Ribosomal Protein rpS2 Is Hypomethylated in PRMT3-deficient Mice

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. 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 (1Bedford M.T. Richard S. Mol. Cell. 2005; 18: 263-272Abstract Full Text Full Text PDF PubMed Scopus (909) Google Scholar). The methylation of arginine residues is catalyzed by at least two different classes of protein arginine methyltransferase (PRMT) 2The abbreviations used are: PRMT, protein arginine N-methyltransferase; rpS2, ribosomal protein S2; GAR motif, glycine/arginine-rich motif; FLAG, peptide epitope DYKDDDDK; ES, embryonic stem cell; GST, glutathione S-transferase; PBS, phosphate-buffered saline; AdoMet, S-adenosyl-l-[methyl-3H]methionine; PVDF, polyvinylidene difluoride; MEF, mouse embryonic fibroblast. 2The abbreviations used are: PRMT, protein arginine N-methyltransferase; rpS2, ribosomal protein S2; GAR motif, glycine/arginine-rich motif; FLAG, peptide epitope DYKDDDDK; ES, embryonic stem cell; GST, glutathione S-transferase; PBS, phosphate-buffered saline; AdoMet, S-adenosyl-l-[methyl-3H]methionine; PVDF, polyvinylidene difluoride; MEF, mouse embryonic fibroblast. enzymes. The Type I enzymes catalyze the formation of asymmetric NG,NG-dimethylarginine residues and the Type II enzyme catalyzes the formation of symmetric NG,N′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 (2Chang F.N. Navickas I.J. Chang C.N. Dancis B.M. Arch. Biochem. Biophys. 1976; 172: 627-633Crossref PubMed Scopus (42) Google Scholar), and further two-dimensional gel electrophoresis studies of purified ribosomes demonstrated that at least six prominent proteins are arginine methylated (3Chang F.N. Navickas I.J. Au C. Budzilowicz C. Biochim. Biophys. Acta. 1978; 518: 89-94Crossref PubMed Scopus (20) Google Scholar). PRMT3 is a ribosome-associated protein (4Bachand F. Silver P.A. EMBO J. 2004; 23: 2641-2650Crossref PubMed Scopus (131) Google Scholar, 5Swiercz R. Person M.D. Bedford M.T. Biochem. J. 2005; 386: 85-91Crossref PubMed Scopus (129) Google Scholar) 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 (6Tang J. Gary J.D. Clarke S. Herschman H.R. J. Biol. Chem. 1998; 273: 16935-16945Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). However, gel filtration analysis of RAT1 cells demonstrated that PRMT3 occurs as a monomer and is not complexed in vivo with PRMT1 (6Tang J. Gary J.D. Clarke S. Herschman H.R. J. Biol. Chem. 1998; 273: 16935-16945Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). PRMT3 also interacts with the tumor suppressor DAL-1 (7Singh V. Miranda T.B. Jiang W. Frankel A. Roemer M.E. Robb V.A. Gutmann D.H. Herschman H.R. Clarke S. Newsham I.F. Oncogene. 2004; 23: 7761-7771Crossref PubMed Scopus (91) Google Scholar). This interaction inhibits PRMT3 enzymatic activity, both in in vitro reactions and in cell lines. At the organ level, PRMT3 is ubiquitously expressed (6Tang J. Gary J.D. Clarke S. Herschman H.R. J. Biol. Chem. 1998; 273: 16935-16945Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), but in the brain it displays higher expression in neurons than in glial cells (8Ikenaka K. Miyata S. Mori Y. Koyama Y. Taneda T. Okuda H. Kousaka A. Tohyama M. Neuroscience. 2006; 141: 1971-1982Crossref PubMed Scopus (18) Google Scholar). PRMT3 is the only type 1 arginine methyltransferase that is localized exclusively to the cytoplasm (6Tang J. Gary J.D. Clarke S. Herschman H.R. J. Biol. Chem. 1998; 273: 16935-16945Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 9Frankel A. Yadav N. Lee J. Branscombe T.L. Clarke S. Bedford M.T. J. Biol. Chem. 2002; 277: 3537-3543Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). 