Structural insights into protein argininesymmetric dimethylation by PRMT5Litao Suna,b,1, Mingzhu Wanga,1, Zongyang Lva,b, Na Yanga, Yingfang Liua, Shilai Baoc, Weimin Gonga,2, and Rui-Ming Xua,2

aNational Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; bGraduate University of ChineseAcademy of Sciences, Beijing 100049, China; and cState Key Laboratory of Molecular and Developmental Biology, Institute of Genetics andDevelopmental Biology, Chinese Academy of Sciences, Beijing 100101, China

Edited by* Dinshaw J. Patel, Memorial Sloan-Kettering Cancer Center, New York, NY, and approved October 19, 2011 (received for review May 2, 2011)

Symmetric and asymmetric dimethylation of arginine are isomericprotein posttranslational modifications with distinct biologicaleffects, evidenced by the methylation of arginine 3 of histone H4(H4R3): symmetric dimethylation of H4R3 leads to repression ofgene expression, while asymmetric dimethylation of H4R3 is asso-ciated with gene activation. The enzymes catalyzing these modifi-cations share identifiable sequence similarities, but the relationshipbetween their catalytic mechanisms is unknown. Here we analyzedthe structure of a prototypic symmetric arginine dimethylase,PRMT5, and discovered that a conserved phenylalanine in theactive site is critical for specifying symmetric addition of methylgroups. Changing it to a methionine significantly elevates theoverall methylase activity, but also converts PRMT5 to an enzymethat catalyzes both symmetric and asymmetric dimethylation ofarginine. Our results demonstrate a common catalytic mechanismintrinsic to both symmetric and asymmetric arginine dimethylases,and show that steric constrains in the active sites play an essentialrole in determining the product specificity of arginine methylases.This discovery also implies a potentially regulatable outcome ofarginine dimethylation that may provide versatile control of eukar-yotic gene expression.

histone methylation ∣ transtriptional regulation ∣ RNA splicing ∣crystal structure

Protein arginine methyltransferase 5 (PRMT5) catalyzes theevenly addition of two methyl groups to the two ω-guanidino

nitrogen atoms of arginine, resulting in ω-NG, N 0G symmetricdimethylation of arginine (sDMA) of the target protein (1–5).PRMT5 functions in the nucleus as well as in the cytoplasm,and its substrates include histones, spliceosomal proteins, tran-scription factors, and proteins involved in piRNA biogenesis(6). Symmetric dimethylation of these proteins profoundly impactmany biological processes; e.g., epigenetic control of gene expres-sion (7), splicing regulation (2, 3, 8, 9), circadian rhythms (9, 10),DNA damage response (11, 12), and germ cell development andpluripotency (13–16). Interestingly, both PRMT5 and a group ofasymmetric (type-I) arginine dimethylases, which add two methylgroups to the same ω-guanidino nitrogen atom (aDMA), sharecommon recognition sequences, and the target arginine can oftenbe symmetrically or asymmetrically dimethylated. Yet, these iso-meric modifications have distinct biological effects. One such ex-ample occurs at arginine-3 of histone H4 (H4R3). Symmetricdimethylation of H4R3 has been linked to repression of geneexpression (17–19), while asymmetric dimethylation of H4R3 isassociated with gene activation (20, 21). The startling differencein biological effects of sDMA and aDMA modifications necessi-tates the understanding of the enzymatic mechanisms differentiat-ing the two chemically isomeric but functionally antagonisticposttranslational modifications.

ResultsOverall Structure. We have determined the crystal structures offull-length PRMT5 from Caenorhabditis elegans, alone and incomplex with S-Adenosyl-L-homocysteine (SAH). The nematode

enzyme shares high sequence homology with its human counter-part, and the recombinant protein displays robust and specificsymmetric arginine dimethylase activity in vitro (Fig. 1). Thestructure shows the PRMT5 is composed of four clearly defineddomains, a previously unsuspected TIM-barrel at the N-terminalend, a middle Rossmann-fold domain, a C-terminal β-barreldomain, and a ∼60 residue dimerization domain inserted be-tween β1 and β2 of the β-barrel domain (Fig. 2A). The first threedomains are packed in a triangular manner, with direct contactsbetween sequential domains, and the oligomerization domainbridges the TIM-barrel and β-barrel domains. The structure ofthe SAH-bound PRMT5 differs from that of free protein inthat a N-terminal loop (L0) and helix (αA) are ordered in theSAH-bound structure. We will use the SAH-bound structure foranalysis unless explicitly noted.

