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

Abstract

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.

Makorin 1 controls embryonic patterning by alleviating Bruno1-mediated repression of oskar translation

“…The extracts were diluted to 1.5 mg protein/ml. Each extract (0.66 ml) was mixed with 24 μg of anti-pAbp Fab antibody (Smibert lab, [61]), 17 μg of α-eIF4G rabbit antibody, or 15 μl of rabbit anti-α−-Tubulin antibody (Abcam). When present, 100 μg RNase A (Qiagen) was added to the samples.…”

The RNA-Binding Protein Rasputin/G3BP Enhances the Stability and Translation of Its Target mRNAs

“…(E and F) Embryo extract treated with either cycloheximide (E) or puromycin (F) was fractionated on sucrose gradients, with the resulting fractions assayed for RIN by western blot. See also Figures S1 and S2 and Table S1. was among the most abundant proteins identified and was greatly enriched in anti-RIN IPs compared with those performed with a control synthetic antibody, C1 (Laver et al, 2012(Laver et al, , 2013(Laver et al, , 2015aNa et al, 2016;Figure 1B).…”

“…To better understand the function of RIN/G3BP in unstressed cells, we have carried out global analyses of the RIN-associated proteome and transcriptome in early Drosophila embryos. Using an anti-RIN synthetic antibody that we isolated from a phagedisplayed library of fragments antigen binding (Fab) (Na et al, 2016;Persson et al, 2013), we immunoprecipitated RIN and then carried out mass spectrometry (IP-MS) to identify RIN's partner proteins. Interactions were found with several RBPs previously shown to interact with G3BP/RIN (e.g., CAPR, FMR1, and LIG), consistent with IP of a biologically relevant RIN-containing complex.…”

Implementation of an antibody characterization process: Application to the major ALS/FTD disease gene C9ORF72

“…Antibodies are vital tools in the biomedical research arsenal and in keeping with their importance there are over a million unique antibodies that are commercially available (Dr. Matt Baker, ThermoFisher, personal communication). In addition are the outputs of numerous, publicly-funded projects, which have generated recombinant or monoclonal antibodies, thus creating renewable antibodies with the potential for high reproducibility (Hornsby et al, 2015;Marcon et al, 2015;Na et al, 2016;Venkataraman et al, 2018;Andrews et al, 2019). The largest public antibody initiative has generated greater than 24,000 antibodies corresponding to almost 17,000 human genes, with a focus on polyclonal antibodies (Uhlen et al, 2015).…”