Combinatorial recognition of clustered RNA elements by the multidomain RNA-binding protein IMP3

Tim Schneider,Anna Wilmen,Lee-Hsueh Hung,Masood Aziz,Andreas Schlundt,Peter Friedhoff,Albrecht Bindereif,Jacqueline Wagner,Stephanie Thaum,Robert Janowski,Simon Müller,Stefan Hüttelmaier,Michael Sattler,Silke Schreiner,Dierk Niessing

doi:10.1038/s41467-019-09769-8

Abstract

How multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We establish an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, allows the prediction of IMP3 targets, and provides a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation.

Highlights

How multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far
We relied on truncated tandem domains for our analyses: RRM1–2, KH1–2, KH3–4, as well as an extended version containing all four KH domains, KH1–4 (Fig. 1a and Supplementary Fig. 1)
We focused on IMP3 to dissect its complex RNA binding through a systematic SELEX-seq approach: We found that all didomains (RRM1–2, KH1–2, and KH3–4) were active in RNA binding, while most previous studies had argued that only the KH domains 3 and 4 guide RNA recognition[5,14,15,16,29,30]

Summary

Introduction

How multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. We establish an integrative systematic strategy, combining singledomain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. The IMP protein family represents a prototypical example of multidomain RBPs and is characterized by a common architecture of six potential RNA-binding units: two N-terminal RNArecognition motifs (RRMs) and four consecutive hnRNP Khomology (KH) domains[1] It has been a long-standing question how multiple RBDs cooperate in specific and high-affinity RNAtarget recognition: Which of the individual domains are involved, what are their contributions, and how flexible is the RNA–protein interaction pattern?

Methods

Results

Conclusion