Fig. 5: RNA binding by A3B and A3H proteins prevents their recognition and ubiquitination by E3 ligases in vitro. | Nature Communications

Fig. 5: RNA binding by A3B and A3H proteins prevents their recognition and ubiquitination by E3 ligases in vitro.

From: Guardian ubiquitin E3 ligases target cancer-associated APOBEC3 deaminases for degradation to promote human genome integrity

Fig. 5: RNA binding by A3B and A3H proteins prevents their recognition and ubiquitination by E3 ligases in vitro.The alternative text for this image may have been generated using AI.

A 10x-His-MBP-A3H-I, 10x-His-MBP-A3H-II and 10xHis-MBP-A3H-II-RBM were expressed in E. coli, purified by HisTrap and subsequent gel filtration, after which purified proteins were analyzed by PAGE and Coomassie staining. B Purified proteins were analyzed by denaturing Urea-TBE PAGE followed by SYBR Gold staining. In vitro ubiquitination assays were performed with recombinant C UBR5, D HUWE1, or E UBR4 and WT A3H-I/II or A3H-RBM as substrates, and in the presence of DyLight488-labeled recombinant ubiquitin. Subsequently, A3H was immunoprecipitated using anti-MBP-coupled beads and the ubiquitination pattern visualized by fluorescent imaging for DyLight488. In vitro ubiquitination assay with recombinant F UBR5, G HUWE1, H UBR4 and A3H-I/II WT or A3H-RBM in the absence or presence of RNase A. Ubiquitinated A3H was visualized as in (C–E). I, J Sucrose gradient binding assays of UBR5 and recombinant A3H proteins. J Recombinant A3H was pre-incubated with RNase A. K Analytical size exclusion chromatography of C-terminally tagged hHUWE1 (inactive) and recombinant A3H. Source data are provided as a Source data file.

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