Structural insights into protein arginine symmetric dimethylation by PRMT5

Abstract

Symmetric and asymmetric dimethylation of arginine are isomeric protein posttranslational modifications with distinct biological effects, evidenced by the methylation of arginine 3 of histone H4 (H4R3): symmetric dimethylation of H4R3 leads to repression of gene expression, while asymmetric dimethylation of H4R3 is associated with gene activation. The enzymes catalyzing these modifications share identifiable sequence similarities, but the relationship between their catalytic mechanisms is unknown. Here we analyzed the structure of a prototypic symmetric arginine dimethylase, PRMT5, and discovered that a conserved phenylalanine in the active site is critical for specifying symmetric addition of methyl groups. Changing it to a methionine significantly elevates the overall methylase activity, but also converts PRMT5 to an enzyme that catalyzes both symmetric and asymmetric dimethylation of arginine. Our results demonstrate a common catalytic mechanism intrinsic to both symmetric and asymmetric arginine dimethylases, and show that steric constrains in the active sites play an essential role in determining the product specificity of arginine methylases. This discovery also implies a potentially regulatable outcome of arginine dimethylation that may provide versatile control of eukaryotic gene expression.histone methylation | transtriptional regulation | RNA splicing | crystal structure P rotein arginine methyltransferase 5 (PRMT5) catalyzes the evenly addition of two methyl groups to the two ω-guanidino nitrogen atoms of arginine, resulting in ω-N G , N 0G symmetric dimethylation 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, transcription factors, and proteins involved in piRNA biogenesis (6). Symmetric dimethylation of these proteins profoundly impact many biological processes; e.g., epigenetic control of gene expression (7), splicing regulation (2,3,8,9), circadian rhythms (9, 10), DNA damage response (11, 12), and germ cell development and pluripotency (13-16). Interestingly, both PRMT5 and a group of asymmetric (type-I) arginine dimethylases, which add two methyl groups to the same ω-guanidino nitrogen atom (aDMA), share common recognition sequences, and the target arginine can often be symmetrically or asymmetrically dimethylated. Yet, these isomeric modifications have distinct biological effects. One such example occurs at arginine-3 of histone H4 (H4R3). Symmetric dimethylation of H4R3 has been linked to repression of gene expression (17-19), while asymmetric dimethylation of H4R3 is associated with gene activation (20, 21). The startling difference in biological effects of sDMA and aDMA modifications necessitates the understanding of the enzymatic mechanisms differentiating the two chemically isomeric but functionally antagonistic posttranslational modifications. ResultsOverall Structure. We have determined the crystal structures of full-length PRMT5 from Caenorhabditis elegans, alone and ...

Recruitment of the NineTeen Complex to the activated spliceosome requires AtPRMT5

“…arginine methylation | protein arginine methyltransferase | AtPRMT5 | pre-mRNA splicing | Prp19C/NTC P rotein arginine methyltransferase 5 (PRMT5), a highly conserved type II protein arginine methyltransferase, transfers methyl groups to arginine residues, generating monomethylarginine and symmetric ω-N G , N′ G -dimethylarginine (SDMA) (1)(2)(3). In mammals, PRMT5 methylates and interacts with diverse proteins, including histones and other proteins, and has long been implicated in the modulation of a variety of processes, such as transcriptional regulation, RNA metabolism (4), apoptosis (5), signal transduction (6), and germ cell development (7).…”

Crystal structure of the human PRMT5:MEP50 complex

“…We have elucidated the crystal structure of the complex bound to an AdoMet analog A9145C (34) and a substrate peptide from histone H4. As we were preparing this article, the crystal structure of PRMT5 from Caenorhabditis elegans, which shares a sequence identity of 31% with human PRMT5, was published (35). In contrast to the dimeric C. elegans PRMT5 structure, our work reveals that human PRMT5 binds MEP50 to form a hetero-octameric complex of molecular weight ∼450 kDa.…”

Protein Arginine Methyltransferase Product Specificity Is Mediated by Distinct Active-site Architectures

“…It should be noted that the H4 peptide substrates used in our study differ from the GGRGGF-GGRGGFGGRGGFG peptide used previously (24). Additionally, immunoblot analysis revealed that the reverse mutation in the PRMT5 enzymes of humans and Caenorhabditis elegans, where the corresponding wild-type residue is a phenylalanine (F327M and F379M, respectively) caused asymmetric dimethylation of human histone H4 (14). It would be interesting to examine these mutants with our more sensitive amino acid analysis techniques to determine any changes in product specificity more precisely.…”

“…For example, whether a particular arginine residue on histone tails is asymmetrically or symmetrically dimethylated can lead to gene repression or activation (13)(14)(15)(16)(17). However, few studies have been conducted to determine the role of MMA marks (18).…”

“…Although previous studies utilizing site-directed mutagenesis have given some support for this hypothesis, efforts to efficiently change the activity type of PRMTs have not yet been fruitful. Two such studies using moderately sensitive analytical techniques have been reported for PRMT1 (14) and PRMT5 (24), but they have not put forth a general model for the factors that guide product specificity for the three main types of PRMTs.…”