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.

Figure 1. HITS-CLIP genome-wide map of Nova-RNA binding sites

a, Chromosome 2 RefSeq genes and CLIP tags from the neocortex of two mouse littermates (Brain A, 46,106 tags in 10,740 clusters; Brain B, 100,874 tags in 15,805 clusters). b, Cluster Density (#tags/cluster length; black) in the Grin1 transcript; c, (i) Grin1 E4 and (ii) E19 tags, one color per biologic replicate, predict Nova-dependent exon skipping and inclusion, respectively (experimental validation is shown).

Figure 2. Nova-RNA interaction maps associated with the Nova-dependent splicing regulation

a, CLIP tags around all known Nova-regulated cassette exons; one color per transcript. Tags were mapped onto a composite transcript containing an alternative (dark blue/red box) and flanking constitutive (light blue box) exons. Tags are from transcripts showing Nova-dependent exon inclusion (top panel) or exclusion (bottom panel); experimental validation is shown (insets). b, Normalized complexity map (see methods) of Nova-RNA interactions recapitulate predicted maps (insets) for Nova-dependent exon inclusion (red) or exclusion (blue).

Figure 3. Nova CLIP tags cluster near polyadenylation sites

a, Distribution of Nova CLIP tag clusters (RefSeq mm8; CDS refers to introns/exons between UTRs). b, Intergenic clusters were plotted relative to the closest annotated start (5′ end) or stop (3′ end) codon. c, Distribution of 2,465 clusters in 3′ UTRs (defined as distance from stop codon to closest transcript termination site). d, CLIP tag clusters relative to the closest poly(A) site (transcript end in UCSC known genes), plotted as clusters per 50 nt in 2,465 3′ UTRs (green bars) or in 43 Nova-dependent 3′ UTRs identified with exon arrays (black bars).

Figure 4. Nova regulates alternative polyadenylation

a, Nova CLIP tags near Cugbp2 poly(A) site 2 (pA2); each color represents a biologic replicate. Right panel: RPA measuring cleavage at pA2 in WT (+/+), Nova2 heterozygous (+/−) and KO (−/−) P10 neocortex, with actin as a normalization control and ΔC (see text) shown. b, qRT-PCR validation of Nova-dependent poly(A) regulation, using RNA from three WT or Nova2 KO littermates, presented as long 3′ UTR isoform relative to total transcript abundance, normalized to WT (1.0; dotted line); error bars represent standard deviation. c-d, Composite (c) and normalized complexity (d) maps for Nova-regulated alternative polyadenylation sites (as in Fig. 2). e, Nova binds YCAY elements to directly regulate alternative polyadenylation. Left panel: qRT-PCR analysis of cells transfected with WT or Mutant (3 YACY mutations) poly(A) reporter constructs cotransfected with a control (−) or Nova2 expressing (+) plasmid, or WT vs. Nova2 KO neocortex (* = p < 0.02, ** = p < 0.01); error bars represent standard deviation. Right panel: RPA for cleavage of Slc8a1 poly(A) site 2, otherwise as described in (a).