XB-IMG-77510
Xenbase Image ID: 77510
Fig. 3. Sox3 mRNA is a direct binding target of the RNA-binding protein XSeb4R. (A) SDS-PAGE electrophoregram of coomassie stained gel. XSeb4R and XSeb4RδRRM recombinant pGEX plamids were used to transform Bl21 bacterial cells. Cultures were induced (+ IPTG) or not (− IPTG) for 4 h, harvested and sonicated in PBS supplemented with protease inhibitors. GST-XSeb4R and GST-XSeb4δRRM were purified on glutathione affinty columns. The corresponding eluted fractions are indicated. (B) Schematic representation of Sox3 cDNA and the mutant constructs used in UV-crosslinking assays. Numbers indicate the nucleotide positions in the cDNA. (C) Electrophoregram of radioactive labeled GST-XSeb4R sample from UV-crosslinking. The two bands seen in purified proteins shown in A correspond to active GST-XSeb4R. As shown in lane 1, the full length (FL) Sox3 mRNA probe binds to GST-XSeb4R. Signal of this interaction was competed with cold probe from the 3′UTR (lane 2, 1-to-1 and line 3, 1-to-10 molar ratio) but not from the ORF (lane 4, 1-to-10 molar ratio). Labeled probes from the 3′UTR (lane 5) but not from the ORF (lane 6) bind to GST-XSeb4R. Probes from 3 adjacent F1, F2 and F3 3′UTR show that F1 binds strongly compared to F2 and F3 (compare lane 7, 8, 9). A probe from GAPDH shows no significant interaction with GST-XSeb4R (lane 10). (D) The intensity of the 3′UTR but not GAPDH signal is proportional to the amount of protein used. The signal of the highest protein quantity was competed by unlabeled 3′UTR probe. GST-XSeb4RδRRM shows no interaction with the 3′UTR probe. (E) Probe from the region spanning the F1b interacts with highest avidity to GST-XSeb4R. (F) Ribonucleic acid immuno-precipitation (RIP) coupled to RT-PCR shows that Sox3 and VegT mRNAs are enriched in XSeb4R-RNP complexes (F, lane 2). Unspecific interactions, proportional to the amount of target RNA in the samples, were revealed in control assays (F, lane 1, 4 and 5). The RT-PCR products were analyzed on 2.3% agarose gels.
Image published in: Bentaya S et al. (2012) Copyright © 2012. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |