HITS-CLIP yields genome-wide insights into brain alternative RNA processing.
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.
Licatalosi
Donny D
DD
Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.
Mele
Aldo
A
Fak
John J
JJ
Ule
Jernej
J
Kayikci
Melis
M
Chi
Sung Wook
SW
Clark
Tyson A
TA
Schweitzer
Anthony C
AC
Blume
John E
JE
Wang
Xuning
X
Darnell
Jennifer C
JC
Darnell
Robert B
RB
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*
D000951:Antigens, Neoplasm; D003432:Cross-Linking Reagents; D000090682:Neuro-Oncological Ventral Antigen; C515624:Nova2 protein, mouse; D012333:RNA, Messenger; D016601:RNA-Binding Proteins