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The Involvement of SRSF1 in pre-mRNA splicing

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posted on 11.09.2018, 12:41 authored by Andrew Michael Jobbins
The key splicing signals in pre-mRNA, the branch-point, 5’ splice site and 3’ splice site, are exceptionally poorly conserved in mammals. To compensate for this, key regulatory sequences are required to direct the reciprocal factors, U2AF, U1 snRNP and U2 snRNP respectively, to the correct sites. These regulatory sequences function by recruiting activators, of which the main family are the SR proteins, or repressors, of which the main family are the hnRNP proteins, which in turn stimulate or repress the binding of key spliceosomal factors. The archetypal SR protein is SRSF1. SRSF1 was the first non-snRNP factor identified and the first found to control alternative splicing. Its best-understood activity is to stimulate the inclusion of exons by binding to purine-rich exonic splicing enhancer (ESE) sequences. There is also some evidence suggesting its involvement in constitutive splicing, which began with demonstrations that it could compensate for depletion of the U1 snRNP. However, further investigations into both its recruitment via ESEs and its possible role in constitutive splicing have foundered due to apparent nonstoichiometric binding. Single molecule experiments allow us to look at the exact number of SRSF1 proteins that bind. The experiments outlined here indicate that the U1 snRNP can actually recruit SRSF1 in a stoichiometric manner. This implicates a possible recruitment mechanism for SRSF1 which would allow it to play a role in core splicing reactions and exon definition. Furthermore we demonstrate that with increasing numbers of enhancers, which sequentially increase splicing efficiency, the number of SRSF1 proteins bound does not change but the chance of a protein binding event increases. This fits a model in which the initial binding of SRSF1 is weak and transient. The same construct is also used to show that introducing a non RNA link in between an ESE and its target site does not silence the ESEs effect, indicating that ESEs exert their effect via RNA loops.

History

Supervisor(s)

Eperon, Ian; Dominguez, Cyril

Date of award

03/07/2018

Author affiliation

Department of Molecular and Cell Biology

Awarding institution

University of Leicester

Qualification level

Doctoral

Qualification name

PhD

Language

en

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