FUBP1 is a core splicing factor that facilitates 3' splice site recognition and splicing of long introns [In vivo 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. In vivo iCLIP is used to study protein-RNA interactions on individual nucleotide resolution by means of UV crosslinking and immunoprecipitation (PMID: 20601959). For the U2AF2 in vivo iCLIP, data from two iCLIP experiments were combined. The first U2AF2 iCLIP  and PTBP1 iCLIP were performed as previously described in (PMID: 24184352 ). The second U2AF2 iCLIP as well as in vivo iCLIPs for FUBP1, SF1, and SF3B1, were performed using the iCLIP2 protocol as described in (PMID: 31610236). Briefly, HeLa cells were irradiated with 150  mJ/cm2 in a UV-C crosslinker to covalently bind the RNA-binding proteins to the bound nucleic acids. For FUBP1 in vivo iCLIP, cross-linking was achieved by 4sU-mediated cross-linking. HeLa cells were 4sU-labelled by adding a 0.1 M 4-Thiouridine (4sU, Sigma) in DMSO to a final concentration of 100 μM/10 cm cell culture dish. Cells were incubated for 16 h at 37 °C, 5% CO2, under exclusion of light. After incubation, the cells were moved onto ice, shielded from light and irradiated at 365 nm, 800 mJ. During subsequent cell lysis, RNA was partially digested to create 50-200 nt long fragments. Immunoprecipitation of the investigated proteins was performed with antibodies listed in Table S9. The anti-PTBP1 Antibody was a kind gift of Chris Smith (PMID: 17679092). Radioactive labelling at the 3’ end of the precipitated RNA enables visualisation of the RNP-complex by SDS-PAGE and transfer to a nitrocellulose membrane. After recovery of protein-RNA complexes from the membrane, a proteinase K digestion results in protein-free RNA.