Enzolution(R) eIF4A3 ATPase FI Assay System

The Enzolution EIF4A3 ATPase Assay System is intended for use with the Transcreener(R) ADP Assay Kits to measure the RNA-dependent ATPase activity of EIF4A3 (eukaryotic initiation factor 4A-III), a member of the DEAD-box family of RNA helicase family (DDX48). EIF4A3 is a key core factor of the exon junction complex (EJC), a splicing-dependent multiprotein assembly that is deposited on mRNAs at splice junctions. ADP formation by EIF4A3 can be easily measured with the Transcreener ADP2 assay, a far-red, competitive fluorescence assay that enables single addition, mix-and-read detection in a continuous or endpoint format. The assay has been optimized and extensively validated for high throughput screening (HTS) and inhibitor dose response measurements using most multimode plate readers. The Enzolution EIF4A3 ATPase Assay System provides all reagents required to screen and profile EIF4A3 inhibitors, including purified, full-length human EIF4A3 and yeast RNA, when used with the Transcreener ADP2 Assay Kits, which are available with FP, FI and TR-FRET readouts. The protocol is configured for 384-well plates, use of different multi-well plate formats will require adjustment of reagents concentrations utilized in the assay. Key Applications:  Screening for EIF4A3 inhibitors (or activators)  Generating dose response curves and IC50 values for EIF4A3 inhibitors  Kinetic and mechanistic analyses Protein Function: ATP-dependent RNA helicase (PubMed:16170325). Involved in pre-mRNA splicing as component of the spliceosome (PubMed:11991638, PubMed:22961380, PubMed:28076346, PubMed:28502770, PubMed:29301961). Core component of the splicing-dependent multiprotein exon junction complex (EJC) deposited at splice junctions on mRNAs (PubMed:16170325, PubMed:16209946, PubMed:16314458, PubMed:16923391, PubMed:16931718, PubMed:19033377, PubMed:20479275). The EJC is a dynamic structure consisting of core proteins and several peripheral nuclear and cytoplasmic associated factors that join the complex only transiently either during EJC assembly or during subsequent mRNA metabolism. The EJC marks the position of the exon-exon junction in the mature mRNA for the gene expression machinery and the core components remain bound to spliced mRNAs throughout all stages of mRNA metabolism thereby influencing downstream processes including nuclear mRNA export, subcellular mRNA localization, translation efficiency and nonsense-mediated mRNA decay (NMD). Its RNA-dependent ATPase and RNA-helicase activities are induced by CASC3, but abolished in presence of the MAGOH-RBM8A heterodimer, thereby trapping the ATP-bound EJC core onto spliced mRNA in a stable conformation. The inhibition of ATPase activity by the MAGOH-RBM8A heterodimer increases the RNA-binding affinity of the EJC. Involved in translational enhancement of spliced mRNAs after formation of the 80S ribosome complex. Binds spliced mRNA in sequence-independent manner, 20-24 nucleotides upstream of mRNA exon-exon junctions. Shows higher affinity for single-stranded RNA in an ATP-bound core EJC complex than after the ATP is hydrolyzed. Involved in the splicing modulation of BCL2L1/Bcl-X (and probably other apoptotic genes), specifically inhibits formation of proapoptotic isoforms such as Bcl-X(S), the function is different from the established EJC assembly (PubMed:22203037). Involved in craniofacial development (PubMed:24360810) [The Uniprot Consortium]