title: HITS-CLIP yields genome-wide insights into brain alternative RNA processing.
abstract: 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.
authors.affiliation: Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.
authors.forename: Donny D
authors.initials: DD
authors.lastname: Licatalosi
authors.forename: Aldo
authors.initials: A
authors.lastname: Mele
authors.forename: John J
authors.initials: JJ
authors.lastname: Fak
authors.forename: Jernej
authors.initials: J
authors.lastname: Ule
authors.forename: Melis
authors.initials: M
authors.lastname: Kayikci
authors.forename: Sung Wook
authors.initials: SW
authors.lastname: Chi
authors.forename: Tyson A
authors.initials: TA
authors.lastname: Clark
authors.forename: Anthony C
authors.initials: AC
authors.lastname: Schweitzer
authors.forename: John E
authors.initials: JE
authors.lastname: Blume
authors.forename: Xuning
authors.initials: X
authors.lastname: Wang
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Darnell
authors.forename: Robert B
authors.initials: RB
authors.lastname: Darnell
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D005091:Exons / Q000235:genetics; D016678:Genome / Q000235:genetics*; D023281:Genomics; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D019579:Neocortex / Q000166:cytology*; D000090682:Neuro-Oncological Ventral Antigen; D009474:Neurons / Q000378:metabolism*; D009928:Organ Specificity; D026723:Polyadenylation / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000951:Antigens, Neoplasm; D003432:Cross-Linking Reagents; D000090682:Neuro-Oncological Ventral Antigen; C515624:Nova2 protein, mouse; D012333:RNA, Messenger; D016601:RNA-Binding Proteins