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.

SRSF1 protein and U1 snRNPs are closely connected splicing factors. They both stimulate exon inclusion, SRSF1 by binding to exonic splicing enhancer sequences (ESEs) and U1 snRNPs by binding to the downstream 5 0 splice site (SS), and both factors affect 5 0 SS selection. The binding of U1 snRNPs initiates spliceosome assembly, but SR proteins such as SRSF1 can in some cases substitute for it. The mechanistic basis of this relationship is poorly understood. We show here by single-molecule methods that a single molecule of SRSF1 can be recruited by a U1 snRNP. This reaction is independent of exon sequences and separate from the U1-independent process of binding to an ESE. Structural analysis and cross-linking data show that SRSF1 contacts U1 snRNA stem-loop 3, which is required for splicing. We suggest that the recruitment of SRSF1 to a U1 snRNP at a 5 0 SS is the basis for exon definition by U1 snRNP and might be one of the principal functions of U1 snRNPs in the core reactions of splicing in mammals.

A. Joutel, H. Chabriat, K. Vahedi, et al. deletion in CADASIL in-frame Notch3 Splice site mutation causing a seven amino acid October 1, 2012 This information is current as of http://www.neurology.org/content/54/9/1874.full.html located on the World Wide Web at:The online version of this article, along with updated information and services, is rights reserved. Print expression of the tumor suppressor protein p53, a marker for programmed cell death. 7 Because cholinomimetics can impair neuronal growth and survival, we conclude that the excessive dose of PYD used during gestation was responsible for our patient's microcephaly, growth retardation, and CNS findings. Use of PYD during pregnancy at less than 600 mg/day remains safe because fetal effects appear dose dependent. It is only when extreme doses are used, such as in our patient, that the infant is put at risk.

SummaryThe use of oligonucleotides to activate the splicing of selected exons is limited by a poor understanding of the mechanisms affected. A targeted bifunctional oligonucleotide enhancer of splicing (TOES) anneals to SMN2 exon 7 and carries an exonic splicing enhancer (ESE) sequence. We show that it stimulates splicing specifically of intron 6 in the presence of repressing sequences in intron 7. Complementarity to the 5′ end of exon 7 increases U2AF65 binding, but the ESE sequence is required for efficient recruitment of U2 snRNP. The ESE forms at least three coexisting discrete states: a quadruplex, a complex containing only hnRNP F/H, and a complex enriched in the activator SRSF1. Neither hnRNP H nor quadruplex formation contributes to ESE activity. The results suggest that splicing limited by weak signals can be rescued by rapid exchange of TOES oligonucleotides in various complexes and raise the possibility that SR proteins associate transiently with ESEs.