A Targeted Oligonucleotide Enhancer of SMN2 Exon 7 Splicing Forms Competing Quadruplex and Protein Complexes in Functional Conditions

Summary The 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.

. The values represent the percentage in each band of the total radioactivity in the identified bands.  Figure 5C. Values were recorded at the wavelengths shown in Figure 5C.

Supplemental
Transition midpoints were determined based on an apparent two-state model.

Supplemental Experimental Procedures
Protein digestion, analysis by mass spectrometry and data processing. Proteomics was carried out by the University of Leicester Proteomics Facility (PNACL, University of Leicester).
Bands of interest were excised from the gel, and in-gel trypsin digestion was carried out upon each (Speicher et al., 2000). Each slice was destained using 200mM ammonium bicarbonate/20% acetonitrile, followed by reduction (10 mM  Proteome Discoverer was further processed using Scaffold Q+S (Searle, 2010) (version 3.6.1, Proteome Software). Upon import, the data was searched using X!Tandem (Craig and Beavis, 2004) (The Global Proteome Machine Organization). PeptideProphet (Keller et al., 2002) and ProteinProphet (Nesvizhskii et al., 2003) (Institute for Systems Biology) probability thresholds of 95% were calculated from the decoy searches and Scaffold was used to calculate an improved 95% peptide and protein probability threshold based on the data from the two different search algorithms. Candidate proteins were only accepted if they contained at least 3 peptides >95%.
Scaffold was used to calculate the Total Ion Count for the 3 most abundant peptides of each protein for the purposes of protein quantitation.
For structural analysis, samples were reconstituted in KCl buffer (100mM KCl, 10mM K 2 HPO 4 /KH 2 PO 4 , pH 7) made with filtered and degassed water. Prior to use samples were heated to 95 o C for 10 minutes and then kept at room temperature for at least an hour to allow secondary structure to form. Unmodified RNA sample concentration was estimated using the nearest neighbour method. CD, UV and thermal denaturation spectra were recorded using a PiStar 180 Spectrophotometer (Applied Photophysics, UK) with strand concentrations between 1-10 µM.
Temperature ramping used a rate of 3 min/0.5 o C steps and a tolerance of 0.2 o C. Data was fitted in OriginPro 8.6 (OriginLab). For NMR, samples were diluted with 10% D 2 O to 0.3 to 0.8 mM. Spectra were recorded using a Bruker Avance 600MHz and Bruker Avance III 800MHz with Cryoprobe.
The pulse program used was a 1D sequence with water suppression and excitation sculpting with gradients (Hwang and Shaka, 1995). Data was processed in Bruker Topspin 3.1. Mass spectrometry was done in negative ion mode using a Synapt High Definition Mass Spectrometer with Quadrupole TOF mass analyzer (Waters, UK). Samples used in mass spectrometry were buffer-exchanged into 150 mM NH 4 OAc, either using a polyacrylamide desalting column (Thermo Scientific) followed by spin concentration to 20-40 µM (23-mer), or dialysis (Slide-a-Lyzer G2, Thermo Scientific, 2K MWCO) to 31 µM (15-mer). Samples were mixed with 10% MeOH immediately before analysis. An injection rate of 4-10 uL.min -1 was used and spectra recorded with capillary voltage 1.5-2.5 kV. Data was processed with MassLynx V4.1 (Waters) with smoothing and baseline subtraction.