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Membraneless organelles are RNA–protein assemblies which have been implicated in post‐transcriptional control. Germ cells form membraneless organelles referred to as germ granules, which contain conserved proteins including Tudor domain‐containing scaffold polypeptides and their partner proteins that interact with Tudor domains. Here, we show that in Drosophila, different germ granule proteins associate with the multi‐domain Tudor protein using different numbers of Tudor domains. Furthermore, these proteins compete for interaction with Tudor in vitro and, surprisingly, partition to distinct and poorly overlapping clusters in germ granules in vivo. This partition results in minimization of the competition. Our data suggest that Tudor forms structurally different configurations with different partner proteins which dictate different biophysical properties and phase separation parameters within the same granule. [ABSTRACT FROM AUTHOR]

Biomolecular condensates (BMCs) play an essential role in development, physiological processes, stress response, and pathogenesis. Tudor-domain-containing proteins contribute to biomolecular condensation via interactions with ligands methylated at arginine or lysine residues. The well-studied Tudor-domain-containing survival motor neuron (SMN) protein participates in multiple cellular events due to interactions with various ligands. Emerging roles of SMN protein include regulation of messenger ribonucleoprotein (mRNP) trafficking and translation. New approaches to sample preparation and advanced mass spectrometry (MS) analysis may distinguish between isobaric asymmetric and symmetric dimethylarginine. Biomolecular condensates (BMCs) can facilitate or inhibit diverse cellular functions. BMC formation is driven by noncovalent protein–protein, protein–RNA, and RNA–RNA interactions. Here, we focus on Tudor domain-containing proteins – such as survival motor neuron protein (SMN) – that contribute to BMC formation by binding to dimethylarginine (DMA) modifications on protein ligands. SMN is present in RNA-rich BMCs, and its absence causes spinal muscular atrophy (SMA). SMN's Tudor domain forms cytoplasmic and nuclear BMCs, but its DMA ligands are largely unknown, highlighting open questions about the function of SMN. Moreover, DMA modification can alter intramolecular interactions and affect protein localization. Despite these emerging functions, the lack of direct methods of DMA detection remains an obstacle to understanding Tudor–DMA interactions in cells. [ABSTRACT FROM AUTHOR]

Throughout metazoa, germ cells assemble RNA-protein organelles (germ granules). In Drosophila ovaries, perinuclear nuage forms in the nurse cells, while compositionally similar polar granules form in the oocyte. A similar system appears to exist in the distantly related (∼350 million years) wasp Nasonia, with some surprising divergences. Nuage is similarly formed in Nasonia, except that anterior nurse cells accumulate significantly more nuage, in association with high levels of DNA double-strand breaks, suggesting that increased transposon activity anteriorly is silenced by high nuage levels. In the oocyte, the germ plasm forms a single granule that is 40 times larger than a homologous Drosophila polar granule. While conserved germ granule proteins are recruited to the oosome, they show unusual localization: Tudor protein forms a shell encapsulating the embryonic oosome, while small Oskar/Vasa/Aubergine granules coalesce interiorly. Wasp Vasa itself is unusual since it has an alternative splice form that includes a previously unreported nucleoporin-like phenylalanine-glycine repeat domain. Our work is consistent with the high degree of evolutionary plasticity of membraneless organelles, and describes a new experimental model and resources for studying biomolecular condensates., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Hosts are continually selected to evolve new defenses against an ever-changing array of pathogens. To understand this process, we examined the genetic basis of resistance to the Drosophila A virus in Drosophila melanogaster. In a natural population, we identified a polymorphic transposable element (TE) insertion that was associated with an ~19,000-fold reduction in viral titers, allowing flies to largely escape the harmful effects of infection by this virulent pathogen. The insertion occurs in the protein-coding sequence of the gene Veneno, which encodes a Tudor domain protein. By mutating Veneno with CRISPR-Cas9 in flies and expressing it in cultured cells, we show that the ancestral allele of the gene has no effect on viral replication. Instead, the TE insertion is a gain-of-function mutation that creates a gene encoding a novel resistance factor. Viral titers remained reduced when we deleted the TE sequence from the transcript, indicating that resistance results from the TE truncating the Veneno protein. This is a novel mechanism of virus resistance and a new way by which TEs can contribute to adaptation. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is a disease that results from mutations in the Survival of Motor Neuron (SMN) gene 1, leading to muscle atrophy due to motor neurons degeneration. SMN plays a crucial role in the assembly of spliceosomal small nuclear ribonucleoprotein complexes via binding to the arginine-glycine rich C-terminal tails of Sm proteins recognized by SMN Tudor domain. E134K Tudor mutation, cause of the more severe type I SMA, compromises the SMN-Sm interaction without a perturbation of the domain fold. By molecular dynamics simulations, we investigated the mechanism of Tudor-SmD1 interaction, and the effects on it of E134K mutation. It was observed that E134 is crucial to catch the positive dimethylated arginines (DMRs) of the SmD1 tail that, wrapping around the acidic Tudor surface, enters a central DMR into an aromatic cage. The flexible cage residue Y130 must be blocked from the wrapped tail to assure a stable binding. The charge inversion in E134K mutation causes the loss of a critical anchor point, disfavoring the tail wrapping and leaving Y130 free to swing, leading to DMR detachments and exposition of the C-terminal region of the tail. This could suggest new hypotheses regarding a possible autoimmune response by anti-Sm autoantibodies. [ABSTRACT FROM AUTHOR]

