Tudor domains bind symmetrical dimethylated arginines

The Tudor domain is an approximately 60-amino acid structure motif in search of a function. Herein we show that the Tudor domains of the spinal muscular atrophy gene product SMN, the splicing factor 30 kDa (SPF30), and the Tudor domain-containing 3 (TDRD3) proteins interacted with arginine-glycine-rich motifs in a methylarginine-dependent manner. The Tudor domains also associated with methylarginine-containing cellular proteins, providing evidence that methylated arginines represent physiological ligands for this protein module. In addition, we report that spliceosomal small nuclear ribonucleoprotein particles core Sm proteins accumulated in the cytoplasm when arginine methylation was inhibited with adenosine dialdehyde or in the presence of an excessive amount of unmethylated arginine-glycine-rich peptides. These data provide in vivo evidence in support of a role for arginine methylation in the proper assembly and localization of spliceosomal Sm proteins.     

TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution

R-loops, which consist of a DNA/RNA hybrid and a displaced single-stranded DNA (ssDNA), are increasingly recognized as critical regulators of chromatin biology. R-loops are particularly enriched at gene promoters, where they play important roles in regulating gene expression. However, the molecular mechanisms that control promoter-associated R-loops remain unclear. The epigenetic ‘reader’ Tudor domain-containing protein 3 (TDRD3), which recognizes methylarginine marks on histones and on the C-terminal domain of RNA polymerase II, was previously shown to recruit DNA topoisomerase 3B (TOP3B) to relax negatively supercoiled DNA and prevent R-loop formation. Here, we further characterize the function of TDRD3 in R-loop metabolism and introduce the DExH-box helicase 9 (DHX9) as a novel interaction partner of the TDRD3/TOP3B complex. TDRD3 directly interacts with DHX9 via its Tudor domain. This interaction is important for recruiting DHX9 to target gene promoters, where it resolves R-loops in a helicase activity-dependent manner to facilitate gene expression. Additionally, TDRD3 also stimulates the helicase activity of DHX9. This stimulation relies on the OB-fold of TDRD3, which likely binds the ssDNA in the R-loop structure. Thus, DHX9 functions together with TOP3B to suppress promoter-associated R-loops. Collectively, these findings reveal new functions of TDRD3 and provide important mechanistic insights into the regulation of R-loop metabolism.     

The multiple Tudor domain-containing protein TDRD1 is a molecular scaffold for mouse Piwi proteins and piRNA biogenesis factors

Piwi-interacting RNAs (piRNAs) are small noncoding RNAs expressed in the germline of animals. They associate with Argonaute proteins of the Piwi subfamily, forming ribonucleoprotein complexes that are involved in maintaining genome integrity. The N-terminal region of some Piwi proteins contains symmetrically dimethylated arginines. This modification is thought to enable recruitment of Tudor domain-containing proteins (TDRDs), which might serve as platforms mediating interactions between various proteins in the piRNA pathway. We measured the binding affinity of the four individual extended Tudor domains (TDs) of murine TDRD1 protein for three different methylarginine-containing peptides from murine Piwi protein MILI. The results show a preference of TD2 and TD3 for consecutive MILI peptides, whereas TD4 and TD1 have, respectively, lower and very weak affinity for any peptide. The affinity of TD1 for methylarginine peptides can be restored by a single-point mutation back to the consensus aromatic cage sequence. These observations were confirmed by pull-down experiments with endogenous Piwi and Piwi-associated proteins. The crystal structure of TD3 bound to a methylated MILI peptide shows an unexpected orientation of the bound peptide, with additional contacts of nonmethylated residues being made outside of the aromatic cage, consistent with solution NMR titration experiments. Finally, the molecular envelope of the four tandem Tudor domains of TDRD1, derived from small angle scattering data, reveals a flexible, elongated shape for the protein. Overall, the results show that TDRD1 can accommodate different peptides from different proteins, and can therefore act as a scaffold protein for complex assembly in the piRNA pathway.     

