FUBP1 is a core splicing factor that facilitates 3' splice site recognition and splicing of long introns [RNA-Seq]

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. "imb_koenig_2018_18: NGS library prep was performed with Illumina's TruSeq stranded total RNA LT Ribo Zero Gold Sample Prep Kit following Illumina?s standard protocol (Part # 15031048 Rev. E). Libraries were prepared with a starting amount of 1000 ng and amplified in 10 PCR cycles. Libraries were profiled in a DNA 1000 Chip on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All 4 samples were pooled in equimolar ratio and sequenced on an Illumina NextSeq 500 sequencing machine as 84 nt single-end reads. imb_koenig_2020_12: NGS library prep was performed with Illumina's TruSeq stranded Total RNA LT Sample Prep Kit following Illumina?s standard protocol (Part # 15031048 Rev. E). Libraries were prepared with a starting amount of 200 ng and amplified in 12 PCR cycles. In addition, 1 µl of ERCC spike-ins (1:250 dilution) was added to every sample. Libraries were profiled in a DNA 1000 kit on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All 12 samples were pooled in equimolar ratio and sequenced on an Illumina NextSeq 500 sequencing machine as 159 nt single-end reads.