Marcin Ziemniak

Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5′,5′-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m27,3′-OGpppG. Higher expression of cancer antigens would make mRNAs containing m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2 favorable for anticancer immunization.

Potential therapeutic applications of RNA cap analogs

Cap analogs are chemically modified derivatives of the unique cap structure present at the 5´ end of all eukaryotic mRNAs and several non-coding RNAs. Until recently, cap analogs have served primarily as tools in the study of RNA metabolism. Continuing advances in our understanding of cap biological functions (including RNA stabilization, pre-mRNA splicing, initiation of mRNA translation, as well as cellular transport of mRNAs and snRNAs) and the consequences of the disruption of these processes - resulting in serious medical disorders - have opened new possibilities for pharmaceutical applications of these compounds. In this review, the medicinal potential of cap analogs in areas, such as cancer treatment (including eIF4E targeting and mRNA-based immunotherapy), spinal muscular atrophy treatment, antiviral therapy and the improvement of the localization of nucleus-targeting drugs, are highlighted. Advances achieved to date, challenges, plausible solutions and prospects for the future development of cap analog-based drug design are described.

Structural basis of mRNA-cap recognition by Dcp1-Dcp2

Removal of the 5′ cap on mRNA by the decapping enzyme Dcp2 is a critical step in 5′-to-3′ mRNA decay. Understanding the structural basis of Dcp2 activity has been a challenge because Dcp2 is dynamic and has weak affinity for the cap substrate. Here we present a 2.6-Å-resolution crystal structure of a heterotrimer of fission yeast Dcp2, its essential activator Dcp1, and the human NMD cofactor PNRC2, in complex with a tight-binding cap analog. Cap binding is accompanied by a conformational change in Dcp2, thereby forming a composite nucleotide-binding site comprising conserved residues in the catalytic and regulatory domains. Kinetic analysis of PNRC2 revealed that a conserved short linear motif enhances both substrate affinity and the catalytic step of decapping. These findings explain why Dcp2 requires a conformational change for efficient catalysis and reveals that coactivators promote RNA binding and the catalytic step of decapping, possibly through different conformational states.

Synthesis and evaluation of fluorescent cap analogues for mRNA labelling

We describe the synthesis and properties of five dinucleotide fluorescent cap analogues labelled at the ribose of the 7-methylguanosine moiety with either anthraniloyl (Ant) or N-methylanthraniloyl (Mant), which have been designed for the preparation of fluorescent mRNAs via transcription in vitro. Two of the analogues bear a methylene modification in the triphosphate bridge, providing resistance against either the Dcp2 or DcpS decapping enzymes. All these compounds were prepared by ZnCl2-mediated coupling of a nucleotide P-imidazolide with a fluorescently labelled mononucleotide. To evaluate the utility of these compounds for studying interactions with cap-binding proteins and cap-related cellular processes, both biological and spectroscopic features of those compounds were determined. The results indicate acceptable quantum yields of fluorescence, pH independence, environmental sensitivity, and photostability. The cap analogues are incorporated by RNA polymerase into mRNA transcripts that are efficiently translated in vitro. Transcripts containing fluorescent caps but unmodified in the triphosphate chain are hydrolysed by Dcp2 whereas those containing a α-β methylene modification are resistant. Model studies exploiting sensitivity of Mant to changes of local environment demonstrated utility of the synthesized compounds for studying cap-related proteins.

Structure of the activated Edc1-Dcp1-Dcp2-Edc3 mRNA decapping complex with substrate analog poised for catalysis

The conserved decapping enzyme Dcp2 recognizes and removes the 5′ eukaryotic cap from mRNA transcripts in a critical step of many cellular RNA decay pathways. Dcp2 is a dynamic enzyme that functions in concert with the essential activator Dcp1 and a diverse set of coactivators to selectively and efficiently decap target mRNAs in the cell. Here we present a 2.84 Å crystal structure of K. lactis Dcp1–Dcp2 in complex with coactivators Edc1 and Edc3, and with substrate analog bound to the Dcp2 active site. Our structure shows how Dcp2 recognizes cap substrate in the catalytically active conformation of the enzyme, and how coactivator Edc1 forms a three-way interface that bridges the domains of Dcp2 to consolidate the active conformation. Kinetic data reveal Dcp2 has selectivity for the first transcribed nucleotide during the catalytic step. The heterotetrameric Edc1–Dcp1–Dcp2–Edc3 structure shows how coactivators Edc1 and Edc3 can act simultaneously to activate decapping catalysis.The decapping enzyme Dcp2 removes the 5′ eukaryotic cap from mRNA transcripts and acts in concert with its essential activator Dcp1 and various coactivators. Here the authors present the structure of the fully-activated mRNA decapping complex, which reveals how Dcp2 recognizes the cap substrate and coactivators Edc1 and Edc3 activate catalysis.