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The mechanisms of a mammalian splicing enhancer.

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journal contribution
posted on 2018-02-21, 12:21 authored by Andrew M. Jobbins, Linus F. Reichenbach, Christian M. Lucas, Andrew J. Hudson, Glenn A. Burley, Ian C. Eperon
Exonic splicing enhancer (ESE) sequences are bound by serine & arginine-rich (SR) proteins, which in turn enhance the recruitment of splicing factors. It was inferred from measurements of splicing around twenty years ago that Drosophila doublesex ESEs are bound stably by SR proteins, and that the bound proteins interact directly but with low probability with their targets. However, it has not been possible with conventional methods to demonstrate whether mammalian ESEs behave likewise. Using single molecule multi-colour colocalization methods to study SRSF1-dependent ESEs, we have found that that the proportion of RNA molecules bound by SRSF1 increases with the number of ESE repeats, but only a single molecule of SRSF1 is bound. We conclude that initial interactions between SRSF1 and an ESE are weak and transient, and that these limit the activity of a mammalian ESE. We tested whether the activation step involves the propagation of proteins along the RNA or direct interactions with 3' splice site components by inserting hexaethylene glycol or abasic RNA between the ESE and the target 3' splice site. These insertions did not block activation, and we conclude that the activation step involves direct interactions. These results support a model in which regulatory proteins bind transiently and in dynamic competition, with the result that each ESE in an exon contributes independently to the probability that an activator protein is bound and in close proximity to a splice site.

Funding

Leverhulme Trust [RPG-2014-001 to G.A.B. and I.C.E.]. Funding for open access charge: Universities of Leicester and Strathclyde.

History

Citation

Nucleic Acids Research, 2018, gky056

Author affiliation

/Organisation/COLLEGE OF LIFE SCIENCES/Biological Sciences/Molecular & Cell Biology

Version

  • VoR (Version of Record)

Published in

Nucleic Acids Research

Publisher

Oxford University Press (OUP)

issn

0305-1048

eissn

1362-4962

Acceptance date

2018-01-19

Copyright date

2018

Available date

2018-02-21

Publisher version

https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gky056/4829697

Notes

Supplementary Data are available at NAR Online. https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gky056#supplementary-data

Language

en

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