Chapter 14
Using RNA Affinity Purification Followed by Mass
Spectrometry to Identify RNA-Binding Proteins (RBPs)
Mengge Shan and Brian D. Gregory
Abstract
RNA-binding proteins (RBPs) perform key functions in posttranscriptional regulation, adding complexity
to the RNA life cycle. RNA interactome capture techniques have been applied to various organisms of
interest and detected hundreds of RBPs, some with uncharacterized functions. However, even in many
well-studied organisms, the primary sequence motif for most RBPs remains unknown. Here, we describe a
3-day protocol where users couple an RNA sequence of interest that is known to be bound by an RBP
(s) with agarose beads, incubate the now tagged RNA sequence with protein lysate, and then pull down the
proteins bound to the RNA. Subsequent mass spectrometry allows users to profile the RNA sequenceinteracting proteome and pick out any enriched proteins as RBPs of interest. This protocol allows
researchers to match sequences to their RBPs and even often identify novel RBPs or new functions for
known RBPs.
Key words RNA tagging, RNA-binding proteins
1 Introduction
RNA-binding proteins (RBPs) are key players in posttranscriptional
regulatory processes, and perform diverse functions such as stabilization, localization, splicing, and transport of bound RNAs
[1]. Research has shown that interactions between RBPs and target
RNAs impact every part of the RNA life cycle, from polyadenylation to degradation. This is because RBPs bind to specific target
RNAs to form ribonucleoprotein complexes (RNPs), which then
progress through the events of the RNA life cycle. The binding of
an RBP to its target RNA molecule can depend on the RNA’s
primary nucleotide sequence, its RNA secondary structure, and
oftentimes both of these features. The importance of these interactions makes it no surprise that defects in the RBP-RNA binding
can disrupt important regulatory networks and ultimately result in
diseases such as cancer, autoimmune problems, and metabolic and
neurological disorders [2–4].
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_14, © Springer Science+Business Media, LLC, part of Springer Nature 2020
241
Using RNA Affinity Purification Followed by Mass
Spectrometry to Identify RNA-Binding Proteins (RBPs)
Mengge Shan and Brian D. Gregory
Abstract
RNA-binding proteins (RBPs) perform key functions in posttranscriptional regulation, adding complexity
to the RNA life cycle. RNA interactome capture techniques have been applied to various organisms of
interest and detected hundreds of RBPs, some with uncharacterized functions. However, even in many
well-studied organisms, the primary sequence motif for most RBPs remains unknown. Here, we describe a
3-day protocol where users couple an RNA sequence of interest that is known to be bound by an RBP
(s) with agarose beads, incubate the now tagged RNA sequence with protein lysate, and then pull down the
proteins bound to the RNA. Subsequent mass spectrometry allows users to profile the RNA sequenceinteracting proteome and pick out any enriched proteins as RBPs of interest. This protocol allows
researchers to match sequences to their RBPs and even often identify novel RBPs or new functions for
known RBPs.
Key words RNA tagging, RNA-binding proteins
1 Introduction
RNA-binding proteins (RBPs) are key players in posttranscriptional
regulatory processes, and perform diverse functions such as stabilization, localization, splicing, and transport of bound RNAs
[1]. Research has shown that interactions between RBPs and target
RNAs impact every part of the RNA life cycle, from polyadenylation to degradation. This is because RBPs bind to specific target
RNAs to form ribonucleoprotein complexes (RNPs), which then
progress through the events of the RNA life cycle. The binding of
an RBP to its target RNA molecule can depend on the RNA’s
primary nucleotide sequence, its RNA secondary structure, and
oftentimes both of these features. The importance of these interactions makes it no surprise that defects in the RBP-RNA binding
can disrupt important regulatory networks and ultimately result in
diseases such as cancer, autoimmune problems, and metabolic and
neurological disorders [2–4].
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_14, © Springer Science+Business Media, LLC, part of Springer Nature 2020
241