Abstract
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.
Highlights
The high point in studies on the mechanisms of action of exonic splicing enhancer sequences was reached in 1998
To test whether there was a correlation between the efficacy of an Exonic splicing enhancer (ESE) and binding of SRSF1, four different sequences were tested: ESE-A, the known SRSF1 binding site from Ron exon 12 [60,61]; ESE-B, an SRSF1 binding motif identified by functional SELEX [62]; ESE-C, the Tra2 binding site from SMN exon 7, which has been shown to promote splicing but should not bind SRSF1 [63,64]; ESE-D, an SRSF1 motif established via RNA-seq [34]
The results described here show that tandemly repeated ESEs increased splicing efficiency linearly, but that only a single molecule of SRSF1 was recruited by the ESEs in addition to the one recruited by the 5’SS-bound U1 snRNP
Summary
The high point in studies on the mechanisms of action of exonic splicing enhancer sequences was reached in 1998. SR proteins have one or two RNA recognition motif (RRM)-type RNA-binding domains and a C-terminal RS domain, rich in arginine-serine dipeptides, that is subject to extensive phosphorylation [11,12,13]. The binding of these proteins to ESEs was shown to stimulate splicing, splicing complex assembly and binding of U2 snRNPs and U2-associated proteins U2AF65 and U2AF35 [8,14,15,16]. ESEs that are within 100 nts or so of the 3’SS were proposed to work by binding of an SR protein to the ESE and propagation of the complex by cooperative interactions with further SR proteins until interactions with U2AF were possible; ESEs that are regulated, such as the repeated elements in Drosophila dsx exon 4 (dsxREs) that depend on the proteins Tra and Tra, might form stable multi-protein complexes and interact directly over greater distances by 3D-diffusion (looping) [18]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