Structure and function of eTudor domain containing TDRD proteins

Tudor domain-containing (TDRD) proteins, as a family of evolutionarily conserved proteins, have been studied extensively in recent years in terms of their biological and biochemical functions. A major function of the TDRD proteins is to recognize the N-terminal arginine-rich motifs of the P-element-induced wimpy testis (PIWI) proteins via their conserved extended Tudor (eTudor or eTud) domains, which is essential in piRNA biogenesis and germ cell development. In this review, we summarize recent progress in the study of the TDRD proteins, and discuss the molecular mechanisms for the different binding selectivity of these eTudor domains to PIWI proteins based on the available binding and structural data. Understanding the binding differences of these TDRDs to PIWI proteins will help us better understand their functional differences and aid us in developing the target-specific therapeutics, because overexpression or mutations of the human TDRD proteins have been demonstrated to associate with various diseases.     

High-resolution X-ray and NMR structures of the SMN Tudor domain: conformational variation in the binding site for symmetrically dimethylated arginine residues

The SMN protein, which is linked to spinal muscular atrophy (SMA), plays an important role in the assembly of the spliceosomal small nuclear ribonucleoprotein complexes. This function requires binding of SMN to the arginine-glycine (RG) rich C-terminal tails of the Sm proteins, which contain symmetrically dimethylated arginine residues (sDMA) in vivo. Using NMR titrations, we show that the SMN Tudor domain recognizes these sDMAs in the methylated RG repeats. Upon complex formation a cluster of conserved aromatic residues in the SMN Tudor domain interacts with the sDMA methyl groups. We present two high resolution structures of the uncomplexed SMN Tudor domain, a 1.8A crystal structure and an NMR structure that has been refined against a large number of backbone and side-chain residual dipolar couplings. The backbone conformation of both structures is very similar, however, differences are observed for the cluster of conserved aromatic side-chains in the sDMA binding pocket. In order to validate these variations we introduce a novel application of residual dipolar couplings for aromatic rings. We show that structural information can be derived from aromatic ring residual dipolar couplings, even in the presence of internal motions such as ring flipping. These residual dipolar couplings and ring current shifts independently confirm that the SMN Tudor domain adopts two different conformations in the sDMA binding pocket. The observed structural variations may play a role for the recognition of sDMAs.     

Tudor-related proteins TDRD1/MTR-1, TDRD6 and TDRD7/TRAP: domain composition, intracellular localization, and function in male germ cells in mice

The germ-line cells of many animals possess a characteristic cytoplasmic structure termed nuage or germinal granules. In mice, nuage that is prominent in postnatal male germ cells is also called intermitochondrial cement or chromatoid bodies. TDRD1/MTR-1, which contains Tudor domain repeats, is a specific component of the mouse nuage, analogously to Drosophila Tudor, a constituent of polar granules/nuage in oocytes and embryos. We show that TDRD6 and TDRD7/TRAP, which also contain multiple Tudor domains, specifically localize to nuage and form a ribonucleoprotein complex together with TDRD1/MTR-1. The characteristic co-localization of TDRD1, 6 and 7 was disrupted in a mutant of mouse vasa homologue/DEAD box polypeptide 4 (Mvh/Ddx4), which encodes another evolutionarily conserved component of nuage. In vivo over-expression experiments of the TDRD proteins and truncated forms during male germ cell differentiation showed that a single Tudor domain is a structural unit that localizes or accumulates to nuage, but the expression of the truncated, putative dominant negative forms is detrimental to meiotic spermatocytes. These results indicate that the Tudor-related proteins, which contain multiple repeats of the Tudor domain, constitute an evolutionarily conserved class of nuage components in the germ-line, and their localization or accumulation to nuage is likely conferred by a Tudor domain structure and downstream of Mvh, while the characteristic repeated architecture of the domain is functionally essential for the differentiation of germ cells.     