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 (6Tang J. Gary J.D. Clarke S. Herschman H.R. J. Biol. Chem. 1998; 273: 16935-16945Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 10Frankel A. Clarke S. J. Biol. Chem. 2000; 275: 32974-32982Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Deletion analysis studies demonstrated that PRMT3 lacking the zinc finger domain is still active in vitro, however the zinc finger minus enzyme loses its ability to methylate substrates when presented with a complex mix of hypomethylated proteins isolated from RAT1 cells (10Frankel A. Clarke S. J. Biol. Chem. 2000; 275: 32974-32982Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). This suggests that the zinc finger is the substrate recognition module of PRMT3. Indeed, it was recently found that the 40 S ribosomal protein S2 (rpS2) is a zinc finger-dependent substrate of PRMT3 (5Swiercz R. Person M.D. Bedford M.T. Biochem. J. 2005; 386: 85-91Crossref PubMed Scopus (129) Google Scholar). Importantly, in fission yeast this same enzyme/substrate pair (PRMT3/rpS2) exists (4Bachand F. Silver P.A. EMBO J. 2004; 23: 2641-2650Crossref PubMed Scopus (131) Google Scholar), and the disruption of the rmt3 gene in this organism results in an imbalance in the 40 S:60 S free subunit ratio. Furthermore, rmt3-null cells compensate for the loss of RMT3 by increasing the production of 40 S ribosomal proteins (11Bachand F. Lackner D.H. Bahler J. Silver P.A. Mol. Cell. Biol. 2006; 26: 1731-1742Crossref PubMed Scopus (38) Google Scholar). Taken together, these findings demonstrate a highly conserved role for arginine methylation in ribosomal assembly and protein biosynthesis. To establish the biological role of PRMT3 in mice, we generated a knock-out model system. We investigated the effects of PRMT3 loss on mouse embryo development. In addition, we used cell lines derived from these mice to investigate the methylation status of ribosome-associated proteins. Generation of PRMT3-targeted Mice—The ES cell line (XC265, strain 129/Ola) was kindly provided by BayGenomics. These ES cells were transferred to the M.D. Anderson Cancer Center's genetically engineered mouse facility for blastocyst injection and chimeric mice production. Southern analysis with an “external” probe confirmed three XC265 chimeras successfully transmitted the targeted allele through the germline. The Prmt3+/– mice are fertile and healthy, with no obvious abnormalities. These lines are being maintained on a mixed 129/B6 background and an inbred 129 background. The external probe for Prmt3 knock-out genotyping is 540 bp long and includes exon 14 and surrounding 5′ and 3′ intron sequences. It was generated by PCR, from 129 mouse genomic DNA and subcloned into pCR2.1 TOPO vector (Invitrogen). The PCR primers used are 5′-CGT CAG GTA ATT AAT GAT CAA TTG CGT-3′ and 5′-AAT ACT AAG ATA CTT GAA ACA CAC ACA-3′. Antibodies and Plasmids—All GST fusion proteins were subcloned in pGEX6P1 (Amersham Biosciences). pGEX6P1-PRMT3 and pGEX6P1-rpS2 were generated by PCR using FirstChoice™ PCR-ready mouse kidney cDNA (Ambion, Austin, TX) as described previously (5Swiercz R. Person M.D. Bedford M.T. Biochem. J. 2005; 386: 85-91Crossref PubMed Scopus (129) Google Scholar). The truncated form of GST-PRMT3 (GST-PRMT3Δ34) was generated by PCR using 5′-TCA GGT ACC CTC GAG CGG CCG CAT CGT GAC-3′ and 5′-ACG GGT ACC TCA TTT AAC CGG AAA AGG TTT TTC-3′ oligos, pGEX6P1-PRMT3 as a template, and the ExSite™ site-directed mutagenesis kit (Stratagene, Cedar Creek, TX) according to a manufacturer's protocol. The FLAG-tagged PRMT3 was subcloned into a pTRACER vector (Invitrogen) as described previously (5Swiercz R. Person M.D. Bedford M.T. Biochem. J. 2005; 386: 85-91Crossref PubMed Scopus (129) Google Scholar). The αPRMT3 antibody was raised in rabbits against GST-PRMT3(zf), which encodes amino acids 1 to 183 of mouse PRMT3. The αrpS2 antibody was raised in rabbits against GST-rpS2, which encodes amino acids 1–293 of mouse rpS2. The αasym24 and αPRMT1 antibodies were a gift from Dr. Stephane Richard (University of McGill, Montréal, Canada). αrpS6 antibody was obtained from Cell Signaling Technology, Inc. (Beverly, MA). Anti-βGal was obtained from Cortex Biochem (San Leandro, CA). In Vitro Methylation Assays Using GST-PRMT3—The GST, GST-GAR, GST-rpS2, GST-PRMT3, and GST-PRMT3Δ34 were expressed and purified as described previously (5Swiercz R. Person M.D. Bedford M.T. Biochem. J. 2005; 386: 85-91Crossref PubMed Scopus (129) Google Scholar). In vitro methylation reactions were performed in a final volume of 30 μl of PBS (pH = 7.4). The reaction contained 0.5–1.0 μg of substrate and 1 μg of recombinant GST-PRMT3 of GST-PRMT3Δ34. All