Promiscuous Modification of the Nuclear Poly(A)-binding Protein by Multiple Protein-arginine Methyltransferases Does Not Affect the Aggregation Behavior

Katharina Fronz,Silke Otto,Knut Kölbel,Uwe Kühn,Henning Friedrich,Angelika Schierhorn,Annette G Beck-Sickinger,Antje Ostareck-Lederer,Elmar Wahle

doi:10.1074/jbc.m802329200

Katharina Fronz, Silke Otto + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m802329200

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Abstract

The mammalian nuclear poly(A)-binding protein, PABPN1, carries 13 asymmetrically dimethylated arginine residues in its C-terminal domain. By fractionation of cell extracts, we found that protein-arginine methyltransferases (PRMTs)-1, -3, and -6 are responsible for the modification of PABPN1. Recombinant PRMT1, -3, and -6 also methylated PABPN1. Our data suggest that these enzymes act on their own, and additional polypeptides are not involved in recognizing PABPN1 as a substrate. PRMT1 is the predominant methyltransferase acting on PABPN1. Nevertheless, PABPN1 was almost fully methylated in a Prmt1(-/-) cell line; thus, PRMT3 and -6 suffice for methylation. In contrast to PABPN1, the heterogeneous nuclear ribonucleoprotein (hnRNP) K is selectively methylated only by PRMT1. Efficient methylation of synthetic peptides derived from PABPN1 or hnRNP K suggested that PRMT1, -3, and -6 recognize their substrates by interacting with local amino acid sequences and not with additional domains of the substrates. However, the use of fusion proteins suggested that the inability of PRMT3 and -6 to modify hnRNP K is because of structural masking of the methyl-accepting amino acid sequences by neighboring domains. Mutations leading to intracellular aggregation of PABPN1 cause the disease oculopharyngeal muscular dystrophy. The C-terminal domain containing the methylated arginine residues is known to promote PAPBN1 self-association, and arginine methylation has been reported to inhibit self-association of an orthologous protein. Thus, arginine methylation might be relevant for oculopharyngeal muscular dystrophy. However, in two different types of assays we have been unable to detect any effect of arginine methylation on the aggregation of bovine PABPN1.

Highlights

20408 JOURNAL OF BIOLOGICAL CHEMISTRY proteins modified in this manner are involved in DNA or RNA function; many of them bind nucleic acids directly, and the modification is frequently found in RNA binding domains
PRMT1 Methylates PABPN1—HeLa cell extracts were initially employed in an unbiased approach to identify, by protein fractionation, protein-arginine methyltransferases acting on PABPN1
The two PRMT1 peaks overlapped with the peaks of PRMT3 and -6, whereas PRTM2 and -4 were present in the flow-through and in the earliest fractions, which were devoid of methylation activity

Summary

Introduction

20408 JOURNAL OF BIOLOGICAL CHEMISTRY proteins modified in this manner are involved in DNA or RNA function; many of them bind nucleic acids directly, and the modification is frequently found in RNA binding domains. A knock-out of Prmt abolishes the modification of the cytoplasmic poly(A)-binding protein and the transcriptional cofactor p300 (13), and knockdown of PRMT6 interferes with histone H3 methylation at Arg-2 (14, 15) These data suggest specific enzyme-substrate relationships, and a published survey. A GST-glycine- and arginine-rich fusion protein (17) and synthetic peptides with similar sequences serve as generic methyl acceptor sites, being methylated, in vitro, by PRMT1, -3, -6 and by the type II enzyme PRMT7 (3) but not by PRMT4 (18, 19). This supports the idea that at least some of the PRMTs may be largely redundant with respect to their substrates. The latter is responsible for substrate recognition by binding to the Sm domain (22–24)

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