PRMT5 exists as a homodimer, shown both in the crystal struc-ture and in solution, as determined by analytic ultracentrifugation(Fig. 2B, Fig. S1). The dimeric interface buries a total pair wisesurface area of 2;305 Å2, and the intermolecular interactionsoccur between the dimerization domains and that between theTIM-barrel and β-barrel domains. Human PRMT5 has a shorteroligomerization domain, but extensive conservation of aminoacids involved in intermolecular interactions implies that it alsoforms a dimer, consistent with the report of dimeric and higheroligomeric forms of human PRMT5 (5). In fact, all argininemethy-lases with known structures dimerize via a homologous dimeriza-tion domain, also known as the dimerization “arm” (Fig. 3A),(22–26). Thus, protein dimerization appears to be an evolutionarilyconserved property of arginine methylases, although the functionalsignificance remains poorly understood.

Comparison with Arginine Asymmetric Dimethylases. The overallfold and spatial positioning of the Rossmann-fold and β-barreldomains are similar to that of type-I enzymes, represented byPRMT1 and CARM1 (Fig. 3A) (24–26). The root-mean-squareddeviation of Cα positions between PRMT5 and PRMT1 isapproximately 2.1 Å when both the Rossmann-fold and β-barreldomains are compared, whereas it is 1.4 Å for the Rossmann-folddomain alone. In particular, a segment including a N-terminalloop (L0) and a following helix (αA) (a.a. 359–380) becameordered upon SAH binding. Helix αA is positioned similar to that

Author contributions: L.S., Y.L., S.B., W.G., and R.-M.X. designed research; L.S., M.W., Z.L.,N.Y., and R.-M.X. performed research; S.B. contributed new reagents/analytic tools; L.S.,M.W., Z.L., N.Y., Y.L., S.B., W.G., and R.-M.X. analyzed data; and L.S., M.W., and R.-M.X.wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,www.pdb.org (PDB ID codes 3UA3 and 3UA4).1L.S. and M.W. contributed equally to this work.2To whom correspondence may be addressed. E-mail: rmxu@sun5.ibp.ac.cn or wgong@sun5.ibp.ac.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1106946108/-/DCSupplemental.

20538–20543 ∣ PNAS ∣ December 20, 2011 ∣ vol. 108 ∣ no. 51 www.pnas.org/cgi/doi/10.1073/pnas.1106946108

Dow

nloa

ded

by g

uest

on

May

17,

202

1

found in type-I enzymes, sheltering SAH from exposing to thesolvent and creating a secluded catalytic active site. However,key residues responsible for the disordered-to-ordered conforma-tional transition upon SAH/SAM binding are separately con-served among PRMT5 family members (Fig. 1B). Among which,Tyr376 and Phe379 on αA interact with the ribose and homocys-teine moieties via hydrogen bonds and van der Waals contacts,respectively. Loop L0 contains several amino acids uniquelyconserved in PRMT5s across species. The corresponding regionin PRMT1 is disordered, and that of CARM1 adopts a helicalconformation. This PRMT5 loop has two apparent functions,(i) contact the SAH/SAM molecule via residues conserved in

PRMT5 proteins (Pro366, Leu367, and Leu371) and forms asolvent inaccessible area for catalysis; (ii) the N-terminal endof L0 makes a U-turn and contacts the dimerization domain,which stabilizes the loop in a conformation endowed with theability to influence substrate binding (Figs. 3 A and B). Hence,the highly conserved PRMT5 loop is important for SAH/SAMbinding, as well as in a position to regulate substrate binding.