methylation reactions were carried out in the presence of 0.42 μm [3H]AdoMet (S-adenosyl-l-[methyl-3H]methionine) (79 Ci/mmol from a 7.5 μm stock solution; PerkinElmer Life Sciences). The reaction was incubated at 30 °C for 1 h, then subjected to fluorography by separation on SDS-PAGE, transferred to a PVDF membrane, treated with EN3HANCE™ (PerkinElmer Life Sciences) and exposed to film overnight. In Vitro Methylation of Cell Extracts Using GST-PRMT3—Individual embryos from Prmt3+/– intercrosses at E13.5 were placed into culture as described (12Yadav N. Lee J. Kim J. Shen J. Hu M.C. Aldaz C.M. Bedford M.T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6464-6468Crossref PubMed Scopus (236) Google Scholar). Cells were maintained on a 3T3-culture protocol in which 106 cells were passed onto a gelatinized 10-cm dish every 3 days. MEF extracts were prepared using mild lysis buffer (10 mm Tris-Cl (pH = 7.50), 1% Triton X-100, 150 mm NaCl, 5 mm EDTA). Cell debris was spun down for 10 min at 14,000 rpm at 4 °C. The resulting extract was dialyzed overnight at 4 °C against The protein was by the MEF extracts were at °C further the in vitro methylation shown in the reactions were performed in a final volume of 30 μl of μg of protein MEF extract was mixed with 1 μg of GST-PRMT3 and μl of [3H]AdoMet (79 Ci/mmol from a 7.5 μm stock and incubated for min at 30 °C. proteins were by SDS-PAGE, transferred to a PVDF membrane, and by the cell were incubated with the antibodies to of rpS2. In Vitro Methylation with in vitro methylation shown in 4 used we to out analysis of rpS2 after fluorography to and the αrpS2 antibody that we was raised against a GST fusion of rpS2. this HeLa cells were with 10 μg of using 30 μl of (Invitrogen). At after cells were with PBS, and cell extracts were prepared using mild lysis FLAG-tagged PRMT3 was using overnight at 4 FLAG-tagged PRMT3 to was with PBS and in PBS to a final volume of 60 to cytoplasm 6 and 60 S and and 80 S 10 and ribosomal and were the in vitro methylation 10 μl of proteins were mixed with 10 μl to and μl of [3H]AdoMet (79 Ci/mmol from a 7.5 μm stock The final volume of the reaction was to 30 μl with min of at 30 methylated proteins were by SDS-PAGE, transferred to a PVDF membrane, and by cells from and E13.5 at were incubated for min at °C in modified with and cells were with PBS cell extract was prepared using μl of lysis buffer Triton X-100, mm (pH mm 10 mm 1 mm a on debris was by at 14,000 rpm for min at 4 °C. A of of extracts were onto prepared in lysis buffer and for 3 at rpm at 4 °C in a The were then by with using a to a for of the at were and proteins were to a final volume of μl using molecular MA). The was by of PBS μl The final volume was to μl with proteins were by and transferred to a PVDF and embryos were in and for expression as described previously A Scholar). embryos were in and in were at 3 mm and subjected to of using a antibody Leandro, CA). was performed using the and the was in Prmt3 gene has been targeted in mouse embryonic stem cells using gene by Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The targeted ES cell was obtained from the The ES cell line an in intron 14 of Prmt3 This is to PRMT3 and only the amino acids of this the the that the of PRMT3 is an EMBO J. 2000; PubMed Scopus Google Scholar), and the in the XC265 cells the three of these The of the of yeast is to substrate binding and enzymatic activity Silver P.A. Biol. 2000; PubMed Scopus Google Scholar). it is that the generated by the in the XC265 ES cell line PRMT3. To we generated a truncated GST-PRMT3Δ34 and it to PRMT3 in an in vitro methylation on two rpS2 and GAR GST-PRMT3Δ34 no enzymatic the truncated is in the XC265 ES it be To the biological of PRMT3, we to chimeric mice using XC265 ES cells that were then to establish A probe for Southern analysis was for genotyping these The vector a which results in a of the Southern for mice the allele The of PRMT3 on rpS2 mice were normal and fertile and were mice and To additional studies of PRMT3 we from E13.5 MEF lines and and MEF lines and were The of PRMT3 expression in the MEF lines was confirmed by analysis A of this that there is a at the of the PRMT3 