NAD + capping of RNA in Archaea and Mycobacteria

ABSTRACT Chemical modifications of RNA affect essential properties of transcripts, such as their translation, localization and stability. 5’-end RNA capping with the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD + ) has been discovered in organisms ranging from bacteria to mammals. However, the hypothesis that NAD + capping might be universal in all domains of life has not been proven yet, as information on this RNA modification is missing for Archaea. Likewise, this RNA modification has not been studied in the clinically important Mycobacterium genus. Here, we demonstrate that NAD + capping occurs in the archaeal and mycobacterial model organisms Methanosarcina barkeri and Mycobacterium smegmatis. Moreover, we identify the NAD + -capped transcripts in M. smegmatis, showing that this modification is more prevalent in stationary phase, and revealing that mycobacterial NAD + -capped transcripts include non-coding small RNAs, such as Ms1. Furthermore, we show that mycobacterial RNA polymerase incorporates NAD + into RNA, and that the genes of NAD + -capped transcripts are preceded by promoter elements compatible with σ A /σ F dependent expression. Taken together, our findings demonstrate that NAD + capping exists in the archaeal domain of life, suggesting that it is universal to all living organisms, and define the NAD + -capped RNA landscape in mycobacteria, providing a basis for its future exploration.