PARP14 and PARP9/DTX3L regulate interferon-induced ADP-ribosylation

PARP-catalysed ADP-ribosylation (ADPr) is important in regulating various cellular pathways. Until recently, PARP-dependent mono-ADP-ribosylation has been poorly understood due to the lack of sensitive detection methods. Here, we utilised an improved antibody to detect mono-ADP-ribosylation. We visualised endogenous interferon (IFN)-induced ADP-ribosylation and show that PARP14 is a major enzyme responsible for this modification. Fittingly, this signalling is reversed by the macrodomain from SARS-CoV-2 (Mac1), providing a possible mechanism by which Mac1 counteracts the activity of antiviral PARPs. Our data also elucidate a major role of PARP9 and its binding partner, the E3 ubiquitin ligase DTX3L, in regulating PARP14 activity through protein-protein interactions and by the hydrolytic activity of PARP9 macrodomain 1. Finally, we also present the first visualisation of ADPr-dependent ubiquitylation in the IFN response. These approaches should further advance our understanding of IFN-induced ADPr and ubiquitin signalling processes and could shed light on how different pathogens avoid such defence pathways. Synopsis Mono-ADP-ribosylation has emerged as a crucial factor in innate immune responses, but is understudied due to the lack of sensitive detection methods. This study visualizes endogenous interferon-induced ADP-ribosylation and shows that PARP14 is a major enzyme responsible for this signalling event. Immunity responses induce PARP14-dependent ADP-ribosylation. SARS2-CoV2 Mac1 can remove PARP14-dependent ADP-ribosylation. PARP14, PARP9 and DTX3L regulate the formation of ubiquitin and ADPr foci in the cytoplasm. PARP14 activity is regulated by PARP9/DTX3L, through (1) the hydrolytic activity of PARP9 and (2) PARP14 interaction with DTX3L. Innate immune responses induce PARP14-dependent ADP-ribosylation that is tightly regulated by PARP9 and DTX3L.