It also the of a large molecular that is unique to the MEF line and may the fusion To that this unique is the fusion we PRMT3 from a MEF line (12Yadav N. Lee J. Kim J. Shen J. Hu M.C. Aldaz C.M. Bedford M.T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6464-6468Crossref PubMed Scopus (236) Google Scholar) and a MEF line (3Chang F.N. Navickas I.J. Au C. Budzilowicz C. Biochim. Biophys. Acta. 1978; 518: 89-94Crossref PubMed Scopus (20) Google Scholar) and performed analysis with an The same was and with an αPRMT3 antibodies the same at in this we a at the of the PRMT3 and was performed on the from the and knock-out cell lines. We that the results in a Prmt3 with PRMT3 expression levels in This is of cells that were generated in a through mutagenesis J. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). We previously shown that the ribosomal protein rpS2 interacts with the zinc finger of PRMT3 and that this ribosomal protein is an in vitro substrate for this enzyme (5Swiercz R. Person M.D. Bedford M.T. Biochem. J. 2005; 386: 85-91Crossref PubMed Scopus (129) Google Scholar). To rpS2 is an in vivo substrate for PRMT3, we methylated cell from and and knock-out and cells with GST-PRMT3 and We that a protein the size of rpS2 is methylated in knock-out cells but not in cell extracts This that rpS2 is hypomethylated in Prmt3 knock-out thus it a in vitro substrate. However, in Prmt3 cells rpS2 is heavily with methylation sites thus it a substrate for recombinant PRMT3. The same of rpS2 is in both and knock-out To that this is rpS2, we rpS2 from cell and no methylated GST-PRMT3 methylation of these The is still after with Furthermore, rpS2 from knock-out cells is a substrate for GST-PRMT3 and at the same as the methylated in the cell Taken together, these the that rpS2 is the major PRMT3 substrate. We also show that rpS2 is hypomethylated in Prmt3 knock-out cells in a The rpS2 protein two arginine methylation at its two of an and Deletion analysis has shown that the of rpS2 methylation by PRMT3, in vitro, is the (5Swiercz R. Person M.D. Bedford M.T. Biochem. J. 2005; 386: 85-91Crossref PubMed Scopus (129) Google Scholar). A antibody has been raised to methylate J. M.C. Richard S. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Using this we shown that rpS2 is hypomethylated in PRMT3 knock-out cells A small of is with rpS2 from knock-out which be with The of PRMT3 in these cells A and may for this methylation. mice at the that the levels of PRMT3 found in these mice are not However, when we generated the MEF lines from E13.5 embryos we that the embryos were than It be that the of the is not of the PRMT3 it is to the the ES which were used by to a in the gene on mouse and Prmt3 to this these two usually of from in a To further investigate this we and investigated embryonic at E13.5 and We found that embryos at E13.5 are on the size of this size is not as with on the size of and by there is no size and PRMT3 is ubiquitously expressed in (6Tang J. Gary J.D. Clarke S. Herschman H.R. J. Biol. Chem. 1998; 273: 16935-16945Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). The PRMT3 allele is expressed as a which of embryos to the PRMT3 expression of embryos expression at the of the in of the and in the PRMT3 expression as by antibody The 40 S:60 S in fission the loss of RMT3 PRMT3 results in a ribosomal subunit with the of 40 S and of 60 S ribosomal (11Bachand F. Lackner D.H. Bahler J. Silver P.A. Mol. Cell. Biol. 2006; 26: 1731-1742Crossref PubMed Scopus (38) Google Scholar). To the same occurs in we purified from and by The of these two cell was 4, A and In addition, analysis that both rpS2 and associated with the ribosomal the arginine methylation of rpS2 by PRMT3 is not for its into ribosomes or for the of ribosomal However, it be that rpS2 methylation is by and is not in the thus the of the ribosome may be maintained by this methylation. We also investigated the of protein in by into proteins and found that the protein was by the loss of PRMT3 in not Proteins than rpS2 investigate ribosomal proteins other than rpS2 are methylated by PRMT3, we the and methylated with in methylated substrates are in the ribosomal from that