we have applied RIP to analyze in vivo interaction of an RBP with
single mRNAs by RIP-qPCR [3, 4]. Here, we extend our protocol
to screen target transcripts transcriptome-wide by constructing
RNA libraries for high-throughput sequencing (RIP-seq) from
the immunoprecipitated RNA population.
To preserve the in vivo binding landscape, a fixation step is
applied that stabilizes transient and weak RNA-protein-complexes
and thus allows more stringent washing conditions to be applied.
Moreover, it abrogates reconfiguration or RNA-protein complexes
upon tissue disruption [5]. In Arabidopsis, chemical cross-linking
through formaldehyde fixation has been mostly used. To ensure
that formaldehyde penetrates the plant cell efficiently, vacuum infiltration is applied to displace the gas in the intercellular space. Of
note, formaldehyde also crosslinks proteins so that transcripts may
also coprecipitate due to binding to an interacting protein.
Transgenic expression of an epitope-tagged version of the
RNA-binding protein allows the recovery of RNA-protein complexes by virtue of commercially available antibodies of high specificity and high affinity for the tag. To obtain a realistic picture of the
in vivo targets expressed in the very cell type as the RNA-binding
protein, the endogenous promoter and regulatory sequences
including 5
0 UTR, 3
0 UTR, and introns should be used to drive
expression of the epitope-tagged protein.
Reliable interpretation of the RIP data depends on several
appropriate controls. An aliquot of the cellular lysate can be taken
before preclearing. This “input” sample represents the RNA complement employed for RIP and serves as positive control for the
presence of the transcripts under study. In parallel to the experimental line expressing the RNA-binding protein-GFP fusion protein, control lines can be generated that express an RNA-binding
dead variant of the RNA-binding protein or the GFP moiety by
itself, both driven by the identical regulatory elements of the RBP
gene. A comparative analysis of experimental and control lines is
then performed using GFP-Trap
® beads for samples as well as
controls. Alternatively, control precipitations can be performed
with GFP antibodies in wild-type plants lacking the RBP-GFP
fusion protein [6]. Furthermore, a mock immunoprecipitation of
the cellular lysate can be performed using an unrelated antibody,
e.g., RFP-Trap
® beads in case of GFP fusion proteins. RNAs recovered in this mock IP reflect noise during processing of the extract.
We describe here a protocol to construct libraries from RNAs
co-immunoprecipitated with RBP-GFP fusion proteins. Sequencing of standard RNA-seq libraries prepared from plants harvested in
parallel can be used to determine an enrichment of transcripts in the
RIP samples.
454
Tino Ko ¨ ster and Dorothee Staiger
single mRNAs by RIP-qPCR [3, 4]. Here, we extend our protocol
to screen target transcripts transcriptome-wide by constructing
RNA libraries for high-throughput sequencing (RIP-seq) from
the immunoprecipitated RNA population.
To preserve the in vivo binding landscape, a fixation step is
applied that stabilizes transient and weak RNA-protein-complexes
and thus allows more stringent washing conditions to be applied.
Moreover, it abrogates reconfiguration or RNA-protein complexes
upon tissue disruption [5]. In Arabidopsis, chemical cross-linking
through formaldehyde fixation has been mostly used. To ensure
that formaldehyde penetrates the plant cell efficiently, vacuum infiltration is applied to displace the gas in the intercellular space. Of
note, formaldehyde also crosslinks proteins so that transcripts may
also coprecipitate due to binding to an interacting protein.
Transgenic expression of an epitope-tagged version of the
RNA-binding protein allows the recovery of RNA-protein complexes by virtue of commercially available antibodies of high specificity and high affinity for the tag. To obtain a realistic picture of the
in vivo targets expressed in the very cell type as the RNA-binding
protein, the endogenous promoter and regulatory sequences
including 5
0 UTR, 3
0 UTR, and introns should be used to drive
expression of the epitope-tagged protein.
Reliable interpretation of the RIP data depends on several
appropriate controls. An aliquot of the cellular lysate can be taken
before preclearing. This “input” sample represents the RNA complement employed for RIP and serves as positive control for the
presence of the transcripts under study. In parallel to the experimental line expressing the RNA-binding protein-GFP fusion protein, control lines can be generated that express an RNA-binding
dead variant of the RNA-binding protein or the GFP moiety by
itself, both driven by the identical regulatory elements of the RBP
gene. A comparative analysis of experimental and control lines is
then performed using GFP-Trap
® beads for samples as well as
controls. Alternatively, control precipitations can be performed
with GFP antibodies in wild-type plants lacking the RBP-GFP
fusion protein [6]. Furthermore, a mock immunoprecipitation of
the cellular lysate can be performed using an unrelated antibody,
e.g., RFP-Trap
® beads in case of GFP fusion proteins. RNAs recovered in this mock IP reflect noise during processing of the extract.
We describe here a protocol to construct libraries from RNAs
co-immunoprecipitated with RBP-GFP fusion proteins. Sequencing of standard RNA-seq libraries prepared from plants harvested in
parallel can be used to determine an enrichment of transcripts in the
RIP samples.
454
Tino Ko ¨ ster and Dorothee Staiger