西亚试剂：HITS-CLIP yields genome-wide insights into brain alternativ

HITS-CLIP yields genome-wide insights into brain alternative RNA processing

Donny D. Licatalosi1, Aldo Mele1, John J. Fak1, Jernej Ule3, Melis Kayikci3, Sung Wook Chi1, Tyson A. Clark4, Anthony C. Schweitzer4, John E. Blume4, Xuning Wang2, Jennifer C. Darnell1 & Robert B. Darnell1

1 Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute,
2 Biocomputing, Information Technology, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA
3 MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK
4 Expression Research, Affymetrix, Inc., Santa Clara, California 95051, USA

Protein–RNA interactions have critical roles in all aspects of gene expression. However, applying biochemical methods to understand such interactions in living tissues has been challenging. Here we develop a genome-wide means of mapping protein–RNA binding sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova revealed extremely reproducible RNA-binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova–RNA interactions in 3' untranslated regions, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein–RNA interactions in vivo.

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