the sites are in the PRMT3 substrates. These substrates are hypomethylated in the and are thus for the in vitro methylation reaction we rpS2 as the PRMT3 substrate. However, we also a protein of 40 that is methylated in knock-out but not in the rpS2, this protein is in the 40 S of ribosomal proteins of this size that also a were but no substrate was the of hypomethylated extracts in the of PRMT3 substrates in the PRMT3 and its rpS2, are both ribosome-associated proteins (5Swiercz R. Person M.D. Bedford M.T. Biochem. J. 2005; 386: 85-91Crossref PubMed Scopus (129) Google Scholar). The disruption of the Prmt3 gene results in embryo size and the E13.5 embryos are and protein or the of a are to the loss of rpS2 other this may as a in This size is not as at but is still and by birth we cannot on This is of in ribosomal protein as A. 1998; PubMed Scopus Google Scholar). Indeed, a into the rpS2 to as of has Minute-like PubMed Google Scholar). the 40 S:60 S ribosomal subunit in mouse it is that in the of may in these cells to compensate for the loss of PRMT3, as is the in the rmt3-null cells (11Bachand F. Lackner D.H. Bahler J. Silver P.A. Mol. Cell. Biol. 2006; 26: 1731-1742Crossref PubMed Scopus (38) Google Scholar). be the 40 S ribosomal that are to active in fission yeast It has long been that ribosomal proteins are heavily methylated on both arginine and residues (2Chang F.N. Navickas I.J. Chang C.N. Dancis B.M. Arch. Biochem. Biophys. 1976; 172: 627-633Crossref PubMed Scopus (42) Google Scholar). amino analysis of two-dimensional ribosomal proteins identified six different protein that were arginine-methylated and protein (3Chang F.N. Navickas I.J. Au C. Budzilowicz C. Biochim. Biophys. Acta. 1978; 518: 89-94Crossref PubMed Scopus (20) Google Scholar). using antibodies a of ribosomal proteins were as methylated proteins J. M.C. Richard S. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The role of rpS2 methylation has To this enzyme/substrate pair of is of the enzyme substrate been conserved an and are found from yeast to (4Bachand F. Silver P.A. EMBO J. 2004; 23: 2641-2650Crossref PubMed Scopus (131) Google Scholar, 5Swiercz R. Person M.D. Bedford M.T. Biochem. J. 2005; 386: 85-91Crossref PubMed Scopus (129) Google Scholar, Mol. Cell. Biol. PubMed Scopus Google Scholar, M. V. M. J. M. S. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). The of occurs in to and with the of protein Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). is as yet no that the methylation of rpS2 be by Indeed, to the that rpS2 is methylated in The of rpS2 that is methylated by PRMT3 has been to a of residues at its (5Swiercz R. Person M.D. Bedford M.T. Biochem. J. 2005; 386: 85-91Crossref PubMed Scopus (129) Google Scholar). This is a GAR In vitro this be methylated by PRMT3, and M.T. Clarke S. F. The of Arginine Scholar, J. J. J. Clarke S. Bedford M.T. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). However, in cells derived from Prmt3 knock-out rpS2 is which that in vivo no other for the loss of PRMT3. This is to the that the zinc finger of PRMT3 interacts with rpS2. Indeed, rpS2 was identified as a PRMT3 substrate by using the rpS2 zinc finger as an (5Swiercz R. Person M.D. Bedford M.T. Biochem. J. 2005; 386: 85-91Crossref PubMed Scopus (129) Google Scholar). in it may be for PRMTs and substrates to be the identified been genetically targeted in the are embryonic and after J. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). knock-out embryos survive to birth and (12Yadav N. Lee J. Kim J. Shen J. Hu M.C. Aldaz C.M. Bedford M.T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6464-6468Crossref PubMed Scopus (236) Google Scholar). In to small embryos a cell J. Lee J. Yadav N. C. Richard S. Bedford M.T. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). has recently been out in mice and no T. M. M. H. T. Cell 2006; PubMed Scopus Google Scholar). Prmt3 is the of the arginine methyltransferase to be out in mice, with embryos displaying a size and cells that the ribosomal protein rpS2 is a dedicated PRMT3 substrate. We of the Bedford for this we for of and for blastocyst of

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