FUBP1 is a core splicing factor that facilitates 3' splice site recognition and splicing of long introns [In vitro iCLIP]

Splicing is a central process in metazoans and greatly expands their proteome by alternative splicing of pre-mRNA transcripts. An essential regulatory step during early spliceosome assembly is the recognition of cis-regulatory RNA motifs in pre-mRNAs. Here, we identified the RNA binding protein FUBP1 as a novel core splicing factor with a ubiquitous footprint on pre-mRNAs. FUBP1 binds to a previously unknown cis-regulatory motif upstream of the branch point of human introns. We show that FUBP1 binds and stabilises known 3' splice site components such as the essential splicing factor U2AF2. FUBP1 mutant cell lines and patient data indicate that FUBP1 is particularly relevant for efficient splicing of exons flanked by long introns. In addition to its role at the 3' splice site, FUBP1 shows multiple interactions with U1 snRNP- associated proteins. This demonstrates an important role for FUBP1 in splice site bridging in the context of long introns. Overall design: In vitro iCLIP measures the intrinsic binding affinity of a given RBP. Recombinant protein and minigene in vitro transcripts (PMID: 29643205) or a large-scale oligo library are mixed and subjected to crosslinking and immunoprecipitation of the RBP of interest. For all in vitro iCLIPs, the in vitro transcript pool or oligo library was preheated for 5 min at 70°C to minimise RNA secondary structures.For minigene in vitro iCLIP, a final concentration of 2 nM of in vitro transcript pool were added to 50 nM U2AF2 RRM12 alone, or to 50 nM U2AF2 RRM12 supplemented with either 50 nM of FUBP1FL, FUBP1?N, or FUBP1N74 (imb_koenig_2018_01_sub12). The mixtures were incubated at 37°C for 5 min before UV-irradiation (50 mJ/cm^2). Minigene in vitro iCLIP was spiked with 10 µl crosslinked mixture containing 250 nM U2AF2RRM12 and 6 nM NUP133 in vitro transcript for normalisation.For the oligo in vitro iCLIP, the large-scale oligo library of 200mers was used. Here, 50 nM of the oligo library was incubated with 50 nM U2AF2 RRM12 alone or with either 50 nM or 300 nM FUBP1 (imb_koenig_2018_01_sub16). The mixtures were incubated at 37°C for 5 min before UV-irradiation (50 mJ/cm^2). Partial RNase digestion and L3 linker ligation was skipped as they do not apply here. Oligo in vitro iCLIP was spiked with a mix of ten 150 nt long spike-in oligonucleotides for normalisation.