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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 and 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 bacteria3,4,5,6. 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 caps6,7,8. 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.

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Figure 1: De novo transcription initiation by ATP and NCINs.
Figure 2: Promoter-sequence effects on efficiency of NCIN-mediated transcription initiation.
Figure 3: NCIN-mediated transcription initiation in vivo.
Figure 4: Structural basis of NCIN-mediated transcription initiation.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

Data deposits

The coordinates and structure factors of RPo-pppApC, RPo-NAD+pC, and RPo-dpCoApC have been deposited in the Protein Data Bank under accession numbers 5D4C, 5D4D and 5D4E, respectively.

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Acknowledgements

We thank N. Woychik for MazF-mt3 protein. Work was supported by NSF grant CHE-1361462 (J.K.L.), Welch Foundation Grant A-1763 (C.D.K.), Czech Science Foundation 15-05228S (L.K., N.P.), and NIH grants NIEHS P30 ES005022, GM097260 (C.D.K.), GM041376 (R.H.E.), GM088343 (B.E.N.), GM096454 (B.E.N.), and GM115910 (B.E.N.).

Author information

Authors and Affiliations

Authors

Contributions

L.K., J.K.L., C.D.K., R.H.E. and B.E.N. designed experiments; J.G.B., Y.Z., Y.T., N.P., I.B., L.G. and M.L. performed experiments; B.B. provided resources to perform experiments; J.G.B., Y.Z., Y.T., N.P., I.B., L.G., L.K., J.K.L., C.D.K., R.H.E. and B.E.N. performed data analysis; R.H.E. and B.E.N. wrote the paper.

Corresponding authors

Correspondence to Richard H. Ebright or Bryce E. Nickels.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 De novo transcription initiation by ATP and NCINs.

a, Structures of ATP, NAD+, NADH, and dpCoA. Red, identical atoms. b, Initial RNA products of in vitro transcription reactions with ATP, NAD+, NADH, or dpCoA as initiating nucleotide and [α32P]-CTP as extending nucleotide (E. coli RNAP; PrnaI; see analogous data for PgadY in Fig. 1b). Products were treated with RppH (processes 5′-triphosphate RNA to 5′-monophosphate RNA and 5′-NTP to 5′-NDP/5′-NMP9,14) or NudC (processes 5′-NAD+/NADH-capped RNA to 5′-monophosphate RNA6) as indicated. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 2 LC/MS/MS analysis of initial RNA products of in vitro transcription reactions with NAD+ as initiating nucleotide and CTP as an extending nucleotide.

a, Structure of NAD+pC (red, atoms corresponding to CID-generated fragment ion). b, Extracted ion chromatogram (signal derived from detection of parent ion of m/z = 967 and CID fragment of m/z = 845 corresponding to NAD+pC minus nicotinamide). Reactions contained the indicated components. c, Mass spectrum of CID fragment.

Extended Data Figure 3 Sensitivity of full-length RNA products to alkaline phosphatase treatment.

Full-length RNA products of in vitro transcription reactions with [γ32P]-ATP or [α32P]-NAD+ as initiating nucleotide and CTP, GTP, and UTP as extending nucleotides (E. coli RNAP; PrnaI fused to an A-less cassette). Products were treated with alkaline phosphatase (AP; processes 5′ phosphates) or NudC (processes 5′-NAD+/NADH-capped RNA to 5′-monophosphate RNA6) as indicated. Results indicate that full-length RNA products generated in reactions with [α32P]-NAD+ as initiating nucleotides are not sensitive to AP until they are processed by NudC. M, 100-nucleotide marker. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 4 Promoter-sequence effects on efficiency of NCIN-mediated transcription initiation: NAD+.

a, Templates having rnaI, gadY, N25, and T7A1 promoters used in the assays. b, Representative raw data from experiments of Fig. 2b. Initial RNA products of in vitro transcription reactions performed in the presence of 50 μM ATP and 1 mM NAD+ as initiating nucleotides and [α32P]-CTP as extending nucleotide (E. coli RNAP; PrnaI, PgadY, PN25, or PT7A1). (We note that contaminating AMP in the NAD+ stock results in production of pAp*C.) For gel source data, see Supplementary Fig. 1.

Extended Data Figure 5 Promoter-sequence effects on efficiency of NCIN-mediated transcription initiation: NADH and dpCoA.

a, Left, dependence of NADH capping on [NADH]/[ATP] ratio (mean ± s.e.m. of three determinations). Right, relative efficiencies of NADH capping. (E. coli RNAP; PrnaI, PgadY, PN25, or PT7A1). b, Left, dependence of dpCoA-capping on [dpCoA]/[ATP] ratio (mean ± s.e.m. of three determinations). Right, relative efficiencies of dpCoA capping. (E. coli RNAP; PrnaI, PgadY, PN25, or PT7A1).

Extended Data Figure 6 NCIN-mediated de novo transcription initiation by eukaryotic RNAP II.

Initial RNA products of in vitro transcription reactions with ATP, NAD+, or NADH as initiating nucleotide and [α32P]-UTP as extending nucleotide. Reactions were performed with yeast RNAP II and an artificial bubble transcription initiation template. Products were treated with RppH or NudC as indicated. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 7 Structural basis of NCIN-mediated transcription initiation: stereoviews.

ac, Crystal structures of RPo-pppApC, RPo-NAD+pC, and RPo-dpCoApC. Stereo views of density and fit for initial RNA product. Green mesh, FoFc omit map (contoured at 2.5σ in a, b and 2.2σ in c); red, DNA; pink, RNA product and diphosphate in ‘E site’ (see refs 15, 16, 17); violet spheres, Mg2+(I) and Mg2+(II); grey, RNAP bridge helix.

Extended Data Figure 8 AMP content of dpCoA stock.

HPLC chromatogram of dpCoA stock (Sigma-Aldrich, lot SLBJ2886V; 50 nmol). Green, HPLC chromatogram of AMP (20 nmol). Comparison of chromatograms indicates that the dpCoA stock contains ~2% AMP. The observation that the dpCoA stock contains ~2% AMP in the dpCoA stock accounts for the formation of pApC in reactions performed with dpCoA (Fig. 1b).

Extended Data Table 1 Data collection and refinement statistics

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, Supplementary Table 1 and Supplementary Figure 1, gel source data. (PDF 1294 kb)

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Bird, J., Zhang, Y., Tian, Y. et al. The mechanism of RNA 5′ capping with NAD+, NADH and desphospho-CoA. Nature 535, 444–447 (2016). https://doi.org/10.1038/nature18622

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