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

Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.
Nature (Impact Factor: 42.35). 12/2008; 456(7221):464-9. DOI: 10.1038/nature07488
Source: PubMed

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.

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Available from: John J Fak, Aug 22, 2015
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    • "Both types should change the 3 ′ untranslated region (UTR) but may or may not change the coding sequence. Recent global analyses revealed that Nova1 (Licatalosi et al. 2008) and PABPN1 (Jenal et al. 2012) regulate APA sites of the first type, and U1 snRNP (Kaida et al. 2010; Berg et al. 2012) and CstF64 (Yao et al. 2012) regulate APA sites of the second type. However, all of the molecules associated with APA regulation have yet to be fully elucidated. "
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    • "To perform a genome-wide identification of RNA targets for APC in brain, we next used HITS-CLIP, which combines CLIP with high-throughput sequencing (Figure 1A) (Licatalosi et al., 2008). To reduce potential false positives, two different APC antibodies were used, and for one of them three independent immunoprecipitation replicates were performed, giving a total of four data sets (Figure S1D). "
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    • "Next, we used two different strategies to clone and amplify the isolated RNA fragments (Figure 1A). The first protocol , denoted as standard CLIP, was performed as described previously (Darnell, 2010; Licatalosi et al., 2008; Moore et al., 2014; Ule et al., 2005a). In this protocol, RNA linkers are ligated to the 5 0 and 3 0 ends of the RNA fragments (Figure 1A, left branch) and are later used for RT-PCR amplification. "
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