Highly efficient 5\u27 capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly. © 2018, Bird et al

Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells

Capped RNAs synthesized by in vitro transcription have found wide utility for studying mRNA function and metabolism and for producing proteins of interest. We characterize here a recently synthesized series of cap analogs with improved properties that contain a sulfur substitution for a nonbridging oxygen in either the α-, β-, or γ-phosphate moieties, m2 7,2′-OGpppSG, m2 7,2′-OGppSpG, and m2 7,2′-OGpSppG, respectively. The new compounds were also modified at the 2′-O position of the m7Guo to make them anti-reverse cap analogs (ARCAs), i.e., they are incorporated exclusively in the correct orientation during in vitro transcription. Each of the S-ARCAs exists in two diastereoisomeric forms (D1 and D2) that can be resolved by reverse-phase HPLC. A major in vivo pathway for mRNA degradation is initiated by removal of the cap by the pyrophosphatase Dcp1/Dcp2, which cleaves between the α- and β-phosphates. Oligonucleotides capped with m2 7,2′-OGppSpG (D2) were completely resistant to hydrolysis by recombinant human Dcp2 in vitro, whereas those capped with m2 7,2′-OGppSpG (D1) and both isomers of m2 7,2′-OGpppSG were partially resistant. Luciferase mRNA capped with m2 7,2′-OGppSpG (D2) had a t 1/2 of 257 min in cultured HC11 mammary epithelial cells compared with 86 min for m7Gp3G-capped mRNA. Luciferase mRNAs capped with m2 7,2′-OGppSpG (D1) and m2 7,2′-OGppSpG (D2) were translated 2.8-fold and 5.1-fold, respectively, more efficiently in HC11 cells than those capped with m7Gp3G. The greater yield of protein due to combining higher translational efficiency with longer t 1/2 of mRNA should benefit applications that utilize RNA transfection such as protein production, anti-cancer immunization, and gene therapy