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Nat Struct Mol Biol. 2019 Sep 30. doi: 10.1038/s41594-019-0300-4. [Epub ahead of print]

A systems view of spliceosomal assembly and branchpoints with iCLIP.

Author information

1
MRC Laboratory of Molecular Biology, Cambridge, UK.
2
Institute of Clinical Neurobiology, University of Wuerzburg, Wuerzburg, Germany.
3
The Francis Crick Institute, London, UK.
4
Department of Neuromuscular Disease, UCL Institute of Neurology, London, UK.
5
Division of Brain Sciences, Department of Medicine, Imperial College London, London, UK.
6
Institute of Quantitative Biology, Biochemistry and Biotechnology, Edinburgh University, Edinburgh, UK.
7
Department of Genetics, Environment and Evolution, UCL Genetics Institute, London, UK.
8
Institute of Molecular Biology GmbH, Mainz, Germany.
9
MRC Cancer Unit at the University of Cambridge, Cambridge, UK.
10
RNA Biology and Cancer Laboratory, Peter MacCallum Cancer Centre, Melbourne, Australia.
11
Okinawa Institute of Science & Technology Graduate University, Okinawa, Japan.
12
Center for Motor Neuron Biology and Disease, Department of Pathology and Cell Biology, Columbia University, New York, NY, USA.
13
Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UK.
14
Department of Biochemistry, University of Cambridge, Cambridge, UK.
15
Faculty of Computer and Information Science, University of Ljubljana, Ljubljana, Slovenia.
16
MRC Laboratory of Molecular Biology, Cambridge, UK. jernej.ule@crick.ac.uk.
17
The Francis Crick Institute, London, UK. jernej.ule@crick.ac.uk.
18
Department of Neuromuscular Disease, UCL Institute of Neurology, London, UK. jernej.ule@crick.ac.uk.

Abstract

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.

PMID:
31570875
DOI:
10.1038/s41594-019-0300-4

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