Chapter 15
Plant Individual Nucleotide Resolution Cross-Linking
and Immunoprecipitation to Characterize RNA-Protein
Complexes
Tino Ko ¨ ster and Dorothee Staiger
Abstract
In recent years, it has become increasingly recognized that regulation at the RNA level pervasively shapes
the transcriptome in eukaryotic cells. This has fostered an interest in the mode of action of RNA-binding
proteins that, via interaction with specific RNA sequence motifs, modulate gene expression. Understanding
such posttranscriptional networks controlled by an RNA-binding protein requires a comprehensive identification of its in vivo targets. In metazoans and yeast, methods have been devised to stabilize RNA-protein
interactions by UV cross-linking before isolating RNA-protein complexes using antibodies, followed by
identification of associated RNAs by next-generation sequencing. These methods are collectively referred to
as CLIP-Seq (cross-linking immunoprecipitation-high-throughput sequencing). Here, we present a version
of the individual nucleotide resolution cross-linking and immunoprecipitation procedure that is suitable for
use in the model plant Arabidopsis thaliana.
Key words RNA immunoprecipitation, iCLIP, Posttranscriptional, RNA-binding protein
1 Introduction
Upon transcription, pre-mRNAs undergo a suite of processing
steps including alternative splicing, 3
0 end formation, modification,
export from the nucleus, and degradation. RNA-binding proteins
(RBPs) control each of these steps through dynamic interaction
with cis-regulatory motifs on their RNA targets [1–6]. To identify
RNAs associated with RBPs in vivo, endogenously assembled
RNA-protein complexes are stabilized by cross-linking and captured from cellular extracts with immobilized antibodies against
the RBP. Subsequently, target mRNAs are identified in the RNA
fraction isolated from the immunoprecipitated complexes.
In vivo cross-linking stabilizes transient and weak RNA-protein
complexes and thus allows more stringent washing conditions to be
applied. Moreover, it prevents reconfiguration or RNA-protein
complexes upon tissue disruption [7]. In Arabidopsis, chemical
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_15, © Springer Science+Business Media, LLC, part of Springer Nature 2020
255
Plant Individual Nucleotide Resolution Cross-Linking
and Immunoprecipitation to Characterize RNA-Protein
Complexes
Tino Ko ¨ ster and Dorothee Staiger
Abstract
In recent years, it has become increasingly recognized that regulation at the RNA level pervasively shapes
the transcriptome in eukaryotic cells. This has fostered an interest in the mode of action of RNA-binding
proteins that, via interaction with specific RNA sequence motifs, modulate gene expression. Understanding
such posttranscriptional networks controlled by an RNA-binding protein requires a comprehensive identification of its in vivo targets. In metazoans and yeast, methods have been devised to stabilize RNA-protein
interactions by UV cross-linking before isolating RNA-protein complexes using antibodies, followed by
identification of associated RNAs by next-generation sequencing. These methods are collectively referred to
as CLIP-Seq (cross-linking immunoprecipitation-high-throughput sequencing). Here, we present a version
of the individual nucleotide resolution cross-linking and immunoprecipitation procedure that is suitable for
use in the model plant Arabidopsis thaliana.
Key words RNA immunoprecipitation, iCLIP, Posttranscriptional, RNA-binding protein
1 Introduction
Upon transcription, pre-mRNAs undergo a suite of processing
steps including alternative splicing, 3
0 end formation, modification,
export from the nucleus, and degradation. RNA-binding proteins
(RBPs) control each of these steps through dynamic interaction
with cis-regulatory motifs on their RNA targets [1–6]. To identify
RNAs associated with RBPs in vivo, endogenously assembled
RNA-protein complexes are stabilized by cross-linking and captured from cellular extracts with immobilized antibodies against
the RBP. Subsequently, target mRNAs are identified in the RNA
fraction isolated from the immunoprecipitated complexes.
In vivo cross-linking stabilizes transient and weak RNA-protein
complexes and thus allows more stringent washing conditions to be
applied. Moreover, it prevents reconfiguration or RNA-protein
complexes upon tissue disruption [7]. In Arabidopsis, chemical
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_15, © Springer Science+Business Media, LLC, part of Springer Nature 2020
255