The Active Site.The active site of PRMT5 is identified by the loca-tion of the sulfur atom of SAH and a pair of invariant glutamateresidues, Glu499 and Glu508, found in all protein argininemethylases (Fig. 3B). These two residues are located on a hairpin

Fig. 1. Structural and functional conservation of PRMT5. (A) A schematic representation of domain structures of C. elegans and human PRMT5, and a re-presentative type-I arginine methylase, PRMT1 of rat. The lengths of the boxes are approximately drawn in scale with the protein lengths, and the residuenumbers at domain boundaries are labeled. Areas filled in tan, cyan, green, and yellow represent TIM-barrel, Rossmann-fold, β-barrel, and oligomerizationdomains, respectively. Levels of amino acid identity and similarity of the individual domains between C. elegans and human PRMT5s, and that between humanPRMT5 and rat PRMT1 are shown. (B) Sequence alignment. The full-length sequences of C. elegans and human PRMT5s, and the regions of the solved structuresof rat PRMT1 andmouse CARM1 are aligned. Residues conserved in all four proteins are shown in white letters over purple background, and similar residues areindicated with red letters. Residues conserved in PRMT5 proteins and type-I arginine methylases are highlighted tan and yellow, respectively. Blue stars markthe CePRMT5 residues subjected to mutagenesis. At the top of the sequences, a schematic representation of the secondary structure elements of CePRMT5 isshown. Every ten residues are indicated with a “·” sign. (C) Enzymatic activity assay. Top box, coomassie-stained gel of enzymes and substrate (histone H4) used.GST-tagged rat PRMT1 and poly(His)-tagged C. elegans PRMT5 were expressed in E. coli, and flag-tagged human PRMT5 was purified from HEK293 cells.Approximately 5 μg of enzymes and histone H4 each were used in the assay. Top 2nd box, autoradiograph generated with the use of 0.25 mCi of SAM withtritiated methyl group. Top 3rd box, Western blot detection of asymmetrically dimethylated histone H4R3. Bottom box, Western blot detection of symme-trically dimethylated histone H4R3.

Sun et al. PNAS ∣ December 20, 2011 ∣ vol. 108 ∣ no. 51 ∣ 20539

BIOCH

EMISTR

Y

Dow

nloa

ded

by g

uest

on

May

17,

202

1

loop connecting β4 and αF, also known as the “double-E”loop (24), and are absolutely required for enzymatic activities(Fig. S2). An examination of the active site reveals that thereare four residues separately conserved among PRMT5 proteins.These four residues are Phe379, Lys385, Ser503, and Ser669(Fig. 1B). The corresponding residues in type-I arginine methy-

lases are Met, Arg, Tyr, and His, respectively, and they are alsoconserved among type-I enzymes. We reasoned that some ofthese differences might be important for PRMT5’s catalytic ac-tivity, and carried out analyses of these residues by mutagenesis.Interestingly, changing Phe379 to a methionine (F379M) resultedin a more active enzyme, while an F279Y mutant is inactive, and

Fig. 2. Overall structure of PRMT5. (A) A ribbon representation of PRMT5monomer. Domains are colored as in Fig. 1A. Helices and strands are labeled TA to THand T1 to T8, respectively, for the TIM-barrel domain; αA to αF and β1 to β5, respectively, for the Rossmann-fold domain; and OA-OB and O1-O2, respectively, forthe oligomerization domain. Strands in the β-barrel domain are labeled b1 to b10. L0 indicates the N-terminal loop of the Rossmann-fold domain, and the SAHmolecule is shown in a stick model. (B) A PRMT5 dimer is shown as a ribbon model superimposed onto a surface representation. The surface for onemonomer iscolored light green and the other in light blue. The red line approximately traces the dimeric interface.

Fig. 3. Structural features of the PRMT5 active site. (A) Structural comparison with type-I arginine methylases PRMT1 (Pdb id: 1OR8; magenta) and CARM1(Pdb id: 2V74; light blue). For visual clarity, only three regions of major differences, enclosed in red circles and labeled I, II and III, are superimposed onto thestructure of PRMT5. (B) An up-close view of the active site. Key residues of PRMT5 (carbon: yellow; oxygen: red; nitrogen: blue) and the SAHmolecule (carbon:orange; sulfur: gold) are shown in a stick model superimposed with a ribbon representation of PRMT5. The double-E loop of PRMT1 (while ribbon), Glu153 onit, and the bound substrate arginine (stick model; carbon: white) are also shown. (C) Methylase activities of Phe379 mutants. Top box: coomassie-stained gel ofenzymes and substrate used. Bottom box: autoradiography detection. (D) Circular dichroism spectra of the wild-type and Phe379 mutants of PRMT5.

