ADP-ribosylation of RNA and DNA: from in vitro characterization to in vivo function

Abstract

The functionality of DNA, RNA and proteins is altered dynamically in response to physiological and pathological cues, partly achieved by their modification. While the modification of proteins with ADP-ribose has been well studied, nucleic acids were only recently identified as substrates for ADP-ribosylation by mammalian enzymes. RNA and DNA can be ADP-ribosylated by specific ADP-ribosyltransferases such as PARP1-3, PARP10 and tRNA 2'-phosphotransferase (TRPT1). Evidence suggests that these enzymes display different preferences towards different oligonucleotides. These reactions are reversed by ADP-ribosylhydrolases of the macrodomain and ARH families, such as MACROD1, TARG1, PARG, ARH1 and ARH3. Most findings derive from in vitro experiments using recombinant components, leaving the relevance of this modification in cells unclear. In this Survey and Summary, we provide an overview of the enzymes that ADP-ribosylate nucleic acids, the reversing hydrolases, and the substrates' requirements. Drawing on data available for other organisms, such as pierisin1 from cabbage butterflies and the bacterial toxin-antitoxin system DarT-DarG, we discuss possible functions for nucleic acid ADP-ribosylation in mammals. Hypothesized roles for nucleic acid ADP-ribosylation include functions in DNA damage repair, in antiviral immunity or as non-conventional RNA cap. Lastly, we assess various methods potentially suitable for future studies of nucleic acid ADP-ribosylation.

Figure 1.

ADP-ribosyltransferases and their substrates. The ADP-ribosyltransferases (ARTs) can be subdivided in two subclasses based on key amino acids present in their catalytic domain: either H–Y–E or a derivate thereof for the ARTDs (including mammalian PARPs and the two tankyrases (TNKSs)), or a motif based on R–S–E for the ARTCs (including mammalian ecto-ARTs). The PARP family is most divergent and different members modify different amino acids. Some PARPs generate poly(ADP-ribose), whereas others are limited to mono(ADP-ribosyl)ation. The ecto-ARTs are more restricted and modify arginine with ADP-ribose. Several bacterial toxins of the ARTC subfamily with an R-S-E motif were shown to modify DNA internally. ARTDs show a much broader spectrum of nucleic acid substrates but appear to be dependent on a phosphate for modification, with the exception of DarT, as summarized in more detail in Table 1. Examples of relevant enzymes and substrates are shown; for PARP10, PARP11 and PARP15 only catalytic domains have shown activity towards nucleic acids.

Figure 2.

Substrate specificities of proteins that reverse ADP-ribosylation. (A) ARH1 cleaves the N-glycosidic bond in modified arginine residues. (B) The O-glycosidic bond in PAR-chains is broken by PARG and ARH3 or (C) the O-glycosidic bond present in serine-ADPr is broken by ARH3, respectively. (D) PARG, MACROD1, MACROD2, TARG1 and ARH3 have all been demonstrated to remove ADPr from DNA and RNA by cleaving the phosphoester type O-glycosidic bond (E) ADP-ribosylation of acidic amino acids is resolved by MACROD1, MACROD2 and TARG1.

Figure 3.

Detection of ADP-ribosylated RNA by immunostaining. (A) Mono-phosphorylated ssRNA (44 nucleotides) was incubated with PARP10(818–1025) and MACROD1 as indicated, followed by proteinase K treatment and RNA extraction. Samples were analysed on an urea–PAGE stained with SYBR-gold. (B) ADP-ribosylated oligonucleotides were blotted and incubated with the indicated detection reagents, followed by secondary antibodies and chemiluminescence detection. Ten ng ssRNA was loaded per slot. Controls: auto-modified PARP10(818–1025) without nucleic acid; buffer alone. Detection reagents used: anti-PAR/MAR, Cell Signalling Technology (CST) E6F6A; murine Parp14 macro2/macro3 wildtype or mutant (GE) fused to an Fc-tag for detection (166). PARP10(818–1025) corresponds to the catalytic domain of the enzyme (7). Data were generated in our laboratory (L. Weixler).

Figure 4.

Potential in vivo functions of nucleic acid ADP-ribosylation. ADP-ribosyltransferases, hydrolases and their substrates are schematically displayed. Possible consequences of ADP-ribosylation are indicated with a question mark. The modification of double-stranded DNA by DarT leads to inhibition of replication, which is released by reversal of the modification by DarG. DNA modification in eukaryotes possibly play a role in the regulation of transcription, DNA damage repair and in the recruitment of PAR-binding proteins. Mono(ADP-ribosyl)ation by PARP3 can possibly be used as primer for PARP1/2 to generate poly(ADP-ribose). RNA modification may modify any RNA property, such as stability, translation, localization and interactome. This might not only apply to cellular RNAs, but also to foreign nucleic acids, such as viral RNA. These are the key questions that need to be addressed by future studies.