1H, 15N and 13C resonance assignments for the three LOTUS RNA binding domains of Tudor domain-containing protein TDRD7

The LOTUS or OST-HTH domain is a recently discovered motif of about 80 amino acids and is found in several germline-specific proteins including the Tudor domain-containing proteins TDRD5 and TDRD7, which are important for germ cell development. The LOTUS domain is an RNA binding domain but its exact function is unknown. Here, we report the ¹H, ¹³C and ¹⁵N resonance assignments for the three LOTUS domains present in mouse TDRD7. These assignments will allow three-dimensional structure determination of the LOTUS domains and mapping of their interaction with RNA, steps toward deciphering the function of TDRD7.     

Different methylation characteristics of protein arginine methyltransferase 1 and 3 toward the Ewing Sarcoma protein and a peptide

The multifunctional Ewing Sarcoma (EWS) protein, a member of a large family of RNA-binding proteins, is extensively asymmetrically dimethylated at arginine residues within RGG consensus sequences. Using recombinant proteins we examined whether type I protein arginine methyltransferase (PRMT)1 or 3 is responsible for asymmetric dimethylations of the EWS protein. After in vitro methylation of the EWS protein by GST-PRMT1, we identified 27 dimethylated arginine residues out of 30 potential methylation sites by mass spectrometry-based techniques (MALDI-TOF MS and MS/MS). Thus, PRMT1 recognizes most if not all methylation sites of the EWS protein. With GST-PRMT3, however, only nine dimethylated arginines, located mainly in the C-terminal region of EWS protein, could be assigned, indicating that structural determinants prevent complete methylation. In contrary to previous reports this study also revealed that trypsin is able to cleave after methylated arginines. Pull-down experiments showed that endogenous EWS protein binds efficiently to GST-PRMT1 but less to GST-PRMT3, which is in accordance to the in vitro methylation results. Furthermore, methylation of a peptide containing different methylation sites revealed differences in the site selectivity as well as in the kinetic properties of GST-PRMT1 and GST-PRMT3. Kinetic differences due to an inhibition effect of the methylation inhibitor S-adenosyl-L-homocysteine could be excluded by determining the corresponding K(i) values of the two enzymes and the K(d) values for the methyl donor S-adenosyl-L-methionine. The study demonstrates the strength of MS-based methods for a qualitative and quantitative analysis of enzymic arginine methylation, a posttranslational modification that becomes more and more the object of investigations.     

Toward an assembly line for U7 snRNPs: interactions of U7-specific Lsm proteins with PRMT5 and SMN complexes

The survival of motor neurons (SMN) complex mediates the assembly of small nuclear ribonucleoproteins (snRNPs) involved in splicing and histone RNA processing. A crucial step in this process is the binding of Sm proteins onto the SMN protein. For Sm B/B', D1, and D3, efficient binding to SMN depends on symmetrical dimethyl arginine (sDMA) modifications of their RG-rich tails. This methylation is achieved by another entity, the PRMT5 complex. Its pICln subunit binds Sm proteins whereas the PRMT5 subunit catalyzes the methylation reaction. Here, we provide evidence that Lsm10 and Lsm11, which replace the Sm proteins D1 and D2 in the histone RNA processing U7 snRNPs, associate with pICln in vitro and in vivo without receiving sDMA modifications. This implies that the PRMT5 complex is involved in an early stage of U7 snRNP assembly and hence may have a second snRNP assembly function unrelated to sDMA modification. We also show that the binding of Lsm10 and Lsm11 to SMN is independent of any methylation activity. Furthermore, we present evidence for two separate binding sites in SMN for Sm/Lsm proteins. One recognizes Sm domains and the second one, the sDMA-modified RG-tails, which are present only in a subset of these proteins.     

