CLIP and RNA interactome studies to unravel genome-wide RNA-protein interactions in vivo in Arabidopsis thaliana
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.
Section snippets
Protein-centric approaches to identify RNA-binding protein targets: Individual nucleotide resolution crosslinking immunoprecipitation
For the comprehensive identification of in vivo targets of RNA-binding proteins, UV crosslinking immunoprecipitation (CLIP) techniques have been developed for metazoa and yeast. CLIP relies on crosslinking of RNA and bound proteins to preserve RNA-protein interactions in vivo. Irradiation with UV-C light of 254 nm generates a covalent bond between RNA and bound proteins due to the photoreactivity of the aromatic ring system of the nucleobases [12]. Covalent bonds are formed to most amino acids
Experimental systems
The experimental systems to comprehensively characterize in vivo RNA-protein interactions by and large reflect the systems used to study post-transcriptional regulation. In mammals, mechanistic studies on post-transcriptional regulation are predominantly performed in cultured cell lines. Accordingly, most CLIP experiments are also performed in cultured cells. More recently, CLIP is also increasingly used with whole tissue, especially in a disease context (reviewed in [22]). RNA-binding proteins
Conclusions and outlook on iCLIP
The development of techniques to identify target transcripts bound by selected RNA-binding proteins in vivo has overcome a major limitation in research on plant RNA-based regulation [54]. Nevertheless, we are still far from understanding the binding landscape of most proteins.
Since the first application of CLIP in human brain tissue, many variants have been developed to increase the efficiency of crosslinking and target recovery, to increase the signal to noise ratio, to eliminate the use of
RNA-centric approaches: mRNA interactome capture
In 2012, mRNA interactome capture was reported to comprehensively identify proteins interacting with mRNAs in mammalian cells [65], [66]. This technique employs in vivo crosslinking of mRNA and bound proteins by UV light irradiation. The RNA-protein complexes are recovered by pulldown of polyadenylated RNAs using magnetic beads coated with oligo(dT). Proteins are released by RNase treatment, subjected to tryptic digest and identified via tandem mass spectrometry. A minimal core mRNA bound
UV crosslinking
In plants, UV crosslinking has traditionally been challenging due to the presence of UV-absorbing pigments such as chlorophyll (see above). To overcome this limitation, Reichel et al. used 4-day-old etiolated seedlings [69]. In higher plants, chlorophyll biosynthesis is strictly dependent on light, and thus these seedlings grown in the dark lack chlorophyll. In comparison to animal cell cultures, which are usually crosslinked with 254 nm UV light at 150 mJ/cm2, plant tissue generally requires a
Conclusions and outlook of mRNA interactome capture
The three mRNA interactome studies have determined the first plant RNA-bound proteomes and shown that the technique can be successfully applied to Arabidopsis [68], [69], [70]. The unbiased nature of the mRNA interactome capture method enabled detection of a large number of proteins potentially involved in RNA metabolism in plants beyond the proteins predicted solely on sequence information. Only seventy-nine of the mRNA interacting proteins were present in all three Arabidopsis mRNA
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
We thank Dr. Claudius Marondedze for comparisons of the mRNA interactome data sets.
Funding
Our research is funded by the German Research Foundation (KO 5364/1-1; STA653/13-1).
References (76)
- et al.
Beyond transcription: RNA-binding proteins as emerging regulators of plant response to environmental constraints
Plant Sci.
(2012) - et al.
Emerging roles of RNA-binding proteins in plant growth, development, and stress responses
Mol. Cells
(2016) Role of plant RNA-binding proteins in development, stress response and genome organization
Trends Plant Sci.
(2009)- et al.
Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP
Cell
(2010) - et al.
CLIP: a method for identifying protein-RNA interaction sites in living cells
Methods
(2005) - et al.
A two-tracked approach to analyze RNA–protein crosslinking sites in native, nonlabeled small nuclear ribonucleoprotein particles
Methods
(2002) - et al.
Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements
Cell
(2013) - et al.
fCLIP-seq for transcriptomic footprinting of dsRNA-binding proteins: Lessons from DROSHA
Methods
(2019) - et al.
iCLIP: Protein–RNA interactions at nucleotide resolution
Methods
(2014) - et al.
Metacaspase-8 modulates programmed cell death induced by ultraviolet light and H2O2 in Arabidopsis
J. Biol. Chem.
(2008)
Resilient Ribonucleases
Plant ribonomics: proteins in search of RNA partners
Trends Plant Sci.
Denaturing CLIP, dCLIP, pipeline identifies discrete RNA footprints on chromatin-associated proteins and reveals that CBX7 Targets 3′ UTRs to regulate mRNA expression
Cell Systems
Advances in CLIP technologies for studies of protein-RNA interactions
Mol. Cell
Insights into RNA biology from an atlas of mammalian mRNA-binding proteins
Cell
The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts
Mol. Cell
UV crosslinked mRNA-binding proteins captured from leaf mesophyll protoplasts
Plant Methods
RNA-binding proteins revisited – the emerging Arabidopsis mRNA interactome
Trends Plant Sci.
Circadian rhythms and post-transcriptional regulation in higher plants
Front. Plant Sci.
Emerging role for RNA-based regulation in plant immunity
New Phytol.
RNA-binding proteins and circadian rhythms in Arabidopsis thaliana
Philo. Trans. R. Soc. Lond. B. Biol. Sci.
RNA regulons: coordination of post-transcriptional events
Nat. Rev. Genet.
Alternative splicing at the intersection of biological timing, development, and stress responses
Plant Cell
Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays
PNAS
iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution
Nat. Struct. Mol. Biol.
Photochemical addition of amino acids and peptides to polyuridylic acid
Photochem Photobiol
Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins
Nat Meth
CLIP identifies Nova-regulated RNA networks in the brain
Science
CLIP: crosslinking and immunoprecipitation of in vivo RNA targets of RNA-binding proteins
Methods Mol. Biol.
Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions
Genome Biol.
Insights into the design and interpretation of iCLIP experiments
Genome Biol.
Crosslinking-immunoprecipitation (iCLIP) analysis reveals global regulatory roles of hnRNP L
RNA Biol.
The future of Cross-Linking and Immunoprecipitation (CLIP)
Cold Spring Harb. Perspect. Biol.
Elongator and SPT4/SPT5 complexes as proxy to study RNA polymerase II transcript elongation control of plant development
Proteomics
Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions
J. Mass Spectrom.
Ultraviolet-B radiation-mediated responses in plants
. Balancing damage and protectionPlant Physiology
Reversible photoswitchable DRONPA-s monitors nucleocytoplasmic transport of an RNA-binding protein in transgenic plants
Traffic
The small glycine-rich RNA-binding protein AtGRP7 promotes floral transition in Arabidopsis thaliana
Plant J.
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