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Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells
Published on: August 7, 2021
Poly-GR Impairs PRMT1-Mediated Arginine Methylation of Disease-Linked RNA-Binding Proteins by Acting as a Substrate
Saskia Hutten1, Jia-Xuan Chen2, Adrian M Isaacs3,4
1Institute of Molecular Physiology, Johannes Gutenberg-Universität, 55128 Mainz, Germany.
Abstract:
Dipeptide repeat proteins (DPRs) are aberrant protein species found in C9orf72-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two neurodegenerative diseases characterized by the cytoplasmic mislocalization and aggregation of RNA-binding proteins (RBPs). In particular, arginine (R)-rich DPRs (poly-GR and poly-PR) have been suggested to promiscuously interact with multiple cellular proteins and thereby exert high cytotoxicity. Components of the protein arginine methylation machinery have been identified as modulators of DPR toxicity and/or potential cellular interactors of R-rich DPRs; however, the molecular details and consequences of such an interaction are currently not well understood. Here, we demonstrate that several members of the family of protein arginine methyltransferases (PRMTs) can directly interact with R-rich DPRs in vitro and in the cytosol. In vitro, R-rich DPRs reduce solubility and promote phase separation of PRMT1, the main enzyme responsible for asymmetric arginine-dimethylation (ADMA) in mammalian cells, in a concentration- and length-dependent manner. Moreover, we demonstrate that poly-GR interferes more efficiently than poly-PR with PRMT1-mediated arginine methylation of RBPs such as hnRNPA3. We additionally show by two alternative approaches that poly-GR itself is a substrate for PRMT1-mediated arginine dimethylation. We propose that poly-GR may act as a direct competitor for arginine methylation of cellular PRMT1 targets, such as disease-linked RBPs.
Insights
Arginine-rich dipeptide repeat proteins (DPRs) found in ALS and FTD interact with protein arginine methyltransferases (PRMTs). Poly-GR DPRs disrupt PRMT1 function and are methylated by PRMT1, potentially competing with RNA-binding proteins.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Dipeptide repeat proteins (DPRs) are implicated in C9orf72-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
- Arginine-rich DPRs (poly-GR, poly-PR) are cytotoxic and interact with cellular proteins.
- Protein arginine methyltransferases (PRMTs) modulate DPR toxicity, but molecular details are unclear.
Purpose of the Study:
- To investigate the molecular interactions between arginine-rich DPRs and PRMTs.
- To determine the functional consequences of these interactions on PRMT1 activity and DPRs.
Main Methods:
- In vitro binding assays to detect interactions between DPRs and PRMTs.
- Solubility and phase separation assays for PRMT1 in the presence of DPRs.
- In vitro methylation assays to assess PRMT1 activity on RNA-binding proteins and DPRs.
- Mass spectrometry to confirm PRMT1-mediated methylation of poly-GR.
Main Results:
- Several PRMT family members directly interact with R-rich DPRs in vitro and in the cytosol.
- R-rich DPRs, particularly poly-GR, reduce PRMT1 solubility and promote its phase separation.
- Poly-GR more potently inhibits PRMT1-mediated arginine methylation of RNA-binding proteins (e.g., hnRNPA3) than poly-PR.
- Poly-GR is itself a substrate for PRMT1-mediated arginine dimethylation.
Conclusions:
- Arginine-rich DPRs directly interact with and inhibit PRMT1, a key enzyme in arginine methylation.
- Poly-GR acts as a substrate and competitor for PRMT1, disrupting methylation of essential cellular targets like RBPs.
- This interaction provides a molecular mechanism for DPR-induced neurotoxicity in ALS and FTD.
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