SMN tudor domain structure and its interaction with the Sm proteins

Spinal muscular atrophy (SMA) is a common motor neuron disease that results from mutations in the Survival of Motor Neuron (SMN) gene. The SMN protein plays a crucial role in the assembly of spliceosomal uridine-rich small nuclear ribonucleoprotein (U snRNP) complexes via binding to the spliceosomal Sm core proteins. SMN contains a central Tudor domain that facilitates the SMN-Sm protein interaction. A SMA-causing point mutation (E134K) within the SMN Tudor domain prevents Sm binding. Here, we have determined the three-dimensional structure of the Tudor domain of human SMN. The structure exhibits a conserved negatively charged surface that is shown to interact with the C-terminal Arg and Gly-rich tails of Sm proteins. The E134K mutation does not disrupt the Tudor structure but affects the charge distribution within this binding site. An intriguing structural similarity between the Tudor domain and the Sm proteins suggests the presence of an additional binding interface that resembles that in hetero-oligomeric complexes of Sm proteins. Our data provide a structural basis for a molecular defect underlying SMA.     

Determinants of the interaction of the spinal muscular atrophy disease protein SMN with the dimethylarginine-modified box H/ACA small nucleolar ribonucleoprotein GAR1

Deletion or mutation of the SMN1 (survival of motor neurons) gene causes the common, fatal neuromuscular disease spinal muscular atrophy. The SMN protein is important in small nuclear ribonucleoprotein (snRNP) assembly and interacts with snRNP proteins via arginine/glycine-rich domains. Recently, SMN was also found to interact with core protein components of the two major families of small nucleolar RNPs, fibrillarin and GAR1, suggesting that SMN may also function in the assembly of small nucleolar RNPs. Here we present results that indicate that the interaction of SMN with GAR1 is mediated by the Tudor domain of SMN. Single point mutations within the Tudor domain, including a spinal muscular atrophy patient mutation, impair the interaction of SMN with GAR1. Furthermore, we find that either of the two arginine/glycine-rich domains of GAR1 can provide for interaction with SMN, but removal of both results in loss of the interaction. Finally, we have found that unlike the interaction of SMN with the Sm snRNP proteins, interaction with GAR1 and fibrillarin is not enhanced by arginine dimethylation. Our results argue against post-translational arginine dimethylation as a general requirement for SMN recognition of proteins bearing arginine/glycine-rich domains.     

Symmetrical dimethylation of arginine residues in spliceosomal Sm protein B/B'' and the Sm-like protein LSm4, and their interaction with the SMN protein.

Arginine residues in RG-rich proteins are frequently dimethylated posttranslationally by protein arginine methyltransferases (PRMTs). The most common methylation pattern is asymmetrical dimethylation, a modification important for protein shuttling and signal transduction. Symmetrically dimethylated arginines (sDMA) have until now been confined to the myelin basic protein MBP and the Sm proteins D1 and D3. We show here by mass spectrometry and protein sequencing that also the human Sm protein B/B' and, for the first time, one of the Sm-like proteins, LSm4, contain sDMA in vivo. The symmetrical dimethylation of B/B', LSm4, D1, and D3 decisively influences their binding to the Tudor domain of the "survival of motor neurons" protein (SMN): inhibition of dimethylation by S-adenosylhomocysteine (SAH) abolished the binding of D1, D3, B/B', and LSm4 to this domain. A synthetic peptide containing nine sDMA-glycine dipeptides, but not asymmetrically modified or nonmodified peptides, specifically inhibited the interaction of D1, D3, B/B', LSm4, and UsnRNPs with SMN-Tudor. Recombinant D1 and a synthetic peptide could be methylated in vitro by both HeLa cytosolic S100 extract and nuclear extract; however, only the cytosolic extract produced symmetrical dimethylarginines. Thus, the Sm-modifying PRMT is cytoplasmic, and symmetrical dimethylation of B/B', D1, and D3 is a prerequisite for the SMN-dependent cytoplasmic core-UsnRNP assembly. Our demonstration of sDMAs in LSm4 suggests additional functions of sDMAs in tri-UsnRNP biogenesis and mRNA decay. Our findings also have interesting implications for the understanding of the aetiology of spinal muscular atrophy (SMA).