20540 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1106946108 Sun et al.

Dow

nloa

ded

by g

uest

on

May

17,

202

1

mutations to an alanine or a glycine pronouncedly reduced theenzymatic activity (Fig. 3C). Circular dichroism spectra show thatthe observed differences in enzymatic activities are not due togross structural alterations caused by mutations (Fig. 3D).

Mutation of other conserved residues near the active site, suchas changing Ser503 to a tyrosine (S503Y) or a double substitutionof Val668 and Ser669 to a threonine and a histidine (V668T/S669H), greatly diminished the enzymatic activity (Fig. S2).These residues are unlikely to be directly involved in catalysis, assuggested by their distances from the SAH molecule. Val668 andSer669 are situated on a β-barrel domain loop between strands b5and b6 that interacts with critical elements of the Rossmann-folddomain: the αA-αB junction and the double-E loop. The confor-mation of this loop is considerably different from those in type-Iarginine methylases (Fig. 3A, region II). Ser503 is located on thetip of the double-E loop and makes hydrogen bonds with the car-bonyl and amide groups of Phe671 of this β-barrel domain loop.Thus, the mutagenesis data of Ser503 and Val88/Ser669 indicatethat interdomain contacts mediated by the b5–b6 loop is criticalfor precise positioning of key residues for catalysis.

Determinants for Arginine Symmetric Dimethylation. An in-depthevaluation of the enzymatic property of the F379M mutant showsthat it is more active than the wild-type enzyme over a broadrange of enzyme concentrations (Fig. 4A). Enzyme kinetic mea-surements indicate that a drop of the Km value is largely respon-sible for the elevated enzymatic activity of the mutant enzyme

(Fig. 4C). Furthermore, we probed whether the F379M mutantexclusively carries out symmetric dimethylation of arginine. Sur-prisingly, both symmetric and asymmetric dimethylation of H4R3were detected (Fig. 4B). This phenomenon appears to be specificto the change to a methionine, as no such activity was detectedfor the F379A and F379G mutants (Fig. S3). These observationimply that: (i) symmetric and asymmetric dimethylation of argi-nine shares a common catalytic mechanism, as the same activesite is involved; (ii) Phe379 occupies a key position for PRMT5’ssDMA product specificity. The above observation should holdfor all type-II PRMTs, as the phenylalanine is absolutely con-served among them. The corresponding mutation (F327M) in hu-man PRMT5 also resulted in the gaining of asymmetric argininedimethylase activity (Fig. 4D).

DiscussionA surprising finding of this study is the role of Phe379 in specify-ing sDMA specificity of PRMT5. Phe379’s conformation is tightlyfixed in the structure, and its closest distance to the sulfur atom ofSAH is ∼4 Å. The structure suggests that Phe379 will be juxta-posed with the methyl group of SAM and the substrate arginine,thus, serving as a steric factor restricting the reaction product tosDMA. A methionine in this position has more conformationalflexibility, as seen from the structures of PRMT1 and CARM1,which may allow the production of both sDMA and aDMA.We also mutated the corresponding methionine in PRMT1 toa phenylalanine to see if the reverse is true, but the result is in-conclusive due to a much lowered level of the mutants’s overall

