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 σA/σF 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.

NAD is a key component of cellular metabolism and also serves as an alternative 5'cap on short noncoding RNAs. The function of NAD in RNA, however, remains poorly understood. We investigated NAD capping of RNAs in HIV-1 infected cells, as HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causes intracellular pellagra. We used NAD captureSeq on HIV-1 infected/noninfected cells and revealed that four snRNAs (U1, U4ATAC, U5E, and U7) and four snoRNAs (SNORD3G, SNORD102, SNORA50A, and SNORD3B) lost their NAD cap when infected with HIV-1. Here, we provide evidence that the loss of the NAD cap increases the stability of the U1-HIV-1 pre-mRNA duplex. We also show that decreasing the amount of NAD-capped U1 snRNA by overexpressing the NAD RNA decapping enzyme DXO leads to a marked increase in HIV-1 infectivity. Moreover, an experimental increase of NAD capped RNAs improves the efficiency of splicing of HIV-1 and cellular RNAs and lowers HIV-1 infectivity. Our experiments show a dual role of U1 snRNA in HIV-1 infection and present the first clear role of NAD-capped RNAs in eukaryotic antiviral responses with a potential overlap into cellular splicing.

The Picornavirales include viruses that infect vertebrates, insects, and plants. It was believed that they pack only their genomic mRNA in the particles; thus, we envisaged these viruses as excellent model systems for studies of mRNA modifications. We used LC–MS to analyze digested RNA isolated from particles of the sacbrood and deformed wing iflaviruses as well as of the echovirus 18 and rhinovirus 2 picornaviruses. Whereas in the picornavirus RNAs we detected only N6‐methyladenosine and 2’‐O‐methylated nucleosides, the iflavirus RNAs contained a wide range of methylated nucleosides, such as 1‐methyladenosine (m1A) and 5‐methylcytidine (m5C). Mapping of m1A and m5C through RNA sequencing of the SBV and DWV RNAs revealed the presence of tRNA molecules. Both modifications were detected only in tRNA. Further analysis revealed that tRNAs are present in form of 3’ and 5’ fragments and they are packed selectively. Moreover, these tRNAs are typically packed by other viruses.

NAD is a key component of cellular metabolism and also serves as an alternative 5’cap on short noncoding RNAs. The function of NAD in RNA, however, remains poorly understood. We investigated NAD capping of RNAs in HIV-1 infected cells, as HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causes intracellular pellagra. We used NAD captureSeq on HIV-1 infected/noninfected cells and revealed that four snRNAs (U1, U4ATAC, U5E, and U7) and four snoRNAs (SNORD3G, SNORD102, SNORA50A, and SNORD3B) lost their NAD cap when infected with HIV-1. Here, we provide evidence that the loss of the NAD cap increases the stability of the U1-HIV-1 pre-mRNA duplex. We also show that decreasing the amount of NAD-capped U1 snRNA by overexpressing the NAD RNA decapping enzyme DXO leads to a marked increase in HIV-1 infectivity. Moreover, an experimental increase of NAD capped RNAs improves the efficiency of splicing of HIV-1 and cellular RNAs and lowers HIV-1 infectivity. Our experiments show a potential dual role of U1 snRNA in HIV-1 infection and present the first possible function of NAD-capped RNAs in eukaryotic antiviral responses.