Novel RNA-binding Proteins Bind U1 snRNA to Promote Splicing of Weak 5' Splice Sites

Removal of introns during pre-mRNA splicing, which is central to gene expression, initiates by base pairing of U1 snRNA with a 5' splice site (5'SS). In mammals, many introns contain weak 5'SSs that are not efficiently recognized by the canonical U1 snRNP, suggesting alternative mechanisms exist. Here, we develop a cross-linking immunoprecipitation coupled to a high-throughput sequencing method, BCLIP-seq, to identify NRDE2 (Nuclear RNAi defective-2) and CCDC174 (Coiled-Coil Domain-Containing 174) as novel RNA-binding proteins in mouse ES cells that associate with U1 snRNA and unspliced 5'SSs. Both proteins bind directly to U1 snRNA independently of canonical U1 snRNP specific proteins, and they are required for the selection and effective processing of weak 5'SSs. Our results reveal that mammalian cells use non-canonical splicing factors bound directly to U1 snRNA to effectively select suboptimal 5'SS sequences in hundreds of genes, promoting proper splice site choice and accurate pre-mRNA splicing. All BCLIP-seq, RNA-seq and Ribo-seq experiments were performed in two independent replicates for each sample. BCLIP-seq was done with proteins endogenously tagged with a composite 3xFLAG+AviTag (3A tag). Nrde2 and Mtrex knockout were induced by treating the conditional knockout mES cells with 100 nM 4-hydroxytamoxifen for 4 and 3 days, respectively. Depletion of all the proteins fused to FKBP12F36V degron was achieved by treating the cells with 500 nM dTAG-13 compound for 24 hours. The indicated RNA-seq samples were treated for 4 hours with 0.1 mg/ml cycloheximide (CHX).