Abstract
SMN (Survival motor neuron protein) was characterized as a dimethyl-arginine binding protein over ten years ago. TDRD3 (Tudor domain-containing protein 3) and SPF30 (Splicing factor 30 kDa) were found to bind to various methyl-arginine proteins including Sm proteins as well later on. Recently, TDRD3 was shown to be a transcriptional coactivator, and its transcriptional activity is dependent on its ability to bind arginine-methylated histone marks. In this study, we systematically characterized the binding specificity and affinity of the Tudor domains of these three proteins quantitatively. Our results show that TDRD3 preferentially recognizes asymmetrical dimethylated arginine mark, and SMN is a very promiscuous effector molecule, which recognizes different arginine containing sequence motifs and preferentially binds symmetrical dimethylated arginine. SPF30 is the weakest methyl-arginine binder, which only binds the GAR motif sequences in our library. In addition, we also reported high-resolution crystal structures of the Tudor domain of TDRD3 in complex with two small molecules, which occupy the aromatic cage of TDRD3.
Highlights
Arginine methylation is an abundant covalent post-translational modification, which regulates diverse cellular processes, including transcriptional regulation, RNA processing, signal transduction and DNA repair [1]
In order to characterize its binding specificity and affinity quantitatively, we performed a series of fluorescence polarization (FP) binding assays using our fluorescein-labeled peptide library, which includes glycine and arginine-rich (GAR) motif-containing SmD3 and PIWIL1 peptides, PGM motifcontaining SmB peptides, and histone H3R2 peptides (Table 1 and Fig. 1)
TDRD3 has a different binding selectivity than SND1, which we have previously established that the extended Tudor domain of SND1 preferentially binds symmetrically dimethylated arginine PIWIL1 peptides [20]
Summary
Arginine methylation is an abundant covalent post-translational modification, which regulates diverse cellular processes, including transcriptional regulation, RNA processing, signal transduction and DNA repair [1]. TDRD3 has a different binding selectivity than SND1, which we have previously established that the extended Tudor domain of SND1 preferentially binds symmetrically dimethylated arginine PIWIL1 peptides [20]. Our ITC (Isothermal Titration Calorimetry) binding results show that TDRD3 preferentially recognizes H3R17me2a over H3R17me2s (Fig. 2), which is consistent with our fluorescence polarization results for other methyl-arginine peptides (Table 1).
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