Dominant mutations in five tRNA synthetases cause Charcot–Marie–Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNAGly aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT.

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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 ...