To date, research has identified the binding sequences of more
than 400 RBPs in humans and mice model systems [5]. Exploratory
computational analysis in human cells puts the number of human
RBPs at over 1500, or approximately 7.5% of the human proteome
[6]. In Arabidopsis, interactome capture techniques have tentatively identified 1145 RBPs, with 550 of them known to have
RNA binding potential and 595 novel candidates [7]. Many identified RBPs have functions that are tissue or timepoint specific,
prompting further research to categorize their functions in posttranscriptional regulatory processes.
The development of recent techniques such as genome-wide
UV cross-linking immunoprecipitation followed by sequencing
(gCLIP-seq) [8] and protein interaction profile sequencing
(PIP-seq) [9] enable researchers to identify the interaction sites of
RBPs on a transcriptome-wide scale. However, the proteins that
interact with the identified RBP-bound sequences are not known.
Therefore, subsequent RNA-tagging-based techniques can help
identify the specific RBPs that bind to unique target sequences
identified by these high-throughput techniques. To do this, the
identified protein-interacting sequences are used as bait to form
RNP complexes with RBPs that bind these sequences in a protein
lysate of interest. These probe-bound RBPs are then eluted and
identified using mass spectrometry. The process is based on the
propensity of RBPs to bind to specific sequences, which enables
researchers to identify proteins that are enriched in a purification
experiment as likely RBP candidates for further validation.
The protocol described in this chapter (Fig. 1) is one such
approach for identifying RBPs where RNA sequences of interest
are covalently tagged to adipic acid dihydrazide agarose beads
through their 3
0 hydroxyl groups. Each tagged RNA sequence is
then combined with a protein lysate so that the RBPs that recognize and interact with the RNA sequence of interest are isolated and
then subsequently identified using mass spectrometry. We have
successfully used this technique multiple times with results from
the model plant Arabidopsis to identify a novel nuclear function for
an RBP that had previously been annotated to be localized specifically in the chloroplast [10] as well as SERRATE and GLYCINERICH RNA-BINDING PROTEIN8 (GRP8) as RBPs with novel
functions in regulating hair root cell development [11].
2 Materials
2.1 Equipment
1. Centrifuge.
2. Microcentrifuge.
3. SpeedVac.
242
Mengge Shan and Brian D. Gregory
than 400 RBPs in humans and mice model systems [5]. Exploratory
computational analysis in human cells puts the number of human
RBPs at over 1500, or approximately 7.5% of the human proteome
[6]. In Arabidopsis, interactome capture techniques have tentatively identified 1145 RBPs, with 550 of them known to have
RNA binding potential and 595 novel candidates [7]. Many identified RBPs have functions that are tissue or timepoint specific,
prompting further research to categorize their functions in posttranscriptional regulatory processes.
The development of recent techniques such as genome-wide
UV cross-linking immunoprecipitation followed by sequencing
(gCLIP-seq) [8] and protein interaction profile sequencing
(PIP-seq) [9] enable researchers to identify the interaction sites of
RBPs on a transcriptome-wide scale. However, the proteins that
interact with the identified RBP-bound sequences are not known.
Therefore, subsequent RNA-tagging-based techniques can help
identify the specific RBPs that bind to unique target sequences
identified by these high-throughput techniques. To do this, the
identified protein-interacting sequences are used as bait to form
RNP complexes with RBPs that bind these sequences in a protein
lysate of interest. These probe-bound RBPs are then eluted and
identified using mass spectrometry. The process is based on the
propensity of RBPs to bind to specific sequences, which enables
researchers to identify proteins that are enriched in a purification
experiment as likely RBP candidates for further validation.
The protocol described in this chapter (Fig. 1) is one such
approach for identifying RBPs where RNA sequences of interest
are covalently tagged to adipic acid dihydrazide agarose beads
through their 3
0 hydroxyl groups. Each tagged RNA sequence is
then combined with a protein lysate so that the RBPs that recognize and interact with the RNA sequence of interest are isolated and
then subsequently identified using mass spectrometry. We have
successfully used this technique multiple times with results from
the model plant Arabidopsis to identify a novel nuclear function for
an RBP that had previously been annotated to be localized specifically in the chloroplast [10] as well as SERRATE and GLYCINERICH RNA-BINDING PROTEIN8 (GRP8) as RBPs with novel
functions in regulating hair root cell development [11].
2 Materials
2.1 Equipment
1. Centrifuge.
2. Microcentrifuge.
3. SpeedVac.
242
Mengge Shan and Brian D. Gregory