Visible Light Activates Coumarin-Caged mRNA for Cytoplasmic Cap Methylation in Cells.

Protein synthesis is important and regulated by various mechanisms in the cell. Translation initiation in eukaryotes starts at the 5' cap and is the most complex of the three phases of mRNA translation. It requires methylation of the N7 position of the terminal guanosine (m7 G). The canonical capping occurs in the nucleus, however, cytoplasmic recapping has been discovered. It functions in switching mRNAs between translating and non-translating states, but the individual steps are difficult to dissect. We targeted cytoplasmic cap methylation as the ultimate step of cytoplasmic recapping. We present an N7G photocaged 5' cap that can be activated for cytoplasmic methylation by visible light. We report chemical and chemo-enzymatic synthesis of this 5' cap with 7-(diethylamino)-4-methyl-coumarin (DEACM) at the N7G and validate that it is not bound by translation initiation factor 4E (eIF4E). We demonstrate incorporation into mRNA, the release of unmethylated cap analog and enzymatic remethylation to functional cap 0 after irradiation at 450 nm. In cells, irradiation triggers translation of mRNAs with the N7G photocaged 5' cap via cytoplasmic cap methylation.

Enzymatic or In Vivo Installation of Propargyl Groups in Combination with Click Chemistry for the Enrichment and Detection of Methyltransferase Target Sites in RNA.

m6 A is the most abundant internal modification in eukaryotic mRNA. It is introduced by METTL3-METTL14 and tunes mRNA metabolism, impacting cell differentiation and development. Precise transcriptome-wide assignment of m6 A sites is of utmost importance. However, m6 A does not interfere with Watson-Crick base pairing, making polymerase-based detection challenging. We developed a chemical biology approach for the precise mapping of methyltransferase (MTase) target sites based on the introduction of a bioorthogonal propargyl group in vitro and in cells. We show that propargyl groups can be introduced enzymatically by wild-type METTL3-METTL14. Reverse transcription terminated up to 65 % at m6 A sites after bioconjugation and purification, hence enabling detection of METTL3-METTL14 target sites by next generation sequencing. Importantly, we implemented metabolic propargyl labeling of RNA MTase target sites in vivo based on propargyl-l-selenohomocysteine and validated different types of known rRNA methylation sites.

Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2.

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in the regulation of metabolic activity in cancer and immune cells, and affects whole-body metabolism by regulating ghrelin-signalling in the hypothalamus. This has led to efforts to develop specific CaMKK2 inhibitors, and STO-609 is the standardly used CaMKK2 inhibitor to date. We have developed a novel fluorescence-based assay by exploiting the intrinsic fluorescence properties of STO-609. Here, we report an in vitro binding constant of KD ∼17 nM between STO-609 and purified CaMKK2 or CaMKK2:Calmodulin complex. Whereas high concentrations of ATP were able to displace STO-609 from the kinase, GTP was unable to achieve this confirming the specificity of this association. Recent structural studies on the kinase domain of CaMKK2 had implicated a number of amino acids involved in the binding of STO-609. Our fluorescent assay enabled us to confirm that Phe(267) is critically important for this association since mutation of this residue to a glycine abolished the binding of STO-609. An ATP replacement assay, as well as the mutation of the 'gatekeeper' amino acid Phe(267)Gly, confirmed the specificity of the assay and once more confirmed the strong binding of STO-609 to the kinase. In further characterising the purified kinase and kinase-calmodulin complex we identified a number of phosphorylation sites some of which corroborated previously reported CaMKK2 phosphorylation and some of which, particularly in the activation segment, were novel phosphorylation events. In conclusion, the intrinsic fluorescent properties of STO-609 provide a great opportunity to utilise this drug to label the ATP-binding pocket and probe the impact of mutations and other regulatory modifications and interactions on the pocket. It is however clear that the number of phosphorylation sites on CaMKK2 will pose a challenge in studying the impact of phosphorylation on the pocket unless the field can develop approaches to control the spectrum of modifications that occur during recombinant protein expression in Escherichia coli.

Data for the co-expression and purification of human recombinant CaMKK2 in complex with calmodulin in Escherichia coli.

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. Here, we describe the expression in Escherichia coli and the purification protocol for the following constructs: full-length CaMKK2 in complex with CaM, CaMKK2 'apo', CaMKK2 (165-501) in complex with CaM, and the CaMKK2 F267G mutant. The protocols described have been optimized for maximum yield and purity with minimal purification steps required and the proteins subsequently used to develop a fluorescence-based assay for drug binding to the kinase, "Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2" [1].