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• Engineering of sequence-specific alternative splicing modulators. • Robust exclusion of endogenous FAS exon 6 and inclusion of endogenous SMN2 exon 7. • Underscoring U1 snRNA as a candidate therapeutic and research tool. Alternative splicing accounts for a considerable portion of transcriptomic diversity, as most protein-coding genes are spliced into multiple mRNA isoforms. However, errors in splicing patterns can give rise to mis-splicing with pathological consequences, such as the congenital diseases familial dysautonomia, Duchenne muscular dystrophy, and spinal muscular atrophy. Small nuclear RNA (snRNA) components of the U snRNP family have been proposed as a therapeutic modality for the treatment of mis-splicing. U1 snRNAs offer great promise, with prior studies demonstrating in vivo efficacy, suggesting additional preclinical development is merited. Improvements in enabling technologies, including screening methodologies, gene delivery vectors, and relevant considerations from gene editing approaches justify further advancement of U1 snRNA as a therapeutic and research tool. With the goal of providing a user-friendly protocol, we compile and demonstrate a methodological toolkit for sequence-specific targeted perturbation of alternatively spliced pre-mRNA with engineered U1 snRNAs. We observe robust modulation of endogenous pre-mRNA transcripts targeted in two contrasting splicing contexts, SMN2 exon 7 and FAS exon 6, exhibiting the utility and applicability of engineered U1 snRNA to both inclusion and exclusion of targeted exons. We anticipate that these demonstrations will contribute to the usability of U1 snRNA in investigating splicing modulation in eukaryotic cells, increasing accessibility to the broader research community. [ABSTRACT FROM AUTHOR]

RNA-binding proteins (RBPs) are involved in all aspects of RNA metabolism, and RNA-RBP interactions are important for cell homeostasis and viral replication. The global RNA-binding proteome was recently reported; however, little is known about the proteins that bind to specific RNAs. In this study, we describe a novel CRISPR-based RNA interaction proteomics method in live cells. In brief, dCas13a with an HA tag was expressed in cells and bound to an RNA of interest with the help of gRNA. The RNA-protein complexes physically bound to dCas13a-HA were crosslinked using UV light and captured using anti-HA beads, after which the proteins were purified and identified using mass spectrometry. We optimized this system and subsequently applied it to U1 small nuclear RNA, which revealed 226 proteins. • dCas13a based RNA binding protein detecting method in vivo. • The all-in-one plasmid simplifies the operation process. • CBRIP identified RBPs on U1 snRNA are highly specified. • RPL7 is verified to be an U1 snRNA binding protein. [ABSTRACT FROM AUTHOR]

Abstract: The importance of U1 snRNA binding free energy in the regulation of alternative splicing has been studied in some genes with site-directed mutagenesis. Here we report a large-scale analysis of its impact on 5′ splice site (5′ss) selection in human genome. The results show that free energy exerts different effects on alternative 5′ss choice in different situations and −8.1kcal/mol is a threshold. When both free energies of two competing 5′ss are larger than −8.1kcal/mol, the 5′ss with lower free energy is more frequently used. However, in other pairs of 5′ss, lower-free-energy 5′ss does not seem to be favored and even the other 5′ss is used more frequently, which suggests that very low binding free energy would impair splicing. Some observations hold true only for those alternative 5′ splicing with short alternative exons (<50nt), which implies a complex mechanism of 5′ss selection involving both U1 snRNA binding free energy and regulatory factors. [Copyright &y& Elsevier]