Fig. 4. Enzymatic properties of the F379M mutant. (A) Top box: coomassie-stained gel showing varying amounts of the wide-type and the F379M mutant ofPRMT5, and a constant amount of histone H4 (5.0 μg) used for enzymatic assay. “C” indicates no enzyme added. Bottom box: autoradiography detection with0.25 mCi of [3H]-SAM used in each reaction. (B) Western blot detection of asymmetric (middle box) and symmetric (bottom box) dimethylation of histone H4R3.Top box is a coomassie-blue stained gel showing proteins used in the activity assays. Please note, for a comparable level of Werstern blot signal, the amount ofthe F379M protein is adjusted to ∼1∕10 of the wild-type protein, and the amount of PRMT1 is even smaller. (C) Double reciprocal plot analysis of the wild-typeand F379 mutant of PRMT5. Derived kinetic parameters are tabulated. (D) Conserved property of the Phe-to-Met mutants of human and nematode PRMT5s.Top two boxes: coomassie staining of the wild-type and mutant enzymes and the substrate used. The right pointing arrow indicates the position of humanPRMT5 proteins; an asterisk indicates the position of rat PRMT1; and the left pointing arrow marks the position of C. elegans PRMT5. Third box: Western blotdetection of the flag-tagged wild-type and mutant human PRMT5. Fourth box: Western blot detection of asymmetrically dimethylated H4R3. Bottom box:Western blot detection of symmetrically dimethylated H4R3.

Sun et al. PNAS ∣ December 20, 2011 ∣ vol. 108 ∣ no. 51 ∣ 20541

BIOCH

EMISTR

Y

Dow

nloa

ded

by g

uest

on

May

17,

202

1

enzymatic activity. It should be noted that changing Phe379 ofPRMT5 to a methionine appears to relax PRMT5's sDMA con-straint rather than a complete switch to aDMA. It is conceivablethat other factors are needed in conjunction with Phe379 toachieve a complete switch. For example, Lys385 and Tyr386 arein the vicinity of Phe379 and SAH, and they are exclusively con-served in PRMT5 proteins (Fig, 3B). The two amino acids makehydrogen bonds with the carboxylate group of SAH and the cat-alytic Glu499. In type-I enzymes, a conserved arginine serves thefunctions of the pair of residues in PRMT5. Further structure andfunction studies are needed to fully delineate requirements forsymmetric vs. asymmetric arginine dimethylation. Another inter-esting point is that Phe379 is located on αA, which undergoeslarge conformational changes upon SAH binding. It is conceiva-ble that the conformation of αA may be regulated, such as byprotein-protein interactions, and the conformational dynamics ofαA is likely to impact the enzyme activity and product specificityof the arginine dimethylases.

This first structure of a symmetric arginine dimethylase alsoreveals that the N-terminal domain of PRMT5 has a TIM-barrelfold, and this domain is important for homodimerization. Asnoted previously, human PRMT5 lacking the N-terminal regionencompassing the TIM-barrel domain is still capable of forming adimer at high protein concentrations, but the catalytic activity isseverely compromised (27). This observation suggests that theTIM-barrel domain has other essential functions in addition tobeing important for PRMT5 dimerization. Human PRMT5 waspurified as a large protein complex, known as the methylosome,that contains pICLn and MEP50 (2, 3, 28). pICLn interacts withthe PRMT5 region now known to have a TIM-barrel structure,and it stimulates PRTM5’s activity towards Sm proteins (27). An-other protein, RioK1 competes with pICLn for binding toPRMT5 and directs PRMT5’s activity towards the RNA-bindingprotein nucleolin (29). Thus, the TIM-barrel domain of PRMT5also serves as a scaffold for the binding of adaptor proteins, suchas pICLn and RioK1. Hence, this domain is important for theassembly of PRMT5 complexes and their substrate selectivities.

In summary, the structural and biochemical properties of thePRMT5 uncovered here will serve as a guide for understandingthe biochemical mechanisms of all type-II arginine methylases,and advance the understanding of symmetric arginine dimethyla-tion in a wide-spectrum of biological processes.

Materials and MethodsExpression, Purification, and Crystallization of PRMT5. A bacterial expressionplasmid of C. elegans PRMT5 was constructed by cloning the full-length cDNAinto a pET21a vector (Novagen). The poly(His)-tagged recombinant proteinwas produced in the BL21(DE3) strain of Escherichia coli. Protein expressionwas induced with 0.4 mM IPTG when the cell density reached OD600 ¼ 0.8,after which point the temperature was shifted to 16 °C for 30 h. The poly(His)-tagged PRMT5 was first purified using Ni-IDA resins, followed by anionexchange and gel-filtration column chromatography. High purity fractionswere pooled and concentrated to ∼8–10 mg∕mL for crystallization. PRMT5crystals were grown by vapor diffusion in hanging drops at 16 °C in a condi-tion with 100 mM Tris (pH 7.0), 9% PEG-5000MME and 5% Tacsimate.

