Highly efficient 5' 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.