Skipper analysis of RNA-protein interactions highlights depletion of genetic variation in translation factor binding sites

Abstract

Technology for crosslinking and immunoprecipitation followed by sequencing (CLIP-seq) has identified the transcriptomic targets of hundreds of RNA-binding proteins in cells. To increase the power of existing and future CLIP-seq datasets, we introduce Skipper, an end-to-end workflow that converts unprocessed reads into annotated binding sites using an improved statistical framework. Compared to existing methods, Skipper on average calls 3.1-4.2 times more transcriptomic binding sites and sometimes >10 times more sites, providing deeper insight into post-transcriptional gene regulation. Skipper also calls binding to annotated repetitive elements and identifies bound elements for 99% of enhanced CLIP experiments. We perform nine translation factor enhanced CLIPs and apply Skipper to learn determinants of translation factor occupancy including transcript region, sequence, and subcellular localization. Furthermore, we observe depletion of genetic variation in occupied sites and nominate transcripts subject to selective constraint because of translation factor occupancy. Skipper offers fast, easy, customizable analysis of CLIP-seq data.

Proteomic discovery of chemical probes that perturb protein complexes in human cells

“…DDX42-dTAG HCT-116 cells were treated with WX-02-23 (5 µM), WX-02-43 (5 µM), or Pladienolide B (100 nM) for 3 h. Data are average values from two independent experiments. eCLIP enriched windows (FDR<0.2) were called and annotated using the Skipper analysis pipeline (Boyle et al ., 2022) and are depicted as percent binding relative to coding sequence (CDS). Proximal denotes within 500 bases and adjacent denotes within 100 bases from the annotated splice site (SS).…”

“…To test this hypothesis more directly, we evaluated DDX42-mRNA interactions by eCLIP-seq (enhanced crosslinking and immunoprecipitation followed by sequencing) (Van Nostrand et al, 2016) using the hemagluttinin (HA) epitope tag component of the dTAG fusion. These eCLIP-seq experiments revealed that, in untreated cells, DDX42 mainly bound to the coding region of (pre-)mRNAs, but, following exposure of cells to SF3B1 ligands WX-02-23 or pladienolide B, the DDX42-RNA interaction preferences strongly shifted towards regions within or near splice sites (Figure 7F and G and Dataset S2) (Boyle et al, 2022;Van Nostrand et al, 2020). Our results, taken together, thus suggest that DDX42 dynamically binds SF3B1 as part of an A-like spliceosome complex that is in close contact with (pre-)mRNAs and splice sites to facilitate branch point selection.…”

“…- Data analysis : After standard NovaSeq demultiplexing, eCLIP libraries processed with Skipper (https://github.com/YeoLab/skipper/) (Boyle et al ., 2022). Briefly, reads were adapter trimmed with skewer (Jiang et al, 2014) (https://github.com/relipmoc/skewer) and reads less than 20 bp were discarded.…”