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: 41.46). 12/2008; 456(7221):464-9. DOI: 10.1038/nature07488
Source: PubMed


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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    • "The hnRNP proteins were originally identified by their coassociation with pre-mRNA (Pinol-Roma et al. 1988), although these proteins share little sequence homology with one another other than containing one or more RRM or KH-type RNA-binding domains. Most hnRNP family members have been shown to regulate alternative splicing as well as other RNA processing steps such as 3 ′ end processing , mRNA translation, and mRNA stability (Bomsztyk et al. 2004; Martinez-Contreras et al. 2007; Licatalosi et al. 2008). Moreover, global comparisons of transcriptomewide binding of individual hnRNPs with their effect on transcriptome expression have led to the general model that the functional consequence of a particular hnRNP on a given transcript is highly dependent on where it binds within an RNA (Licatalosi and Darnell 2009; Fu and Ares 2014; Shi and Manley 2015). "
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    • "Splicing regulatory maps are useful to uncover positional effects of splicing-factor binding sites in exons and introns (Huelga et al., 2012; Licatalosi et al., 2008; Pandit et al., 2013). Briefly, these are motif-density profiles derived from CLIP-seq data or in silico predictions, which after normalization (e.g., by the sequence size) and smoothing (e.g., by moving average) are visualized as density units (e.g., normalized complexity). "
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    ABSTRACT: Splicing factor SRSF1 is upregulated in human breast tumors, and its overexpression promotes transformation of mammary cells. Using RNA-seq, we identified SRSF1-regulated alternative splicing (AS) targets in organotypic three-dimensional MCF-10A cell cultures that mimic a context relevant to breast cancer. We identified and validated hundreds of endogenous SRSF1-regulated AS events. De novo discovery of the SRSF1 binding motif reconciled discrepancies in previous motif analyses. Using a Bayesian model, we determined positional effects of SRSF1 binding on cassette exons: binding close to the 5′ splice site generally promoted exon inclusion, whereas binding near the 3′ splice site promoted either exon skipping or inclusion. Finally, we identified SRSF1-regulated AS events deregulated in human tumors; overexpressing one such isoform, exon-9-included CASC4, increased acinar size and proliferation, and decreased apoptosis, partially recapitulating SRSF1’s oncogenic effects. Thus, we uncovered SRSF1 positive and negative regulatory mechanisms, and oncogenic AS events that represent potential targets for therapeutics development.
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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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