Chapter 23
RNA-Binding Protein Immunoprecipitation
and High-Throughput Sequencing
Tino Ko ¨ ster and Dorothee Staiger
Abstract
The RNA-binding proteome plays a key role in controlling every step in the life of RNA molecules.
Through interaction with dedicated sequence motifs, RNA-binding proteins coordinate processing of
cohorts of genes. Understanding such posttranscriptional networks controlled by an RNA-binding protein
requires a comprehensive identification of its in vivo targets. In Arabidopsis thaliana, RNA immunoprecipitation followed by reverse transcription-PCR has been widely used to test the association of candidate
targets with RNA-binding proteins. The detection of unknown target transcripts requires methods
operating at the level of the entire transcriptome. Here, we describe a protocol for RNA immunoprecipitation coupled to the generation of libraries from the co-purified RNAs for high-throughput sequencing.
This allows determining RNAs associated with RNA-binding proteins in planta at a global scale.
Key words RNA immunoprecipitation, RIP-seq, Posttranscriptional, RNA-binding protein, Crosslinking
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
control each of these steps through dynamic interaction with cisregulatory motifs on their RNA targets.
To identify RNAs associated with RNA-binding proteins
in vivo, endogenously assembled RNA-protein complexes are stabilized by cross-linking and captured from cellular extracts with
immobilized antibodies against the RNA-binding protein. Subsequently, target mRNAs are identified in the RNA fraction isolated
from the immunoprecipitated complexes.
We have established a fast and straightforward protocol for RIP
using Green Fluorescent Protein (GFP) fusion proteins that relies
on the use of total cellular extracts without prior isolation of nuclei
and immunoprecipitation with GFP-Trap
® beads [1, 2]. Previously,
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_23, © Springer Science+Business Media, LLC, part of Springer Nature 2021
453
RNA-Binding Protein Immunoprecipitation
and High-Throughput Sequencing
Tino Ko ¨ ster and Dorothee Staiger
Abstract
The RNA-binding proteome plays a key role in controlling every step in the life of RNA molecules.
Through interaction with dedicated sequence motifs, RNA-binding proteins coordinate processing of
cohorts of genes. Understanding such posttranscriptional networks controlled by an RNA-binding protein
requires a comprehensive identification of its in vivo targets. In Arabidopsis thaliana, RNA immunoprecipitation followed by reverse transcription-PCR has been widely used to test the association of candidate
targets with RNA-binding proteins. The detection of unknown target transcripts requires methods
operating at the level of the entire transcriptome. Here, we describe a protocol for RNA immunoprecipitation coupled to the generation of libraries from the co-purified RNAs for high-throughput sequencing.
This allows determining RNAs associated with RNA-binding proteins in planta at a global scale.
Key words RNA immunoprecipitation, RIP-seq, Posttranscriptional, RNA-binding protein, Crosslinking
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
control each of these steps through dynamic interaction with cisregulatory motifs on their RNA targets.
To identify RNAs associated with RNA-binding proteins
in vivo, endogenously assembled RNA-protein complexes are stabilized by cross-linking and captured from cellular extracts with
immobilized antibodies against the RNA-binding protein. Subsequently, target mRNAs are identified in the RNA fraction isolated
from the immunoprecipitated complexes.
We have established a fast and straightforward protocol for RIP
using Green Fluorescent Protein (GFP) fusion proteins that relies
on the use of total cellular extracts without prior isolation of nuclei
and immunoprecipitation with GFP-Trap
® beads [1, 2]. Previously,
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_23, © Springer Science+Business Media, LLC, part of Springer Nature 2021
453