ADP-ribosylation is well-known as protein posttranslational modification and was recently also identified as RNA posttranscriptional modification. ADP-ribose is added onto substrates by PARP enzymes and removed by the structurally distinct ADP-ribosylhydrolases (ARH) or macrodomain-containing proteins. When macrodomain proteins were identified as hydrolases a decade ago, many ADP-ribosylation substrates were not yet identified. Therefore, the majority of macrodomain-containing proteins have not been tested towards these additional substrates and were considered to be inactive. Here, we compare in vitro activities of the human macrodomain-containing proteins on a wide range of ADP-ribosylated substrates. We confirm recent findings that PARP9 macro1 and PARP14macro1 can reverse ADP-ribosylation from acidic residues and provide evidence that also PARP14macro2 and PARP15macro2 can function as ADP-ribosylhydrolases. In addition we identified both PARP9macro1 and PARP14macro1 as RNA decapping protein domains. Notwithstanding these in vitro activities, our data further show that PARG is the major RNA decapping enzyme in HEK293 cells. Together, our findings expand on defining catalytic functions of macrodomains including some previously thought to be only readers.
*Lisa Weixler and Roko Žaja are shared first authors.