A systems view of spliceosomal assembly and branchpoints with iCLIP.

Michael Briese,Rupert Faraway,Andrea S Elser,Julian König,Tomaž Curk,Christopher R Sibley,Nejc Haberman,Livio Pellizzoni,Nicholas M Luscombe,Vihandha O Wickramasinghe,David Perera,Christopher W J Smith,Zhen Wang,Ashok R Venkitaraman,Anob M Chakrabarti,Jernej Ule,Luciano Saieva

doi:10.1038/s41594-019-0300-4

Abstract

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we employed spliceosome iCLIP, which immunoprecipitates SmB along with snRNPs and auxiliary RNA binding proteins (RBPs), to map spliceosome engagement with pre-mRNAs in human cell lines. This revealed seven peaks of spliceosomal crosslinking around branchpoints (BPs) and splice sites. We identified RBPs that crosslink to each peak, including known and candidate splicing factors. Moreover, we detected use of over 40,000 BPs with strong sequence consensus and structural accessibility, which align well to nearby crosslinking peaks. We show how the position and strength of BPs affect the crosslinking patterns of spliceosomal factors, which bind more efficiently upstream of strong or proximally located BPs, and downstream of weak or distally located BPs. These insights exemplify spliceosome iCLIP as a broadly applicable method for transcriptomic studies of splicing mechanisms.

Highlights

Splicing is a multi-step process in which small nuclear ribonucleoprotein particles and associated splicing factors bind at specific positions around intron boundaries in order to assemble an active spliceosome through a series of remodeling steps
To examine how BP strength affects spliceosomal assembly we focused on BPs that have been identified both by spliceosome individual nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) and computational modelling, and which are located at 23-28 nt upstream of the 3'ss
We established spliceosome iCLIP to study the interactions of endogenous small nuclear ribonucleoprotein particles (snRNPs) and accessory splicing factors on pre-mRNAs

Summary

Introduction

Splicing is a multi-step process in which small nuclear ribonucleoprotein particles (snRNPs) and associated splicing factors bind at specific positions around intron boundaries in order to assemble an active spliceosome through a series of remodeling steps. Spliceosome assembly begins with ATP-independent binding of U1 snRNP at the 5' splice site (ss), and of U2 small nuclear RNA auxiliary factors 1 and 2 (U2AF1 and U2AF2, known as U2AF35 and U2AF65) to the 3'ss. The actions of many RNA helicases and pre-mRNA processing factor 8 (PRPF8) facilitate rearrangements of snRNP interactions and establishment of the catalytically competent Bact and C complexes. These catalyze the two trans-esterification reactions leading to lariat formation, intron removal and exon ligation[2]

Methods

Results

Conclusion