Rbfox Proteins Regulate Splicing as Part of a Large Multiprotein Complex LASR

Abstract

Summary Rbfox proteins control alternative splicing and posttranscriptional regulation in mammalian brain, and are implicated in neurological disease. These proteins recognize the RNA sequence (U)GCAUG, but their structures and diverse roles imply a variety of protein-protein interactions. We find that nuclear Rbfox proteins are bound within a large assembly of splicing regulators (LASR), a multimeric complex containing the proteins hnRNP M, hnRNP H, hnRNP C, Matrin3, NF110/NFAR-2, NF45, and DDX5, all approximately equimolar to Rbfox. We show that splicing repression mediated by hnRNP M is stimulated by Rbfox. Virtually all the intron-bound Rbfox is associated with LASR, and hnRNP M motifs are enriched adjacent to Rbfox crosslinking sites in vivo. These findings demonstrate that Rbfox proteins bind RNA with a defined set of cofactors, and affect a broader set of exons than previously recognized. The function of this multimeric LASR complex has implications for deciphering the regulatory codes controlling splicing networks.

m6A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover

“…Put another way, how does m 6 A modification in pre-mRNAs occur with respect to "exon definition"? Recent experiments on the regulation of splicing have used a fractionation procedure that produced a high-molecular-weight (HMW) nuclear fraction that was released from chromatin and preserved some protein-protein interactions (Damianov et al 2016). We tested whether antibody precipitation of the proteins in the HMW fraction would connect m 6 A methyltransferases and U1 snRNP and U2AF proteins, since exon definition is often hypothesized to depend on the initial U1 RNA binding at the 5 ′ splice site and U2AF at the 3 ′ splice site.…”

RNA-binding protein PSPC1 promotes the differentiation-dependent nuclear export of adipocyte RNAs

“…iCLIP assays were performed according to a modified protocol (37,68). Briefly, 10T1/2 cells expressing Flag-PSPC1 were irradiated with 254 nm UV-C light at 800 mJ/cm 2 on ice to cross-link proteins to nucleic acids.…”

“…We therefore postulated that PSPC1 might drive adipogenesis through interacting with mRNA transcripts of differentiation-relevant genes. To test this idea, we used individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP) coupled with high-throughput sequencing to explore whether PSPC1 binds RNA, and if so, which species (36,37). We used 10T1/2 stable cells expressing vector or Flag-tagged PSPC1 (Fl-Pspc1).…”

RNA Sequence Context Effects Measured In Vitro Predict In Vivo Protein Binding and Regulation

“…Considering intronic binding, several features may be relevant, and examples of each are known (listed below). These features may include: 1) whether the local concentration or activity of the RBP (van der Houven van Oordt et al, 2000), or of its binding partners (Damianov et al, 2016), near the transcribed locus is sufficient; 2) whether or not access to the motif is blocked by local RNA structure (Kazan et al, 2010; Li et al, 2010); 3) whether the motif occurs in a sequence context that has other (non-structural) features favorable for binding (Agarwal et al, 2015); or 4) whether or not access to the motif is sterically blocked by other RBPs (HafezQorani et al, 2016; Liu et al, 2015; Zarnack et al, 2013). Other factors such as RNA modifications may influence binding in some cases, but pre-mRNA modifications are thought to be fairly rare (Carlile et al, 2014; Geula et al, 2015).…”