Selenyl-methionine (SeMet) substituted PRMT5 was expressed using adefined medium supplemented with 25 mg∕L of SeMet. SeMet PRMT5was purified and crystallized in a manner identical to the wild-type protein.

Single or multiple point mutations of PRMT5 were generated by PCR andverified by DNA sequencing. Mutant proteins were expressed and purifiedfollowing the same protocol for the wild-type protein.

Data Collection and Structure Solution. X-ray diffraction data were collected atliquid nitrogen cryogenic temperature using a cryoprotectant with the wellsolution supplemented with 30% glycerol. A 3.0 Å single wave length anom-alous dispersion (SAD) dataset was collected at a wave length of 0.9792 Å at

beamline BL17U of Shanghai Synchrotron Radiation Facility (SSRF) using aMar CCD-225 detector (Mar Research). Diffraction data were processed withthe HKL2000 software (30). The crystal belongs to the P212121 space group,and there are two PRMT5 molecules per asymmetric unit. Se sites were foundby SHELXD (31) and PHENIX (32) was used for phasing and generation of aninitial model. Iterative cycles of model building and refinement were carriedout using COOT (33) and PHENIX. The data used for refinement were sub-jected to anisotropic scaling performed using the UCLA Anisotropy Server.Noncrystallography symmetry restrains, secondary structure restrains, andTLS refinement (four TLS groups: A46-330, A359-734, B46-330, B359-734)were applied to improve the electron density map. The coordinates forthe refined models have been deposited in PDB under the accession codes3UA3 and 3UA4. Detailed statistics of data collection and refinement areshown in Table S1.

Analytical Ultracentrifugation. Protein sample prepared after gel-filtrationwas diluted to OD280 ¼ 0.8 in a buffer of 20 mM Tris-HCl (pH 7.4) and150 mM NaCl. Sedimentation velocity experiment was carried out with theProteomeLab XL-I (Beckman Coulter). Experiment was performed at4,800 rpm at 20 °C for 5 h. Velocity data were collected in a continuous scanmode at 280 nm, and sedimentation coefficients were calculated with theprogram Sedfit (34).

Circular Dichroism (CD) Measurements. CD spectra were recorded using anApplied Photophysics Pi-Star 180 spectropolarimeter at 20 °C, with the pro-tein samples at 0.1 mg∕mL in PBS buffer. Measurements were made undernitrogen with a 0.1 cm path length. Data were collected at 1 nm wave lengthinterval from 200 to 260 nm. Mean residue molar ellipticity was calculatedusing the formula ½θ� ¼ θ × 103∕ðc × l × NÞ, where c is the mean protein con-centration in millimolar, θ is the ellipticity in millidegrees (mdeg), l is the pathlength in millimeters, and N is the number of amino acids.

In Vitro Methylation Assay. Wild-type and various mutants of His-tagged C.elegans PRMT5, and GST-tagged rat PRMT1 were expressed in E. coli and pur-ified using a standard protocol. Flag-tagged human PRMT5 (hPRMT5) or itsF327M mutant was prepared from HEK293 cells transfected with a pCMV2bvector carrying the wild-type or mutant cDNA, and the recombinant enzymewas purified by resins conjugated with anti-FLAG antibody. FLAG-pull downof hPRMT5 samples from cells grown to ∼70% confluency in a 60 mm-die-meter dish and transfected with 2 μg of plasmid DNA were divided intotwo equal aliquots for enzymatic reactions. Approximately 5 μg of E. coli ex-pressed proteins, or an aliquot of hPRMT5 was individually incubated with5 μg of histone H4, and 0.25 mCi of adenosyl-L-[methyl-3H] methionine(Amersham Biosciences, Inc.) at 30 °C for 2 h in a final volume of 20 μL. Afterthe reaction, the mixtures were boiled in the SDS sample buffer and sepa-rated by SDS-PAGE. The gels were stained by Coomassie Blue and destained,then treated with Amplify (Amersham Biosciences, Inc.), dried, and exposedto a film.

