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

Nature. 2008 Nov 27;456(7221):464-9. doi: 10.1038/nature07488. Epub 2008 Nov 2.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing / genetics*
  • Animals
  • Antigens, Neoplasm / genetics
  • Antigens, Neoplasm / metabolism*
  • Cell Line
  • Cross-Linking Reagents / chemistry
  • Cross-Linking Reagents / metabolism
  • Exons / genetics
  • Genome / genetics*
  • Genomics
  • Humans
  • Immunoprecipitation
  • Mice
  • Neocortex / cytology*
  • Neurons / metabolism*
  • Organ Specificity
  • Polyadenylation / genetics
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*

Substances

  • Antigens, Neoplasm
  • Cross-Linking Reagents
  • Nova2 protein, mouse
  • RNA, Messenger
  • RNA-Binding Proteins