions, the RNA level of a transcript lacking the intronic consensus 5’SS but having it at the 3’ end was significantly higher compared to that for a transcript lacking any consensus 5’SSs, suggesting that the U1 snRNP protects transcripts from degradation along their length. It is proposed that rapid binding of the U1 snRNPs to the nascent transcripts induces the assembly of RNA binding proteins that protect the RNA. Interestingly, any transcripts tested regardless of the presence of the consensus 5’SSs were stable in the reaction with ongoing transcription, suggesting that Pol II transcription has an additional effect on RNA stability.
Supervisor(s)Eperon, Ian; Dominguez, Cyril
Date of award2016-10-21
Author affiliationDepartment of Molecular and Cell Biology
Awarding institutionUniversity of Leicester