A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes.

Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins.

Genome-wide analysis of Staufen-associated mRNAs identifies secondary structures that confer target specificity.

Despite studies that have investigated the interactions of double-stranded RNA-binding proteins like Staufen with RNA in vitro, how they achieve target specificity in vivo remains uncertain. We performed RNA co-immunoprecipitations followed by microarray analysis to identify Staufen-associated mRNAs in early Drosophila embryos. Analysis of the localization and functions of these transcripts revealed a number of potentially novel roles for Staufen. Using computational methods, we identified two sequence features that distinguish Staufen's target transcripts from non-targets. First, these Drosophila transcripts, as well as those human transcripts bound by human Staufen1 and 2, have 3' untranslated regions (UTRs) that are 3-4-fold longer than unbound transcripts. Second, the 3'UTRs of Staufen-bound transcripts are highly enriched for three types of secondary structures. These structures map with high precision to previously identified Staufen-binding regions in Drosophila bicoid and human ARF1 3'UTRs. Our results provide the first systematic genome-wide analysis showing how a double-stranded RNA-binding protein achieves target specificity.

Synthetic antibodies as tools to probe RNA-binding protein function.

RNA-binding proteins (RBPs) have essential roles in post-transcriptional regulation of gene expression. They bind sequence elements in specific mRNAs and control their splicing, transport, localization, translation, and stability. A complete understanding of RBP function requires identification of the target RNAs that an RBP regulates, the mechanisms by which the RBP regulates these targets, and the biological consequences for the cell in which these transactions occur. Antibodies are key tools in such studies: first, mRNA targets of RBPs can be identified by co-immunoprecipitation of RBPs with their associated RNAs followed by microarray analysis or sequencing; second, partner proteins can be identified by immunoprecipitation of the RBP followed by mass spectrometry; third, the mechanisms and functions of RBPs can be inferred from loss-of-function studies employing antibodies that block RBP-RNA interactions. One potentially powerful approach to making antibodies for such studies is the generation of synthetic antibodies using phage display, which involves in vitro selection using a human-designed antibody library to generate antibodies that recognize a target protein. Using two well-characterized Drosophila RNA-binding proteins, Staufen and Smaug, for proof-of-principle, we demonstrate that synthetic antibodies can be generated and used either to perform RNA-coimmunoprecipitations (RIPs) to identify RBP-bound mRNAs, or to block RBP-RNA interactions. Given that synthetic antibody selection protocols are amenable to high-throughput antibody production, these results demonstrate that synthetic antibodies can be powerful tools for genome-wide studies of RBP function.