Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins

Eric L Van Nostrand,Steven M Blue,Ruth Wang,Xiang-Dong Fu,Brian A Yee,Balaji Sundararaman,Chelsea Gelboin-Burkhart,Gabriel A Pratt,Gene W Yeo,Jasmine Mueller,Emily C Wheeler,Ines Rabano,Rebecca Stanton,Brenton R Graveley,Thai B Nguyen,Samuel S Park,Keri E Garcia

doi:10.1186/s13059-020-01982-9

Eric L Van Nostrand, Steven M Blue + Show 15 more

Open Access

https://doi.org/10.1186/s13059-020-01982-9

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Abstract

BackgroundA critical step in uncovering rules of RNA processing is to study the in vivo regulatory networks of RNA binding proteins (RBPs). Crosslinking and immunoprecipitation (CLIP) methods enable mapping RBP targets transcriptome-wide, but methodological differences present challenges to large-scale analysis across datasets. The development of enhanced CLIP (eCLIP) enabled the mapping of targets for 150 RBPs in K562 and HepG2, creating a unique resource of RBP interactomes profiled with a standardized methodology in the same cell types.ResultsOur analysis of 223 eCLIP datasets reveals a range of binding modalities, including highly resolved positioning around splicing signals and mRNA untranslated regions that associate with distinct RBP functions. Quantification of enrichment for repetitive and abundant multicopy elements reveals 70% of RBPs have enrichment for non-mRNA element classes, enables identification of novel ribosomal RNA processing factors and sites, and suggests that association with retrotransposable elements reflects multiple RBP mechanisms of action. Analysis of spliceosomal RBPs indicates that eCLIP resolves AQR association after intronic lariat formation, enabling identification of branch points with single-nucleotide resolution, and provides genome-wide validation for a branch point-based scanning model for 3′ splice site recognition. Finally, we show that eCLIP peak co-occurrences across RBPs enable the discovery of novel co-interacting RBPs.ConclusionsThis work reveals novel insights into RNA biology by integrated analysis of eCLIP profiling of 150 RBPs with distinct functions. Further, our quantification of both mRNA and other element association will enable further research to identify novel roles of RBPs in regulating RNA processing.

Highlights

A critical step in uncovering rules of RNA processing is to study the in vivo regulatory networks of RNA binding proteins (RBPs)
Large-scale profiling of RNA binding protein binding sites with enhanced CLIP (eCLIP) The eCLIP methodology enabled highly efficient identification of RBP binding sites [18], leading to the generation of the first large-scale database of RNA binding protein targets profiled in the same cell types using a standardized workflow [20]
This analysis confirmed overlap at both the RNA transcript class level, where eCLIP enrichment for ribosomal RNA or retrotransposable elements correlated with specific RBP functions focused around these element types, and the regulatory region level, where enrichment at 5′UTRs or branch point regions corresponded to specific RBP functional roles

Summary

Introduction

A critical step in uncovering rules of RNA processing is to study the in vivo regulatory networks of RNA binding proteins (RBPs). Crosslinking and immunoprecipitation (CLIP) methods enable mapping RBP targets transcriptome-wide, but methodological differences present challenges to large-scale analysis across datasets. RNA binding proteins (RBPs) interact with RNA through a wide variety of primary sequence motifs and RNA structural elements to control all processing steps [3]. To identify direct RNA targets of RBPs, RNA immunoprecipitation (RIP) and crosslinking and immunoprecipitation (CLIP) methods are frequently used. CLIPbased methods utilize UV crosslinking to covalently link an RBP with its bound RNA in live cells, enabling both stringent immunoprecipitation washes and denaturing SDS-PAGE protein gel electrophoresis and nitrocellulose membrane transfer which serves to remove background unbound RNA [4]. Analyses of single RBP binding profiles by CLIP have provided unique insights into basic mechanisms of RNA processing, as well as identified downstream effectors that drive human diseases [5,6,7]. Methodological differences between CLIP approaches, combined with simple experimental variability between labs and variation in acceptable quality control metrics, add significant challenges to interpretation of differences observed

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