Figure 1.
Schematic of RIP-CHIP approach used to identify functionally relevant mRNAs.
(A) Diagram of the cDNA expression vector containing two FLAG tags (“FF”) and two copies of the IgG binding domain of protein A (“ZZ”). The tobacco etch virus (TEV) protease cleavage site is indicated, including an arrowhead to designate cleavage between glutamine and glycine residues. CMV, cytomegalovirus promoter; NeoR, neomycin resistance cassette; pA, polyadenylation site. (B) Schematic of purification strategy using TEV protease cleavage. ZZ-tagged protein is bound to IgG beads, washed, and the protein, along with co-purified mRNAs, are eluted from the beads by TEV protease cleavage. (C) Demonstration of WT1(+KTS)-FF-ZZ expression in differentiated MPC-5 cells (“Input”) and the elution of WT1(+KTS)-FF bound to the beads after 3 washes and TEV protease cleavage as determined by immunoblotting with anti-FLAG antibodies. (D) Flowchart summarizing the RIP-CHIP approach.
Table 1.
Co-purified mRNAs enriched in eluates relative to input extracts.
Figure 2.
Enriched mRNAs identified by microarray analysis are independently validated by qRT-PCR.
Quantitative RT-PCR analysis of a subset of the enriched mRNAs identified in this study. Two independent pulldowns using MPC-5 cell extracts stably WT1(+KTS)-FF-ZZ were completed and total RNAs from input extracts and WT1(+KTS) enriched fractions following TEV protease cleavage were compared. Mean expression values from triplicate assays±SD are shown relative to the input control samples.
Table 2.
Gene Ontogeny (GO) Analysis.
Table 3.
Protein domains enriched in products of identified mRNAs.
Table 4.
Enriched mRNAs that encode proteins with well established and/or have been shown to bind directly to actin.
Figure 3.
The enrichment of the mRNAs in the pulldown assays does not require association with WT1(+KTS).
(A) Elution of Myh10 mRNA from WT1(+KTS)-FF-ZZ containing IgG beads occurs with addition of bovine serum albumen (BSA) as well as TEV protease. Shown is qRT-PCR analysis of Myh10 and Gapdh in IP's using cell extracts from MPC-5 cells stably expressing WT1(+KTS)-FF-ZZ. Mean expression values from triplicate assays±SD are shown relative to Input samples. (B) The same enrichment of Myh10 mRNA shown in (A) in response to TEV protease and BSA using extracts from cells expressing the pIN empty vector. Mean expression values from triplicate assays±SD are shown relative to Input samples. (C) Immunoblot demonstrating that WT1(+KTS)-FF-ZZ elution occurs only upon addition of TEV protease and not with addition of BSA or TEV cleavage buffer alone. Presence of the FLAG tagged WT1(+KTS) protein was determined using anti-FLAG antibodies.
Figure 4.
Protein components of the actin cytoskeleton are present in eluates used to identify the enriched subset of mRNAs.
(A) Coomassie stained gels demonstrating proteins present in the IP eluates using MPC-5 cell extracts stably expressing WT1(+KTS)-FF-ZZ or empty vector expression constructs. Arrows correspond to the bands from TEV eluate lane of the WT1(+KTS)-FF-ZZ gel that were excised and submitted for mass spectrometry analysis. The same arrows also indicate the bands which correspond to tubulin and elongation factor 1α (top arrow) and actin (bottom arrow). * indicates TEV protease and ** indicates BSA bands. The actin (B) and elongation factor 1α (C) proteins are not enriched in the eluates from IgG beads. Immunoblots confirming the presence, but lack of enrichment of β-actin and elongation factor 1α in eluate samples from WT1(+KTS)-FF-ZZ and empty vector control pulldowns with anti-β-actin and anti-elongation factor 1α antibodies.
Figure 5.
Schematic representation of a hypothesized podocyte protein “interactome” encoded by enriched mRNAs.
Large black type indicates proteins encoded by mRNAs identified in this study. Smaller grey type is used to indicate several known podocyte foot process proteins in order to orient the figure. The interactions shown represent published results showing either direct interactions or presence in common protein complexes.