Ribosomal Protein rpS2 Is Hypomethylated in PRMT3-deficient Mice

Abstract

PRMT3 is a type I arginine methyltransferase that resides in the cytoplasm. A large proportion of this cystosolic PRMT3 is found associated with ribosomes. It is tethered to the ribosomes through its interaction with rpS2, which is also its substrate. Here we show that mouse embryos with a targeted disruption of PRMT3 are small in size but survive after birth and attain a normal size in adulthood, thus displaying Minute-like characteristics. The ribosome protein rpS2 is hypomethylated in the absence of PRMT3, demonstrating that it is a bona fide, in vivo PRMT3 substrate that cannot be modified by other PRMTs. Finally, the levels 40 S, 60 S, and 80 S monosomes and polyribosomes are unaffected by the loss of PRMT3, but there are additional as yet unidentified proteins that co-fractionate with ribosomes that are also dedicated PRMT3 substrates.Arginine methylation is a common posttranslational modification that occurs in both the nucleus and the cytoplasm (1). The methylation of arginine residues is catalyzed by at least two different classes of protein arginine methyltransferase (PRMT) 2 enzymes. The Type I enzymes catalyze the formation of asymmetric N G ,N G -dimethylarginine residues and the Type II enzyme catalyzes the formation of symmetric N G ,NЈ G -dimethylarginine residues. In mammals, there are five Type I enzymes (PRMT1,3,4, 6 and 8).Proteins that are substrates for the PRMTs usually contain glycine and arginine-rich patches, GAR motifs, that are the sites of methylation. The major pools of protein that are arginine methylated are the heterogeneous nuclear ribonucleoproteins, histones, and ribosomal proteins. Thirty years ago, HeLa cell ribosomal proteins were shown to be heavily lysine-and arginine-methylated (2), and further two-dimensional gel electrophoresis studies of purified ribosomes demonstrated that at least six prominent proteins are arginine methylated (3). PRMT3 is a ribosome-associated protein (4, 5) and may be responsible for much of the arginine methylation that occurs in this molecular machine.PRMT3 was identified as a PRMT1 binding protein in a yeast two-hybrid screen (6). However, gel filtration analysis of RAT1 cells demonstrated that PRMT3 occurs as a monomer and is not complexed in vivo with PRMT1 (6). PRMT3 also interacts with the tumor suppressor DAL-1 (7). This interaction inhibits PRMT3 enzymatic activity, both in in vitro reactions and in cell lines. At the organ level, PRMT3 is ubiquitously expressed (6), but in the brain it displays higher expression in neurons than in glial cells (8). PRMT3 is the only type 1 arginine methyltransferase that is localized exclusively to the cytoplasm (6, 9). Another unique property of PRMT3 is that it harbors a zinc finger domain at its N terminus. It has been proposed that this domain may play a role in the regulation of PRMT3 activity or in the recognition of PRMT3 substrates (6, 10). Deletion analysis studies demonstrated that PRMT3 lacking the zinc finger domain is still active in vitro, however the zinc finger minus enzyme loses its abil...

Human protein arginine methyltransferases in vivo – distinct properties of eight canonical members of the PRMT family

“…Interestingly, inhibition of methylation by Adox slightly increases the mobility of PRMT6, in contrast to all other PRMTs that were either unaffected within the limits of statistical significance (PRMT1 and 7), or decreased (PRMT2, CARM1/PRMT4, and most significantly, PRMT3). PRMT3 is known to methylate both nuclear and cytoplasmic proteins, such as the nuclear poly(A)-binding protein (PABPN1) (Fronz et al, 2008;Smith et al, 1999) and, most prominently, the cytoplasmic ribosomal protein S2 (rpS2) (Bachand and Silver, 2004;Choi et al, 2008;Swiercz et al, 2007;Swiercz et al, 2005). The localization in both compartments was therefore not a surprise.…”

“…The methylation of arginine residues appears to be a rather stable, but nonetheless dynamic modification (Bedford, 2007;Bedford and Richard, 2005;Fackelmayer, 2005b;Wysocka et al, 2006). It has been implicated in a large variety of important cellular functions, such as signaling (Berthet et al, 2002;Blanchet et al, 2006;Mowen et al, 2004;Xu et al, 2004), DNA repair (for a review, see Lake and Bedford, 2007), maturation and nucleocytoplasmic transport of RNA (Boisvert et al, 2002;Cheng et al, 2007;Shen et al, 1998;Yanagida et al, 2004), protein protection (Fackelmayer, 2005b), ribosomal assembly (Bachand and Silver, 2004;Swiercz et al, 2007;Swiercz et al, 2005) and the regulation of gene expression (e.g. An et al, 2004;Balint et al, 2005;Iberg et al, 2008;Xu et al, 2004).…”

Arabidopsis protein arginine methyltransferase 3 is required for ribosome biogenesis by affecting precursor ribosomal RNA processing

“…PRMT3 contains a unique C2H2 (Cys2His2)-type zinc finger in the N terminus, before the catalytic core (36). The prmt3 null mice displayed reduced embryo size, depicting Minute-like characteristics during organogenesis and fetal growth (37). Perturbation of PRMT3 expression in cultured rat hippocampal neurons caused deformed spines (38).…”

Interplay between chromatin remodelers and protein arginine methyltransferases

“…Using various deletions of rpS2, it was shown that PRMT3 methylates RG repeats in the N-terminus of rpS2, and disruption of the PRMT3 (Rmt3) gene in S. pombe resulted in viable cells with an increased ratio of free 60S ribosome subunit, suggesting a role for PRMT3 in ribosomal assembly Swiercz et al, 2005). Similarly, PRMT3 knockout mice are viable but the embryos are small and exhibit hypomethylation of rpS2 (Swiercz et al, 2007). Additionally, PRMT3 also interacts with the tumor suppressor DAL-1/4.1B (differentially expressed in adenocarcinoma of the Lung), which negatively modulates its methyltrasnferase activity (Singh et al, 2004).…”