Silencing of transposable elements (TEs) by Piwi-interacting RNAs (piRNAs) is crucial for maintaining germline genome integrity and fertility in animals. To repress TEs, PIWI clade Argonaute proteins cooperate with several Tudor domain-containing (Tdrd) proteins at membraneless perinuclear organelles, called nuage, to produce piRNAs to repress transposons. Here, we identify and characterize Kotsubu (Kots), one of the Drosophila Tudor domain-containing protein-1 (Tdrd1) orthologs, encoded by the CG9925 gene, that localizes to the nuage in gonads. We further show the dynamic localization of Kots in the male germline, where it shows perinuclear signals in spermatogonia but forms large cytoplasmic condensates in the spermatocytes that overlap with components of piNG-body, a nuage-associated organelle. The loss of kots results in a notable upregulation of stellate and a corresponding reduction in the suppressor of stellate piRNAs in the mutants. Furthermore, a moderate yet significant reduction of other piRNAs was observed in kots mutant testes. Taken together, we propose that Kots functions in the piRNA pathway, predominantly in the male germline by forming discrete cytoplasmic granules.

Membraneless granules assemble in different cell types and cellular loci and are the focus of intense research due to their fundamental importance for cellular organization. These dynamic organelles are commonly assembled from RNA and protein components and exhibit soft matter characteristics of molecular condensates currently characterized with biophysical approaches and super-resolution microscopy imaging. In addition, research on the molecular mechanisms of the RNA–protein granules assembly provided insights into the formation of abnormal granules and molecular aggregates, which takes place during many neurodegenerative disorders including Parkinson’s diseases (PD), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). While these disorders are associated with formation of abnormal granules, membraneless organelles are normally assembled in neurons and contribute to translational control and affect stability of neuronal RNAs. More recently, a new subtype of membraneless granules was identified in Drosophila glia (glial granules). Interestingly, glial granules were found to contain proteins which are the principal components of the membraneless granules in germ cells (germ granules), indicating some similarity in the functional assembly of these structures in glia and germline. This mini review highlights recent research on glial granules in the context of other membraneless organelles, including their assembly mechanisms and potential functions in the nervous system.

Methylation of arginine (Arg) residues on histones creates a new binding epitope, enabling recognition by aromatic cage binding pockets in Tudor domains; these protein-protein interactions (PPIs) govern gene expression. Despite their biological importance, the molecular details of methylated Arg recognition are poorly understood. While the desolvation, hydrogen bonding, and guanidinium stacking of methylated Arg have been explored in model systems and proposed to contribute to binding, direct interactions between the methyl groups and the aromatic residues in the binding pocket have not previously been investigated. Herein, we mechanistically study the CH 3 -π interactions between the SPIN1 triple Tudor domain and histone asymmetric dimethylarginine. We find that these CH 3 -π interactions are electrostatically tunable, exhibiting cation-π character, albeit attenuated relative to cation-π interactions with quaternary ammonium ions, offering key insight into how methylation of Arg alters its binding epitope to enable new PPIs.

