ADP-ribosylation is a reversible chemical modification catalysed by ADP-ribosyltransferases such as PARPs that utilize nicotinamide adenine dinucleotide (NAD+) as a cofactor to transfer monomer or polymers of ADP-ribose nucleotide onto macromolecular targets such as proteins and DNA. ADP-ribosylation plays an important role in several biological processes such as DNA repair, transcription, chromatin remodelling, host-virus interactions, cellular stress response and many more. Using biochemical methods we identify RNA as a novel target of reversible mono-ADP-ribosylation. We demonstrate that the human PARPs - PARP10, PARP11 and PARP15 as well as a highly diverged PARP homologue TRPT1, ADP-ribosylate phosphorylated ends of RNA. We further reveal that ADP-ribosylation of RNA mediated by PARP10 and TRPT1 can be efficiently reversed by several cellular ADP-ribosylhydrolases (PARG, TARG1, MACROD1, MACROD2 and ARH3), as well as by MACROD-like hydrolases from VEEV and SARS viruses. Finally, we show that TRPT1 and MACROD homologues in bacteria possess activities equivalent to the human proteins. Our data suggest that RNA ADP-ribosylation may represent a widespread and physiologically relevant form of reversible ADP-ribosylation signalling.

HighlightsPARPs are present in representatives from all six major eukaryotic supergroups.Reversible PAR metabolism was established early in eukaryotic evolution.The last common ancestor of all eukaryotes possessed five types of PARP proteins.PARPs are associated to a large variety of different pathways.

Rapid response to environmental changes is achieved by uni‐ and multicellular organisms through a series of molecular events, often involving modification of macromolecules, including proteins, nucleic acids and lipids. Amongst these, ADP‐ribosylation is of emerging interest because of its ability to modify different macromolecules in the cells, and its association with many key biological processes, such as DNA‐damage repair, DNA replication, transcription, cell division, signal transduction, stress and infection responses, microbial pathogenicity and aging. In this review, we provide an update on novel pathways and mechanisms regulated by ADP‐ribosylation in organisms coming from all kingdoms of life.

ADP-ribosylation (ADPr) is a reversible post-translational modification of proteins, which controls major cellular and biological processes, including DNA damage repair, cell proliferation and differentiation, metabolism, stress and immune responses. In order to maintain the cellular homeostasis, diverse ADP-ribosyl transferases and hydrolases are involved in the fine-tuning of ADPr systems. The control of ADPr network is vital, and dysregulation of enzymes involved in the regulation of ADPr signalling has been linked to a number of inherited and acquired human diseases, such as several neurological disorders and in cancer. Conversely, the therapeutic manipulation of ADPr has been shown to ameliorate several disorders in both human and animal models. These include cardiovascular, inflammatory, autoimmune and neurological disorders. Herein, we summarize the recent findings in the field of ADPr, which support the impact of this modification in human pathophysiology and highlight the curative potential of targeting ADPr for translational and molecular medicine.

Ubiquitylation had been considered limited to protein lysine residues, but other substrates have recently emerged. Here, we show that DELTEX E3 ligases specifically target the 3′ hydroxyl of the adenosine diphosphate (ADP)–ribosyl moiety that can be linked to a protein, thus generating a hybrid ADP-ribosyl-ubiquitin modification. Unlike other known hydroxyl-specific E3s, which proceed via a covalent E3~ubiqutin intermediate, DELTEX enzymes are RING E3s that stimulate a direct ubiquitin transfer from E2~ubiquitin onto a substrate. However, DELTEXes follow a previously unidentified paradigm for RING E3s, whereby the ligase not only forms a scaffold but also provides catalytic residues to activate the acceptor. Comparative analysis of known hydroxyl-ubiquitylating active sites points to the recurring use of a catalytic histidine residue, which, in DELTEX E3s, is potentiated by a glutamate in a catalytic triad-like manner. In addition, we determined the hydrolase specificity profile of this modification, identifying human and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enzymes that could reverse it in cells.