Development of a sensitive microplate assay for characterizing RNA methyltransferase activity: Implications for epitranscriptomics and drug development.

RNA methylation is a ubiquitous post-transcriptional modification found in diverse RNA classes and is a critical regulator of gene expression. In this study, we used Zika virus RNA methyltransferase (MTase) to develop a highly sensitive microplate assay that uses a biotinylated RNA substrate and radiolabeled AdoMet coenzyme. The assay is fast, highly reproducible, exhibits linear progress-curve kinetics under multiple turnover conditions, has high sensitivity in competitive inhibition assays, and significantly lower background levels compared with the currently used method. Using our newly developed microplate assay, we observed no significant difference in the catalytic constants of the full-length nonstructural protein 5 enzyme and the truncated MTase domain. These data suggest that, unlike the Zika virus RNA-dependent RNA polymerase activity, the MTase activity is unaffected by RNA-dependent RNA polymerase-MTase interdomain interaction. Given its quantitative nature and accuracy, this method can be used to characterize various RNA MTases, and, therefore, significantly contribute to the field of epitranscriptomics and drug development against infectious diseases.

Steady-state kinetic studies reveal that the anti-cancer target Ubiquitin-Specific Protease 17 (USP17) is a highly efficient deubiquitinating enzyme.

USP17 is a deubiquitinating enzyme that is upregulated in numerous cancers and therefore a drug target. We developed a robust expression, purification, and assay system for USP17 enabling its enzymatic and structural characterization. USP17 was expressed in E. coli as inclusion bodies and then solubilized, refolded, and purified using affinity and size-exclusion chromatography. Milligram quantities of pure USP17 can be produced that is catalytically more efficient (kcat/Km = 1500 (x103) M-1sec-1) than other human USPs studied to date. Analytical size-exclusion chromatography, analytical ultracentrifugation, and dynamic light scattering studies suggest that the quaternary structure of USP17 is a monomer. Steady-state kinetic studies show that USP17 efficiently hydrolyzes both ubiquitin-AMC (kcat = 1.5 sec-1 and Km = 1.0 µM) and ubiquitin-rhodamine110 (kcat = 1.8 sec-1 and Km = 2.0 µM) substrates. Ubiquitin chain cleavage assays reveal that USP17 efficiently cleaves di-ubiquitin chains with Lys11, Lys33, Lys48 and Lys63 linkages and tetra-ubiquitin chains with Lys11, Lys48 and Lys63 linkages but is inefficient in cleaving di-ubiquitin chains with Lys6, Lys27, or Lys29 linkages or linear ubiquitin chains. The substrate specificity of USP17 is most similar to that of USP1, where both USPs display higher specificity than other characterized members of the USP family.