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Comparative analysis of MACROD1, MACROD2 and TARG1 expression, localisation and interactome
R Žaja1, G Aydin2, B E Lippok2
1Institute of Biochemistry and Molecular Biology, RWTH Aachen University, Pauwelsstrasse 30, 52074, Aachen, Germany. rzaja@ukaachen.de.
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.
Insights
Researchers developed new antibodies to study MACROD1, MACROD2, and TARG1 enzymes, which reverse mono(ADP-ribosyl)ation (MARylation). Their distinct cellular locations and functions, particularly in mitochondria and nucleoli, were revealed, offering insights into RNA metabolism.
Area of Science:
- Biochemistry and Molecular Biology
- Cellular Biology
- Posttranslational Modifications
Background:
- Mono(ADP-ribosyl)ation (MARylation) is a reversible posttranslational modification crucial for cellular processes.
- MACROD1, MACROD2, and TARG1 are macrodomain-containing enzymes that reverse MARylation in vitro.
- The distinct cellular roles of these hydrolases remain unclear due to limited validated reagents and contradictory data.
Purpose of the Study:
- To develop validated monoclonal antibodies for studying MACROD1, MACROD2, and TARG1.
- To determine the tissue distribution and intracellular localization of these MARylation hydrolases.
- To investigate the cellular functions and interactomes of MACROD1 and TARG1.
Main Methods:
- Development and application of monoclonal antibodies for protein analysis.
- Tissue distribution and immunofluorescence microscopy for cellular localization.
- BioID proximity labeling for interactome mapping.
- Gene Ontology (GO) analysis of identified interactors.
Main Results:
- MACROD1 localizes to mitochondria in skeletal muscle; MACROD2 is found in neuroblastoma cells (nucleo- and cytoplasm); TARG1 is ubiquitously expressed (nucleoplasm, nucleolus, stress granules).
- Loss of MACROD1 or TARG1 disrupts mitochondrial or nucleolar morphology, respectively.
- Interactome analysis supports compartment-specific localization and suggests roles in RNA metabolism for MACROD1 and TARG1.
Conclusions:
- Validated antibodies enabled detailed characterization of MARylation hydrolase expression, localization, and interactions.
- MACROD1 and TARG1 play distinct, organelle-specific roles, particularly in mitochondrial and nucleolar function and RNA metabolism.
- This study provides a foundation for further research into the physiological functions of these key enzymes.
