ChIP-on-chip
A New Technique for Identifying the RNA Targets of RNA-binding Proteins
A review of Genome-wide analysis of mRNAs bound to the histone stem–loop binding protein.
Note: This is a review of the published article listed below. All information, quotes, figures, methods, and findings mentioned in this review are from that article, and are the property of its authors and/or the publication in which the article originally appeared.
Replication-dependent, histone mRNAs are cell-cycle-regulated and expressed only during S phase, ending in a highly conserved 16-nucleotide stem–loop. The histone mRNA 3' stem–loop is bound by the stem–loop binding protein (SLBP) that is involved in pre-mRNA processing, translation, and stability of histone mRNA. Townley-Tilson et al (2006) deviate from the traditional technique of RNA-binding protein (RBP) immunoprecipitation followed by microarray analysis, using Agilent ChIP-on-chip microarrays with recombinant RBP to identify mRNA targets from total RNA. In this study, the authors use the Agilent ChIP microarrays with recombinant epitope-tagged SLBP RNA-binding proteins, circumventing the antibody generation step and avoiding problems typically associated with the existing method. Using this method, the researchers demonstrate that recombinant SLBP binds exclusively to all five classes of histone mRNA. In addition, the researchers are able to analyze the messages bound to the endogenous SLBP on polyribosomes by immunoprecipitation. This data suggests that replication-dependent histone mRNAs are likely to be the sole target of SLBP.
Figure 1.
Strategy for identifying SLBP targets using the rRIP-Chip method. (A) Histone mRNAs are not polyadenylated but instead end in a conserved stem–loop. The message undergoes a single processing step in the nucleus that involves the SLBP and the U7 snRNP. The mature message ending in a conserved stem–loop structure is then transported to the cytoplasm where it is loaded onto polyribosomes. (B) Recombinant purified SLBP was used to identify mRNA targets. Total RNA is purified from either isolated polyribosomes or whole cells. Purified RBP is mixed with the purified RNA and RNA–protein complexes isolated using either an antibody against SLBP or an antibody against a tag in the recombinant protein. RNA is then purified from the bound and unbound fractions. Purified RNA is converted to either Cy3- or Cy5-labeled cDNA by reverse transcription primed with random hexamer primers and hybridized to whole genome microarrays.
Figure 2.
RIP-Chip analysis of the SLBP on Agilent oligonucleotide microarrays. We used Agilent 44,000 element oligonucleotide microarrays to analyze the RNAs bound to the endogenous SLBP and to compare the results to those obtained by RIP analysis of SLBP on the cDNA microarrays. The genes are ordered by mean percentile rank in the RIP-Chip experiment determined using the Agilent arrays. Only those genes above a percentile rank of 99.30% are shown. The results obtained on the Agilent platform indicate that histone genes are likely to be the primary target of the SLBP. It is notable that the nonhistone genes identified as significantly enriched in the rRIP-Chip and RIP-Chip treatments are not enriched in this analysis.
Title: Genome-wide analysis of mRNAs bound to the histone stem-loop
Authors: Townley-Tilson WH, Pendergrass SA, Marzluff WF, Whitfield ML.
Journal: RNA. 2006 Oct;12(10):1853-67.
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