A systems view of spliceosomal assembly and branchpoints with iCLIP.
journal contributionposted on 28.10.2020, 14:06 by Michael Briese, Nejc Haberman, Christopher R Sibley, Rupert Faraway, Andrea S Elser, Anob M Chakrabarti, Zhen Wang, Julian König, David Perera, Vihandha O Wickramasinghe, Ashok R Venkitaraman, Nicholas M Luscombe, Luciano Saieva, Livio Pellizzoni, Christopher WJ Smith, Tomaž Curk, Jernej Ule
Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we used spliceosome iCLIP, which immunoprecipitates SmB along with small nuclear ribonucleoprotein particles and auxiliary RNA binding proteins, to map spliceosome engagement with pre-messenger RNAs in human cell lines. This revealed seven peaks of spliceosomal crosslinking around branchpoints (BPs) and splice sites. We identified RNA binding proteins that crosslink to each peak, including known and candidate splicing factors. Moreover, we detected the 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.