Enzyme Kinetics Measurements. Enzyme kinetics parameters were determinedusing the Methyltransferase Colorimetric Assay kit (Cayman, 700140).His-tagged PRMT5 and PRMT5-F379M proteins (0.5 μM), in the presenceof saturating SAM (≥100 μM), and varying concentrations of Histone H4wereincubated for 45 min at 37 °C in a 200 μL reaction mixture. During incubation,color formation was assessed at 515 nm with Thermo Scientific VarioskanFlash (Thermo). Initial velocity data, measured as a function of substrateconcentration, were analyzed using the Michaelis-Menten equation,V ¼ Vmax½S�∕ð½S� þ KmÞ; and Kcat ¼ Vmax∕½E�, where ½E� is the total enzymeconcentration. All measurements were done in triplicate.

Western Blot Analysis. Western blot analysis was performed with antibodiesagainst histone H4 with symmetric dimethyl Arg 3 (ab5823/97454, Abcam),H4 asymmetric dimethyl Arg 3 (39705, Active Motif), and an anti-FLAG anti-body (F1804, Sigma).

ACKNOWLEDGMENTS.We thank SSRF beamline scientists for technical supportduring data collection. The work was supported by grants from the Ministryof Science and Technology of China (2009CB825501 and 2010CB944903),the Natural Science Foundation of China (90919029 and 3098801), ChineseAcademy of Sciences (CAS), and the Novo Nordisk-CAS foundation.

1. Branscombe TL, et al. (2001) PRMT5 (Janus kinase-binding protein 1) catalyzes the

formation of symmetric dimethylarginine residues in proteins. J Biol Chem

276:32971–32976.

2. Friesen WJ, et al. (2001) The methylosome, a 20S complex containing JBP1 and pICln,

produces dimethylarginine-modified Sm proteins. Mol Cell Biol 21:8289–8300.

3. Meister G, et al. (2001) Methylation of Sm proteins by a complex containing PRMT5

and the putative U snRNP assembly factor pICln. Curr Biol 11:1990–1994.

20542 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1106946108 Sun et al.

Dow

nloa

ded

by g

uest

on

May

17,

202

1

4. Pollack BP, et al. (1999) The human homologue of the yeast proteins Skb1 and Hsl7pinteracts with Jak kinases and contains protein methyltransferase activity. J Biol Chem274:31531–31542.

5. Rho J, et al. (2001) Prmt5, which forms distinct homo-oligomers, is a member of theprotein-arginine methyltransferase family. J Biol Chem 276:11393–11401.

6. Bedford MT, Clarke SG (2009) Protein arginine methylation in mammals: who, what,and why. Mol Cell 33:1–13.

7. Wysocka J, Allis CD, Coonrod S (2006) Histone arginine methylation and its dynamicregulation. Front Biosci 11:344–355.

8. Deng X, et al. (2010) Arginine methylation mediated by the Arabidopsis homologof PRMT5 is essential for proper pre-mRNA splicing. Proc Natl Acad Sci USA107:19114–19119.

9. Sanchez SE, et al. (2010) A methyl transferase links the circadian clock to the regula-tion of alternative splicing. Nature 468:112–116.

10. Hong S, et al. (2010) Type II protein arginine methyltransferase 5 (PRMT5) is requiredfor circadian period determination in Arabidopsis thaliana. Proc Natl Acad Sci USA107:21211–21216.

11. Jansson M, et al. (2008) Arginine methylation regulates the p53 response. Nat Cell Biol10:1431–1439.

12. Yang M, et al. (2009) Caenorhabditis elegans protein arginine methyltransferasePRMT-5 negatively regulates DNA damage-induced apoptosis. PLoS Genet 5:e1000514.

13. Anne J, Ollo R, Ephrussi A, Mechler BM (2007) Arginine methyltransferase Capsuleenis essential for methylation of spliceosomal Sm proteins and germ cell formation inDrosophila. Development 134:137–146.

