Abstract
The posttranslational modification ADP-ribosylation is involved in many cellular processes, with distinct roles for poly- and mono(ADP-ribosyl)ation (PAR- and MARylation, respectively). Reversibility of intracellular MARylation was demonstrated with the discovery of MACROD1, MACROD2 and TARG1, three macrodomain-containing enzymes capable of reversing MARylation of proteins and RNA. While the three enzymes have identical activities in vitro, their roles in cells are unclear and published data are partially contradictory, possibly due to a lack of validated reagents. We developed monoclonal antibodies to study these proteins and analysed their tissue distribution and intracellular localisation. MACROD1 is most prevalent in mitochondria of skeletal muscle, MACROD2 localises to nucleo- and cytoplasm and is found so far only in neuroblastoma cells, whereas the more ubiquitously expressed TARG1 is present in nucleoplasm, nucleolus and stress granules. Loss of MACROD1 or loss of TARG1 leads to disruption of mitochondrial or nucleolar morphology, respectively, hinting at their importance for these organelles. To start elucidating the underlying mechanisms, we have mapped their interactomes using BioID. The cellular localisation of interactors supports the mitochondrial, nucleolar and stress granule localisation of MACROD1 and TARG1, respectively. Gene ontology analysis suggests an involvement of MACROD1 and TARG1 in RNA metabolism in their respective compartments. The detailed description of the hydrolases’ expression, localisation and interactome presented here provides a solid basis for future work addressing their physiological function in more detail.
Highlights
The modification of proteins with ADP-ribose, known as ADP-ribosylation, was discovered over 50 years ago[1]
MACROD1, MACROD2 and TARG1 are highly similar, but to date it is not known which of their substrates are essential for their cellular function
MACROD1 for example has been reported to reside in the nucleus, the cytoplasm and/or mitochondria (Table 1)
Summary
The modification of proteins with ADP-ribose, known as ADP-ribosylation, was discovered over 50 years ago[1]. Three macrodomain-containing proteins, MACROD1, MACROD2 and TARG1 were reported to have hydrolase activity: first in deacetylating O-acetyl-ADP-ribose (OAADPR)[23], in removing ADP-ribose from modified proteins[24,25,26,27] and lastly in removing ADP-ribose from MARylated DNA www.nature.com/scientificreports or RNA28,29. These mostly biochemical studies have not addressed their function in cells, leaving it unclear which of these activities is physiologically relevant. In this work we aim to provide a more comprehensive overview of the hydrolases’ expression, localisation and interactome and thereby provide a solid basis for future research
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