CLIP and RNA interactome studies to unravel genome-wide RNA-protein interactions in vivo in Arabidopsis thaliana

Highlights

• UV light irradiation is suitable to crosslink RNA and bound proteins in Arabidopsis.

• RNA-binding protein targets have been determined by iCLIP, HITS-CLIP and RIP-seq mRNA.

• mRNA interactome capture detects many novel plant RNA-binding proteins.

Abstract

Post-transcriptional regulation makes an important contribution to adjusting the transcriptome to environmental changes in plants. RNA-binding proteins are key players that interact specifically with mRNAs to co-ordinate their fate. While the regulatory interactions between proteins and RNA are well understood in animals, until recently little information was available on the global binding landscape of RNA-binding proteins in higher plants. This is not least due to technical challenges in plants. In turn, while numerous RNA-binding proteins have been identified through mutant analysis and homology-based searches in plants, only recently a full compendium of proteins with RNA-binding activity has been experimentally determined for the reference plant Arabidopsis thaliana. State-of-the-art techniques to determine RNA-protein interactions genome-wide in animals are based on the covalent fixation of RNA and protein in vivo by UV light. This has only recently been successfully applied to plants. Here, we present practical considerations on the application of UV irradiation based methods to comprehensively determine in vivo RNA-protein interactions in Arabidopsis thaliana, focussing on individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) and mRNA interactome capture.

Introduction

Higher plants, as sessile organisms, are confronted with an ever-changing environment. Major threats include extreme ambient temperatures, limited mineral availability or toxic salt concentrations in arid soil, and excess light. Additionally, plants continuously have to defend themselves against pathogenic bacteria or fungi. Plant responses to external cues entail massive changes in gene expression programs. Over the years, it has become clear that regulation at all levels of RNA processing plays an important role in modulating the transcriptome once transcription has been initiated [1], [2]. The specific interaction of RNA-binding proteins with dedicated cis-regulatory RNA motifs represents the language of post-transcriptional regulation, dictating which splice sites are to be used, which polyadenylation sites have to be chosen, and blocking or granting access of miRNAs, to name a few [3], [4], [5], [6]. Deciphering this code requires systems approaches to provide insights into RNA-protein interactions in vivo [7].

Based on sequence similarity to known RNA-binding domains, putative RNA-binding proteins have been predicted in the genome of the reference plant Arabidopsis thaliana, a small weed of the crucifer (Brassicaecae) family with a genome of around 130 Mb. However, only a limited number of RNA-binding proteins has been demonstrated to bind RNA and for only a few have target transcripts been determined globally. Functional characterization of Arabidopsis RNA-binding proteins mainly comes from the analysis of mutant phenotypes. For example, numerous mutants with aberrant response to the stress hormone abscisic acid or an impaired response to cold turned out to be defective in RNA-binding proteins [8].

Genome-wide approaches to elucidate in vivo RNA-protein-interactions come in two complementary flavors. Protein-centric approaches start with an RNA-binding protein and associated RNAs are identified via immunopurification of the RNA-binding protein, followed by high throughput sequencing (HITS) [9], [10], [11]. From the sequence information of the target transcripts, sequence motifs and inferred RNA secondary structure can be deduced that are representative of the binding sites. RNA-centric approaches start with RNA and the complement of associated proteins are recovered by RNA pull-down methods and subsequently identified by tandem mass spectrometry (MS/MS).

Here, we provide an up-to-date view on recent technical advances in the global identification of RNA-protein interactions and the successful use of UV light as a crosslinker in plant tissue. We focus on plant-specific aspects in performing iCLIP and mRNA interactome capture in Arabidopsis.