We have adapted the individual nucleotide resolution and
immunoprecipitation (iCLIP) procedure developed by the Ule
laboratory so that it is suitable for use in the model plant Arabidopsis thaliana [12, 19–22].
Transgenic expression of an epitope-tagged version of the RBP
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 same cell type as the RBP, the endogenous promoter should
be used to drive the expression of the epitope-tagged protein. It is
recommended to include the authentic cis-regulatory sequences
within the transcribed part of the gene, i.e., untranslated regions
and introns. It is imperative to demonstrate that the fusion protein
retains in vivo functionality, most conveniently through complementation of a loss-of-function mutant.
In parallel to the experimental transgenic plant line expressing
the RBP-GFP fusion protein, control lines can be generated that
express an RNA-binding deficient variant of the RBP 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 [8] or by mock treatment of the extract with an unrelated
antibody, e.g., RFP Trap beads.
Here, we describe the adaptation of UV cross-linking, recovery
of RNA-protein complexes, and retrieval of the RNA from
gel-purified complexes for plant tissue. Subsequent generation of
cDNA libraries, high-throughput sequencing, and bioinformatics
determination of binding targets and cross-link sites are done
according to published protocols.
2 Materials
2.1 Plant Growth
1. Agar plates: 2.2 g Murashige-Skoog (MS) powder, 0.5 g morpholinoethane sulfonate (MES), 50 g sucrose. Add water to
1 L, adjust to pH 5.7 using 1 M KOH, add 10 g plant growth
agar, and autoclave. Distribute the sterilized medium into agar
plates and allow to cool under sterile conditions.
2.2 UV Light Fixation
1. Liquid nitrogen.
2. UV cross-linker equipped with 254 nm lamps.
3. Mortar and pestle.
4. 2 mL Tubes with punctured lid.
Plant iCLIP
257
immunoprecipitation (iCLIP) procedure developed by the Ule
laboratory so that it is suitable for use in the model plant Arabidopsis thaliana [12, 19–22].
Transgenic expression of an epitope-tagged version of the RBP
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 same cell type as the RBP, the endogenous promoter should
be used to drive the expression of the epitope-tagged protein. It is
recommended to include the authentic cis-regulatory sequences
within the transcribed part of the gene, i.e., untranslated regions
and introns. It is imperative to demonstrate that the fusion protein
retains in vivo functionality, most conveniently through complementation of a loss-of-function mutant.
In parallel to the experimental transgenic plant line expressing
the RBP-GFP fusion protein, control lines can be generated that
express an RNA-binding deficient variant of the RBP 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 [8] or by mock treatment of the extract with an unrelated
antibody, e.g., RFP Trap beads.
Here, we describe the adaptation of UV cross-linking, recovery
of RNA-protein complexes, and retrieval of the RNA from
gel-purified complexes for plant tissue. Subsequent generation of
cDNA libraries, high-throughput sequencing, and bioinformatics
determination of binding targets and cross-link sites are done
according to published protocols.
2 Materials
2.1 Plant Growth
1. Agar plates: 2.2 g Murashige-Skoog (MS) powder, 0.5 g morpholinoethane sulfonate (MES), 50 g sucrose. Add water to
1 L, adjust to pH 5.7 using 1 M KOH, add 10 g plant growth
agar, and autoclave. Distribute the sterilized medium into agar
plates and allow to cool under sterile conditions.
2.2 UV Light Fixation
1. Liquid nitrogen.
2. UV cross-linker equipped with 254 nm lamps.
3. Mortar and pestle.
4. 2 mL Tubes with punctured lid.
Plant iCLIP
257