The mechanisms of a mammalian splicing enhancer

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.

Interplay between coding and exonic splicing regulatory sequences

“…However, it is in agreement with recent observations indicating that the interaction of a splicing factor with a binding motif depends on the sequence context and on the presence of clusters of related binding motifs (Zhang et al 2013;Cereda et al 2014;Fu and Ares 2014;Dominguez et al 2018;Jobbins et al 2018). For example, increasing the exonic frequency of GGAlike motifs increases the probability of an exon to be regulated by the SRSF1 splicing factor that binds to GGAGGA-like motifs although only one binding site is used (Jobbins et al 2018). In this setting, we observed similar nucleotide and amino acid composition biases when analyzing exons regulated by a splicing factor or exons containing CLIP-related signals for the same splicing factor (Supplemental Fig.…”

“…Indeed, splicing factor binding sites are unlikely to be defined by their sole nucleotide composition bias. For example, it has been recently shown that RNA binding sites are within specific context and that some RNA binding sites correspond to spaced "bipartite" short linear motifs (Zhang et al 2013;Cereda et al 2014;Fu and Ares 2014;Dominguez et al 2018;Jobbins et al 2018). By dissecting each RNA binding site, their flanking nucleotide preferences, their clustering, and the space between them, it would be possible to better characterize the way they can affect codon and amino acid usage.…”

“…First, splicing factors bind to short intronic or exonic motifs (or splicing regulatory sequences) that are often low-complexity sequences composed of the repetition of the same nucleotide or dinucleotide (Fu and Ares 2014). In addition, the interaction of splicing factors with their cognate binding motifs often depends on the sequence context and on the presence of clusters of related binding motifs (Zhang et al 2013;Cereda et al 2014;Fu and Ares 2014;Dominguez et al 2018;Jobbins et al 2018). The second principle states that the splicing outcome (i.e., exon inclusion or skipping) depends on where splicing factors bind on pre-mRNAs with respect to the regulated exons.…”

Splicing Enhancers at Intron–Exon Borders Participate in Acceptor Splice Sites Recognition

“…In line with that notion, individual mutations did not markedly vary in the splicing response to the SRSF1 knockdown. This may have been related to requirement of multiple SRSF1 binding sites to ensure the knockdown's function, as described by Jobbins et al [60]. Therefore, a single nucleotide substitution might not have significantly harmed the SRSF1 binding.…”

Interplay between coding and exonic splicing regulatory sequences

“…However, it is in agreement with recent observations indicating that the interaction of a splicing factor with a binding motif depends on the sequence context and on the presence of clusters of related binding motifs 3–7 . For example, increasing the exonic frequency of GGA-like motifs increases the probability of an exon to be regulated by the SRSF1 splicing factor that binds to GGAGGA-like motifs even though only one binding site is used 6 .…”