introduction/practicalTips.html is geared toward HOMER users,
the general principles discussed regarding background selection,
motif size restriction, etc. is applicable to other motif enrichment
tools.
Once you select the motifs of interest for identifying the specific
interacting RBPs, they need to be synthesized as an RNA molecule
for subsequent tagging with agarose beads and ultimately identification of interacting protein. We tend to choose motif sequences
that are relatively short (i.e., <20 nucleotides) in nature, which
allows us to develop an RNA probe that consists of a duplex of the
motif sequence of interest separated by random nucleotides. We
recommend designating these random nucleotides as a sequence of
Ns (Fig. 2). The inclusion of these random spacer regions is to
minimize the risk of the binding site being masked by the
bead [14].
3.2 Protein Lysate
Collection
The RNA affinity purification protocol is cell culture independent
and can therefore be easily adapted to suit any organism of interest.
Standard protein collection protocol should be employed to collect
protein samples, keeping in mind that each reaction requires
45–50 μg of protein at a minimum concentration of 0.75 μg/μL
(see Note 7).
3.3 RNA Affinity
Purification
The following RNA affinity purification protocol takes 3 days and is
written for a single reaction. If using the same RNA probe on
multiple samples, we recommend treating each probe-lysate pair
separately. For example, if probe A is to be used in four samples,
make four probe A samples as opposed to adding four times the
volumes of buffers to a bigger tube and then splitting the resulting
probe sample four ways.
Though the numerous washing steps should alleviate much of
the nonspecific protein binding that can pose a challenge in the
RNA affinity protocol, suitable controls such as scrambled RNA
probe and/or non-RNA probe should still be included in your
experiments to eliminate any nonspecific proteins that generally
bind RNA or that are found in high concentration and tend to
contaminate mass spectrometry analyses (e.g., ribosomal subunit
proteins). In the downstream analysis, RBPs that are enriched in the
test probe when compared to the control probe(s) should be considered as candidates for further analysis and follow-up studies.
3.3.1 Tagging RNA
with Agarose Beads
In order to make the execution of the protocol smoother, users
should (1) reconstitute the RNA probe in RNase-free water and
(2) make up ~42 mL/probe worth of 0.1 M NaOAc (see Note 8)
before starting. The following steps will oxidize the RNA probe at
the 3
0 end before precipitating the oxidized RNA with ethanol and
then tagging the RNA sequences with agarose beads.
Using Bead-Tagged RNAs to Identify RBPs
247
the general principles discussed regarding background selection,
motif size restriction, etc. is applicable to other motif enrichment
tools.
Once you select the motifs of interest for identifying the specific
interacting RBPs, they need to be synthesized as an RNA molecule
for subsequent tagging with agarose beads and ultimately identification of interacting protein. We tend to choose motif sequences
that are relatively short (i.e., <20 nucleotides) in nature, which
allows us to develop an RNA probe that consists of a duplex of the
motif sequence of interest separated by random nucleotides. We
recommend designating these random nucleotides as a sequence of
Ns (Fig. 2). The inclusion of these random spacer regions is to
minimize the risk of the binding site being masked by the
bead [14].
3.2 Protein Lysate
Collection
The RNA affinity purification protocol is cell culture independent
and can therefore be easily adapted to suit any organism of interest.
Standard protein collection protocol should be employed to collect
protein samples, keeping in mind that each reaction requires
45–50 μg of protein at a minimum concentration of 0.75 μg/μL
(see Note 7).
3.3 RNA Affinity
Purification
The following RNA affinity purification protocol takes 3 days and is
written for a single reaction. If using the same RNA probe on
multiple samples, we recommend treating each probe-lysate pair
separately. For example, if probe A is to be used in four samples,
make four probe A samples as opposed to adding four times the
volumes of buffers to a bigger tube and then splitting the resulting
probe sample four ways.
Though the numerous washing steps should alleviate much of
the nonspecific protein binding that can pose a challenge in the
RNA affinity protocol, suitable controls such as scrambled RNA
probe and/or non-RNA probe should still be included in your
experiments to eliminate any nonspecific proteins that generally
bind RNA or that are found in high concentration and tend to
contaminate mass spectrometry analyses (e.g., ribosomal subunit
proteins). In the downstream analysis, RBPs that are enriched in the
test probe when compared to the control probe(s) should be considered as candidates for further analysis and follow-up studies.
3.3.1 Tagging RNA
with Agarose Beads
In order to make the execution of the protocol smoother, users
should (1) reconstitute the RNA probe in RNase-free water and
(2) make up ~42 mL/probe worth of 0.1 M NaOAc (see Note 8)
before starting. The following steps will oxidize the RNA probe at
the 3
0 end before precipitating the oxidized RNA with ethanol and
then tagging the RNA sequences with agarose beads.
Using Bead-Tagged RNAs to Identify RBPs
247