AI Article Synopsis
- The m G cap is crucial for eukaryotic mRNA, interacting with proteins that support cell growth and function.
- Researchers created fluorescently tagged m G nucleotides to study cap-binding proteins and found that pyrene-labeled compounds enhanced fluorescence significantly when bound to eIF4E or cleaved by DcpS.
- This led to the development of assays for measuring binding and enzymatic activity, which were successfully validated using known libraries for eIF4E ligands and DcpS inhibitors in human cell extracts.
Article Abstract
The m G cap is a unique nucleotide structure at the 5'-end of all eukaryotic mRNAs. The cap specifically interacts with numerous cellular proteins and participates in biological processes that are essential for cell growth and function. To provide small molecular probes to study important cap-recognizing proteins, we synthesized m G nucleotides labeled with fluorescent tags via the terminal phosph(on)ate group and studied how their emission properties changed upon protein binding or enzymatic cleavage. Only the pyrene-labeled compounds behaved as sensitive turn-on probes. A pyrene-labeled m GTP analogue showed up to eightfold enhanced fluorescence emission upon binding to eukaryotic translation initiation factor 4E (eIF4E) and over 30-fold enhancement upon cleavage by decapping scavenger (DcpS) enzyme. These observations served as the basis for developing binding- and hydrolytic-activity assays. The assay utility was validated with previously characterized libraries of eIF4E ligands and DcpS inhibitors. The DcpS assay was also applied to study hydrolytic activity and inhibition of endogenous enzyme in cytoplasmic extracts from HeLa and HEK cells.
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| http://dx.doi.org/10.1002/chem.201900051 | DOI Listing |
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