Fig 2: Mutational analysis of the RBM5 OCRE – SmN/B/B’ interaction.Specific mutations of conserved residues in the RBM5 OCRE domain impair the activity of the protein in FAS alternative splicing regulation ex vivo. HeLa cells were co-transfected with a FAS alternative splicing reporter and T7-RBM5 expression plasmids (wild type or mutations of tyrosine residues as indicated). RNA and proteins were isolated 24 hr after transfection. Patterns of alternative splicing were studied by RT-PCR using specific primers. (A) Inclusion and skipping products are annotated. (B) Protein expression was detected by western blot with an anti-T7 epitope antibody. (C) Quantification of the activity of RBM5 OCRE domain mutants in FAS alternative splicing regulation of 3 to 10 replicates of the experiment. The percentage of inclusion is presented. T-test (two-tailed distribution, homoscedastic) results are mentioned (**<0,01; ***<0,001).

Fig 3: Spliceosome iCLIP identifies protein interactions with snRNAs and splicing substrates.(a) Schematic representation of the spliceosome iCLIP method performed under conditions of varying purification stringency.(b) Autoradiogram of crosslinked RNPs immunopurified from HeLa cells under medium conditions by a SmB/B' antibody following digestion with high (++) or low (+) amounts of RNase I. The dotted line depicts the region typically excised from the nitrocellulose membrane for spliceosome iCLIP. As control, the antibody (Ab) was omitted during immunopurification.(c) Genomic distribution of spliceosome iCLIP cDNAs produced under stringent, medium and mild conditions from HEK293 cells. Data was mapped first to snRNAs, allowing multiple mapping reads, and then to the genome, allowing only uniquely mapped reads. Proportions of cDNAs mapping to snRNAs, introns, coding sequence of mRNAs (CDS), untranslated regions of mRNAs (UTR) and long non-coding RNAs (lncRNAs) are shown (but not the intergenic reads and other types of RNAs). Data are shown as mean±s.e.m from three independent experiments for the medium and mild purification condition and two independent experiments for the stringent purification condition. Source data for panel c are available online.

Fig 4: OCRE is a novel PRM-binding domain that interacts with snRNPs.(A) Comparison of proline-rich motif (PRM) recognition by PRM binding domains.Cartoon representations of various PRM binding domains (green) with side chains of important residues shown as sticks and annotated. PRM ligands are shown by stick representation (yellow). PDB accession codes: GYF (PDB 1L2Z), SH3 (PDB accession 1ABO), WW (PDB 1I5H), and RBM5 OCRE. (B) The C-terminal tail of SmN/B/B’ comprising RG-rich regions and proline-rich motifs (PRM) extends far from the core Sm core fold in the seven-membered Sm ring of assembled U snRNPs. The crystal structure of U4 snRNP core (PDB 4WZJ) (Leung et al., 2011) is shown, the C-terminal tails of SmD1/D3, which harbour RG-rich regions and SmN/B/B’ comprising RG and PRM regions are not visible in the crystal structure and indicated schematically. (C) The seven-membered Sm core rings of U4 and U5 snRNPs are located at the outside of the assembled U4/U6.U5 tri-snRNP (Nguyen et al., 2015). Thus, also in the tri snRNP the SmN/B/B’ tails are accessible for interactions with the RBM5 OCRE domain.

Fig 5: Proline-rich motif sequence requirements for OCRE binding.(A) Sequence of the C-terminal tail of human SmN/B/B’ and mutations of proline-rich-motifs (PRMs) tested. Conserved three or four-proline motifs and flanking arginine residues are highlighted. The binding affinities of these tails to the RBM5 OCRE domain determined by ITC (Table 2) are indicated on the right. Note, that association constants are shown here. (B) Binding of various PRM peptides to RBM5 OCRE monitored by NMR titrations. Relative binding affinities of PRM peptides to RBM5 OCRE based on a normalized chemical shift perturbation score are shown.