DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on nucleic acids.

Although ubiquitylation had traditionally been considered limited to proteins, the discovery of non-proteinaceous substrates (e.g. lipopolysaccharides and adenosine diphosphate ribose (ADPr)) challenged this perspective. Our recent study showed that DTX2 E3 ligase efficiently ubiquitylates ADPr. Here, we show that the ADPr ubiquitylation activity is also present in another DELTEX family member, DTX3L, analysed both as an isolated catalytic fragment and the full-length PARP9:DTX3L complex, suggesting that it is a general feature of the DELTEX family. Since structural predictions show that DTX3L possesses single-stranded nucleic acids binding ability and given the fact that nucleic acids have recently emerged as substrates for ADP-ribosylation, we asked whether DELTEX E3s might catalyse ubiquitylation of an ADPr moiety linked to nucleic acids. Indeed, we show that DTX3L and DTX2 are capable of ubiquitylating ADP-ribosylated DNA and RNA synthesized by PARPs, including PARP14. Furthermore, we demonstrate that the Ub-ADPr-nucleic acids conjugate can be reversed by two groups of hydrolases, which remove either the whole adduct (e.g. SARS-CoV-2 Mac1 or PARP14 macrodomain 1) or just the Ub (e.g. SARS-CoV-2 PLpro). Overall, this study reveals ADPr ubiquitylation as a general function of the DELTEX family E3s and presents the evidence of reversible ubiquitylation of ADP-ribosylated nucleic acids.

PARP14 is a PARP with both ADP-ribosyl transferase and hydrolase activities.

PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription, and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. We show that PARP14 is a dual-function enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a marked increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13, and displays specific cellular phenotypes. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response.

Updated protein domain annotation of the PARP protein family sheds new light on biological function.

AlphaFold2 and related computational tools have greatly aided studies of structural biology through their ability to accurately predict protein structures. In the present work, we explored AF2 structural models of the 17 canonical members of the human PARP protein family and supplemented this analysis with new experiments and an overview of recent published data. PARP proteins are typically involved in the modification of proteins and nucleic acids through mono or poly(ADP-ribosyl)ation, but this function can be modulated by the presence of various auxiliary protein domains. Our analysis provides a comprehensive view of the structured domains and long intrinsically disordered regions within human PARPs, offering a revised basis for understanding the function of these proteins. Among other functional insights, the study provides a model of PARP1 domain dynamics in the DNA-free and DNA-bound states and enhances the connection between ADP-ribosylation and RNA biology and between ADP-ribosylation and ubiquitin-like modifications by predicting putative RNA-binding domains and E2-related RWD domains in certain PARPs. In line with the bioinformatic analysis, we demonstrate for the first time PARP14's RNA-binding capability and RNA ADP-ribosylation activity in vitro. While our insights align with existing experimental data and are probably accurate, they need further validation through experiments.

DELTEX2 C-terminal domain recognizes and recruits ADP-ribosylated proteins for ubiquitination.

Cross-talk between ubiquitination and ADP-ribosylation regulates spatiotemporal recruitment of key players in many signaling pathways. The DELTEX family ubiquitin ligases (DTX1 to DTX4 and DTX3L) are characterized by a RING domain followed by a C-terminal domain (DTC) of hitherto unknown function. Here, we use two label-free mass spectrometry techniques to investigate the interactome and ubiquitinated substrates of human DTX2 and identify a large proportion of proteins associated with the DNA damage repair pathway. We show that DTX2-catalyzed ubiquitination of these interacting proteins requires PARP1/2-mediated ADP-ribosylation and depends on the DTC domain. Using a combination of structural, biochemical, and cell-based techniques, we show that the DTX2 DTC domain harbors an ADP-ribose-binding pocket and recruits poly-ADP-ribose (PAR)-modified proteins for ubiquitination. This PAR-binding property of DTC domain is conserved across the DELTEX family E3s. These findings uncover a new ADP-ribose-binding domain that facilitates PAR-dependent ubiquitination.

Structural insights into ADP-ribosylation of ubiquitin by Deltex family E3 ubiquitin ligases.

Cellular cross-talk between ubiquitination and other posttranslational modifications contributes to the regulation of numerous processes. One example is ADP-ribosylation of the carboxyl terminus of ubiquitin by the E3 DTX3L/ADP-ribosyltransferase PARP9 heterodimer, but the mechanism remains elusive. Here, we show that independently of PARP9, the conserved carboxyl-terminal RING and DTC (Deltex carboxyl-terminal) domains of DTX3L and other human Deltex proteins (DTX1 to DTX4) catalyze ADP-ribosylation of ubiquitin's Gly76 Structural studies reveal a hitherto unknown function of the DTC domain in binding NAD+ Deltex RING domain recruits E2 thioesterified with ubiquitin and juxtaposes it with NAD+ bound to the DTC domain to facilitate ADP-ribosylation of ubiquitin. This ubiquitin modification prevents its activation but is reversed by the linkage nonspecific deubiquitinases. Our study provides mechanistic insights into ADP-ribosylation of ubiquitin by Deltex E3s and will enable future studies directed at understanding the increasingly complex network of ubiquitin cross-talk.

The structure of the binary methyltransferase-SAH complex from Zika virus reveals a novel conformation for the mechanism of mRNA capping.

Zika virus, a flavivirus like Dengue and West Nile viruses, poses a significant risk as a pathogen in the category of emerging infectious diseases. Zika infections typically cause nonspecific, mild symptoms, but can also manifest as a neurological disorder like Guillain-Barré syndrome. Infection in pregnant women is linked to microcephaly in newborn infants. The methyltransferase domain of the non-structural protein 5 is responsible for two sequential methylations of the 5'-RNA cap. This is crucial for genome stability, efficient translation, and escape from the host immune response. Here we present the crystal structures of the Zika methyltransferase domain in complex with the methyl-donor SAM and its by-product SAH. The methyltransferase-SAH binary complex presents a new conformation of a "closed" or "obstructed" state that would restrict the binding of new RNA for capping. The combination and comparison of our new structures with recently published Zika methyltransferase structures provide a first glimpse into the structural mechanism of Zika virus mRNA capping.