Noncanonical RNA‐capping: Discovery, mechanism, and physiological role debate

Focus Article

Christina Julius, Yulia Yuzenkova

Published Online: Oct 23 2018

DOI: 10.1002/wrna.1512

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Recently a new type of 5′‐RNA cap was discovered. In contrast to the specialized eukaryotic m7G cap, the novel caps are abundant cellular cofactors like NAD+. RNAs capped with cofactors are found in prokaryotes and eukaryotes. Unlike m7G cap, installed by specialized enzymes, cofactors are attached by main enzyme of transcription, RNA polymerase (RNAP). Cofactors act as noncanonical initiating substrates, provided cofactor's nucleoside base‐pairs with template DNA at the transcription start site. Adenosine—containing NAD(H), flavin adenine dinucleotide (FAD), and CoA modify transcripts on promoters starting with +1A. Similarly, uridine‐containing cell wall precursors, for example, uridine diphosphate‐N‐acetylglucosamine were shown to cap RNA in vitro on +1U promoters. Noncanonical capping is a universal feature of evolutionary unrelated RNAPs—multisubunit bacterial and eukaryotic RNAPs, and single‐subunit mitochondrial RNAP. Cellular concentrations of cofactors, for example, NAD(H) are significantly higher than their Km in transcription. Yet, only a small proportion of a given cellular RNA is noncanonically capped (if at all). This proportion is a net balance between capping, seemingly stochastic, and decapping, possibly determined by RNA folding, protein binding and transcription rate. NUDIX hydrolases in bacteria and eukaryotes, and DXO family proteins eukaryotes act as decapping enzymes for noncanonical caps. The physiological role of noncanonical RNA capping is only starting to emerge. It was demonstrated to affect RNA stability in vivo in bacteria and eukaryotes and to stimulate RNAP promoter escape in vitro in Escherichia coli. NAD+/NADH capping ratio may connect transcription to cellular redox state. Potentially, noncanonical capping affects mRNA translation, RNA‐protein binding and RNA localization. This article is categorized under: RNA Processing > Capping and 5′ End Modifications RNA Export and Localization > RNA Localization RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry

Structural features of the capping complex. crystal structure of the initiation complex with NADpC (only nicotinamide adenine dinucleotide [NAD⁺] is shown) product with Thermus thermophilus RNA polymerase (RNAP), adapted from PDB ID: 5D4D (Bird et al., ). The NAD⁺ (pink) is positioned in vicinity to DNA template (gray) −1 position, RNAP catalytic site (indicated by Mg²⁺ ions), region 3.2 of σ (blue) and part of the rifampicin (Rif)‐binding pocket (lilac) of RNAP β subunit. The residues closest to NAD are βF514, βD516, and βT518

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Comparison of classic and noncanonical capping of RNA

Note. From top to bottom: The classic eukaryotic m⁷G cap is attached co‐ or posttranscriptionally by a number of specialized enzymes. The process involves dephosphorylation of 5′‐triphosphorylated RNA transcript, followed by transferase reaction to attach GTP to the 5′‐terminal diphosphate (along with pyrophosphohyrolysis), and finally methylation of guanosine nucleotide. Here, the “inverted” nucleotide serves as the protective structure. Cap removal is performed by NUDIX hydrolases (mainly Dcp2), or DXO proteins. ADP‐containing cofactors, here NAD⁺, FAD, and DP‐CoA, can be utilized by RNAP in transcription initiation to form the 5′‐end of the transcript on promoters where synthesis starts with A (+1 promoters). The rest of the molecule (e.g., NMN of NAD⁺) serves as the protective structure. Likewise, on +1U promoters UDP‐containing cell wall precursors, such as UDP‐Glc or UDP‐GlcNAc, can substitute for UTP in transcription initiation in vitro. The NCIN‐caps are removed by NUDIX hydrolases in bacteria, and NUDIX hydrolases or DXO proteins in eukaryotes. RNAP: RNA polymerase

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Schematic representation of noncanonical capping and decapping. (a) RNA polymerase (RNAP) creates a capped transcript by using the small molecule as initiating substrate instead of canonical nucleoside triphosphate. This small molecule contains a nucleoside diphosphate (here adenosine diphosphate), and another moiety X bound to β‐phosphate. RNAP uses incoming nucleoside triphosphate substrates (pppN) to elongate RNA chain, and moiety X is retained as noncanonical cap. (b) Cap removal in bacteria is performed by NUDIX hydrolase NudC, which hydrolyses the phosphate bond between monophosphate‐moiety and monophosphate‐RNA. DXO decapping enzymes in eukaryotes removes the complete cap (XppA, containing nucleotide moiety of the protein) from the residual RNA molecule. In both cases the product is 5′‐monophosphorylated RNA

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