The mechanism of RNA 5′ capping with NAD+, NADH and desphospho-CoA

Abstract

The chemical nature of the 5′ end of RNA is a key determinant of RNA stability, processing, localization, translation efficiency1,2, and has been proposed to provide a layer of “epitranscriptomic” gene regulation3. Recently it has been shown that some bacterial RNA species carry a 5′-end structure reminiscent of the 5′ 7-methylguanylate “cap” in eukaryotic RNA. In particular, RNA species containing a 5′-end nicotinamide adenine dinucleotide (NAD+) or 3′-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria3–6. It has been proposed that NAD+, reduced NAD+ (NADH), and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps6–8. Here, we show instead that NAD+, NADH, and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated “ab initio capping” may occur in all organisms

Identification of NAD + capped mRNAs in Saccharomyces cerevisiae

“…First, it has been observed in vitro that bacterial RNA polymerases, as well as yeast RNA polymerase II, can use NAD + instead of ATP to initiate transcription when the transcript starts with an adenosine residue (9,19). Second, we observe that NAD-RNA is similarly enriched on the pre-mRNAs and mature mRNAs for mRNAs with introns ( Fig.…”

“…Because RNA polymerases, including yeast RNA polymerase II, can also initiate with other adenosine-containing derivatives, such as dephospho-CoA, FAD + , and NADH (9,19), it remains possible that a number of other 5′ caps of eukaryotic RNAs are formed.…”

“…Because canonical bacterial RNAs contain a 5′ triphosphate terminus, addition of NAD + to the 5′ end of RNAs represents a rudimentary "capping" mechanism, perhaps designed to impart specific properties for these RNAs by granting them a more structurally complex 5′ end. Consistent with this idea, NAD + addition to RNAs appears to occur during transcription initiation (9), as opposed to the more complex eukaryote 5′ capping, which occurs after transcription has commenced (10). Because the NAD + modification defines the 5′ end of NAD-RNAs, this modification can affect RNA stability in Escherichia coli (7,9).…”

“…Consistent with this idea, NAD + addition to RNAs appears to occur during transcription initiation (9), as opposed to the more complex eukaryote 5′ capping, which occurs after transcription has commenced (10). Because the NAD + modification defines the 5′ end of NAD-RNAs, this modification can affect RNA stability in Escherichia coli (7,9).…”

“…In principle, NAD + could be added to the 5′ end of mRNAs after cytoplasmic or nuclear decapping, or during the process of transcription. Previous results suggest that in bacterial cells, NAD + is added to RNAs in a cotranscriptional process, because RNA polymerases can use NAD + instead of ATP at transcription initiation sites that begin with an adenosine residue (9,19). Two observations suggest that this cotranscriptional mechanism is also true in S. cerevisiae.…”