Chapter 26
TRAP-SEQ of Eukaryotic Translatomes Applied
to the Detection of Polysome-Associated Long
Noncoding RNAs
Soledad Traubenik, Flavio Blanco, Marı ´a Eugenia Zanetti,
and Mauricio A. Reynoso
Abstract
Translating ribosome affinity purification (TRAP) technology allows the isolation of polysomal complexes
and the RNAs associated with at least one 80S ribosome. TRAP consists of the stabilization and affinity
purification of polysomes containing a tagged version of a ribosomal protein. Quantitative assessment of the
TRAP RNA is achieved by direct sequencing (TRAP-SEQ), which provides accurate quantitation of
ribosome-associated RNAs, including long noncoding RNAs (lncRNAs). Here we present an updated
procedure for TRAP-SEQ, as well as a primary analysis guide for identification of ribosome-associated
lncRNAs. This methodology enables the study of dynamic association of lncRNAs by assessing rapid
changes in their transcript levels in polysomes at organ or cell-type level, during development, or in
response to endogenous or exogenous stimuli.
Key words Long noncoding RNAs (lncRNAs), Ribosome immunopurification, Polysomes, Celltype-specific gene expression, RNA-seq
1 Introduction
Noncoding RNAs (ncRNAs) constitute a highly diverse class of
RNA molecules that are not translatable into proteins, including
both housekeeping (e.g., ribosomal, transfer, small nuclear, and
small nucleolar RNAs) and regulatory RNAs. The category of
regulatory ncRNAs can be further classified into small (sncRNAs)
and long noncoding RNAs (lncRNAs). sncRNAs are molecules of
less than 50 nucleotides (nts) such as microRNAs (miRNAs), small
interference RNAs (siRNAs), and piwi-associated RNAs (piRNAs),
which control gene expression at the transcriptional and posttranscriptional level by guiding DNA methylases to certain genomic
loci, endonucleases to specific transcripts, or inhibiting translation
of complementary mRNAs [1, 2]. On the other hand, lncRNAs are
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_26, © Springer Science+Business Media, LLC, part of Springer Nature 2020
451
TRAP-SEQ of Eukaryotic Translatomes Applied
to the Detection of Polysome-Associated Long
Noncoding RNAs
Soledad Traubenik, Flavio Blanco, Marı ´a Eugenia Zanetti,
and Mauricio A. Reynoso
Abstract
Translating ribosome affinity purification (TRAP) technology allows the isolation of polysomal complexes
and the RNAs associated with at least one 80S ribosome. TRAP consists of the stabilization and affinity
purification of polysomes containing a tagged version of a ribosomal protein. Quantitative assessment of the
TRAP RNA is achieved by direct sequencing (TRAP-SEQ), which provides accurate quantitation of
ribosome-associated RNAs, including long noncoding RNAs (lncRNAs). Here we present an updated
procedure for TRAP-SEQ, as well as a primary analysis guide for identification of ribosome-associated
lncRNAs. This methodology enables the study of dynamic association of lncRNAs by assessing rapid
changes in their transcript levels in polysomes at organ or cell-type level, during development, or in
response to endogenous or exogenous stimuli.
Key words Long noncoding RNAs (lncRNAs), Ribosome immunopurification, Polysomes, Celltype-specific gene expression, RNA-seq
1 Introduction
Noncoding RNAs (ncRNAs) constitute a highly diverse class of
RNA molecules that are not translatable into proteins, including
both housekeeping (e.g., ribosomal, transfer, small nuclear, and
small nucleolar RNAs) and regulatory RNAs. The category of
regulatory ncRNAs can be further classified into small (sncRNAs)
and long noncoding RNAs (lncRNAs). sncRNAs are molecules of
less than 50 nucleotides (nts) such as microRNAs (miRNAs), small
interference RNAs (siRNAs), and piwi-associated RNAs (piRNAs),
which control gene expression at the transcriptional and posttranscriptional level by guiding DNA methylases to certain genomic
loci, endonucleases to specific transcripts, or inhibiting translation
of complementary mRNAs [1, 2]. On the other hand, lncRNAs are
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_26, © Springer Science+Business Media, LLC, part of Springer Nature 2020
451