14. Nishida KM, et al. (2009) Functional involvement of Tudor and dPRMT5 in the piRNAprocessing pathway in Drosophila germlines. EMBO J 28:3820–3831.

15. Tee WW, et al. (2010) Prmt5 is essential for early mouse development and acts in thecytoplasm to maintain ES cell pluripotency. Genes Dev 24:2772–2777.

16. Gonsalvez GB, Rajendra TK, Tian L, Matera AG (2006) The Sm-protein methyltransfer-ase, dart5, is essential for germ-cell specification and maintenance. Curr Biol16:1077–1089.

17. Pal S, Vishwanath SN, Erdjument-Bromage H, Tempst P, Sif S (2004) Human SWI/SNF-associated PRMT5 methylates histone H3 arginine 8 and negatively regulates expres-sion of ST7 and NM23 tumor suppressor genes. Mol Cell Biol 24:9630–9645.

18. Wang X, et al. (2007) SKB1-mediated symmetric dimethylation of histone H4R3controls flowering time in Arabidopsis. EMBO J 26:1934–1941.

19. Zhao Q, et al. (2009) PRMT5-mediated methylation of histone H4R3 recruits DNMT3A,coupling histone and DNA methylation in gene silencing. Nat Struct Mol Biol16:304–311.

20. Wang H, et al. (2001) Methylation of histone H4 at arginine 3 facilitating transcrip-tional activation by nuclear hormone receptor. Science 293:853–857.

21. Strahl BD, et al. (2001) Methylation of histone H4 at arginine 3 occurs in vivo and ismediated by the nuclear receptor coactivator PRMT1. Curr Biol 11:996–1000.

22. Zhang X, Zhou L, Cheng X (2000) Crystal structure of the conserved core of proteinarginine methyltransferase PRMT3. EMBO J 19:3509–3519.

23. Weiss VH, et al. (2000) The structure and oligomerization of the yeast arginine methyl-transferase, Hmt1. Nat Struct Biol 7:1165–1171.

24. Zhang X, Cheng X (2003) Structure of the predominant protein arginine methyltrans-ferase PRMT1 and analysis of its binding to substrate peptides. Structure 11:509–520.

25. Yue WW, Hassler M, Roe SM, Thompson-Vale V, Pearl LH (2007) Insights into histonecode syntax from structural and biochemical studies of CARM1 methyltransferase.EMBO J 26:4402–4412.

26. Troffer-Charlier N, Cura V, Hassenboehler P, Moras D, Cavarelli J (2007) Functional in-sights from structures of coactivator-associated arginine methyltransferase 1 domains.EMBO J 26:4391–4401.

27. Pesiridis GS, Diamond E, Van Duyne GD (2009) Role of pICLn in methylation of Smproteins by PRMT5. J Biol Chem 284:21347–21359.

28. Friesen WJ, et al. (2002) A novel WD repeat protein component of the methylosomebinds Sm proteins. J Biol Chem 277:8243–8247.

29. Guderian G, et al. (2011) RioK1, a new interactor of protein arginine methyltransfer-ase 5 (PRMT5), competes with pICln for binding and modulates PRMT5 complex com-position and substrate specificity. J Biol Chem 286:1976–1986.

30. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscilla-tion mode. Methods Enzymol 276:307–326.

31. Sheldrick GM (2008) A short history of SHELX. Acta Crystallogr A 64:112–122.32. Adams PD, et al. (2010) PHENIX: a comprehensive Python-based system for macromo-

lecular structure solution. Acta Crystallogr D 66:213–221.33. Emsley P, Cowtan K (2004) COOT: model-building tools for molecular graphics. Acta

Crystallogr D 60:2126–2132.34. Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity

ultracentrifugation and lamm equation modeling. Biophys J 78:1606–1619.

Sun et al. PNAS ∣ December 20, 2011 ∣ vol. 108 ∣ no. 51 ∣ 20543

BIOCH

EMISTR

Y

Dow

nloa

ded

by g

uest

on

May

17,

202

1