N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance and precise function of PIWI NTR methylation in mammals remains unknown. In mice, PIWI NTRs bind Tudor domain containing proteins (TDRDs) that have essential roles in piRNA biogenesis and the formation of the chromatoid body. Using mouse MIWI (PIWIL1) as paradigm, we demonstrate that the NTRs are essential for spermatogenesis through the regulation of transposons and gene expression. The loss of TDRD5 and TDRKH interaction with MIWI results in attenuation of piRNA amplification. We find that piRNA amplification is necessary for transposon control and for sustaining piRNA levels including select, nonconserved, pachytene piRNAs that target specific mRNAs required for spermatogenesis. Our findings support the notion that the vast majority of pachytene piRNAs are dispensable, acting as self-serving genetic elements that rely for propagation on MIWI piRNA amplification. MIWI-NTRs also mediate interactions with TDRD6 that are necessary for chromatoid body compaction. Furthermore, MIWI-NTRs promote stabilization of spermiogenic transcripts that drive nuclear compaction, which is essential for sperm formation. In summary, the NTRs underpin the diversification of MIWI protein function., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

• SETDB1, a histone methyltransferase (HMT) is primarily involved in H3K9 methylation for transcriptional silencing. • SETDB1 is involved in chromatin remodeling, PML-NB-associated functions, X chromosome inactivation and immune-regulation. • SETDB1 exhibits both oncogenic and tumor suppressive function based on tumor context. • Small molecule SETDB1 inhibitors exhibited anticancer activity via suppressing SETDB1-mediated trimethylation and inducing apoptosis in tumor cells. • Designing more potent and specific SETDB1-TTD small molecule inhibitors may be a promising antitumor therapy and also useful to boost the effects of current cancer radiotherapy and immunotherapy. SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) is an important epigenetic regulator catalyzing histone H3 lysine 9 (H3K9) methylation, specifically di-/tri-methylation. This regulation promotes gene silencing through heterochromatin formation. Aberrant SETDB1 expression, and its oncogenic role is evident in many cancers. Thus, SETDB1 is a valid target with novel therapeutic benefits. In this review, we explore the structural and biochemical features of SETDB1, its regulatory mechanisms, and its role in various cancers. We also discuss recent discoveries in small molecules targeting SETDB1 and provide suggestions for future research. [ABSTRACT FROM AUTHOR]

SET domain bifurcated protein 1 (SETDB1) is a histone lysine methyltransferase that promotes the silencing of some tumour suppressor genes and is overexpressed in many cancers. SETDB1 contains a unique tandem tudor domain (TTD) that recognizes histone H3 sequences containing both methylated and acetylated lysines. Beginning with the identification of a hit compound (Cpd1), we discovered the first potent and selective small molecule SETDB1‐TTD inhibitor (R,R)‐59 through stepwise structure‐guided optimization. (R,R)‐59 showed a KD value of 0.088±0.045 μM in the ITC assay. The high potency of (R,R)‐59 was well explained by the cocrystal structure of the (R,R)‐59‐TTD complex. (R,R)‐59 is an endogenous binder competitive inhibitor. Evidence has also demonstrated its cellular target engagement. Interestingly, the enantiomer (S,S)‐59 did not show activity in all the assays, highlighting the potential of (R,R)‐59 as a tool compound in exploring the biological functions of SETDB1‐TTD. [ABSTRACT FROM AUTHOR]

The methyl-lysine reader protein SPIN1 plays important roles in various human diseases. However, targeting methyl-lysine reader proteins has been challenging. Very few cellularly active SPIN1 inhibitors have been developed. We previously reported that our G9a/GLP inhibitor UNC0638 weakly inhibited SPIN1. Here, we present our comprehensive structure-activity relationship study that led to the discovery of compound 11 , a dual SPIN1 and G9a/GLP inhibitor, and compound 18 (MS8535), a SPIN1 selective inhibitor. We solved the cocrystal structure of SPIN1 in complex with 11 , confirming that 11 occupied one of the three Tudor domains. Importantly, 18 displayed high selectivity for SPIN1 over 38 epigenetic targets, including G9a/GLP, and concentration dependently disrupted the interactions of SPIN1 and H3 in cells. Furthermore, 18 was bioavailable in mice. We also developed 19 (MS8535N), which was inactive against SPIN1, as a negative control of 18 . Collectively, these compounds are useful chemical tools to study biological functions of SPIN1.

Spindlin1 is a histone reader with three Tudor-like domains and its transcriptional co-activator activity could be attenuated by SPINDOC. The first two Tudors are involved in histone methylation readout, while the function of Tudor 3 is largely unknown. Here our structural and binding studies revealed an engagement mode of SPINDOC-Spindlin1, in which a hydrophobic motif of SPINDOC, DOCpep3, stably interacts with Spindlin1 Tudor 3, and two neighboring K/R-rich motifs, DOCpep1 and DOCpep2, bind to the acidic surface of Spindlin1 Tudor 2. Although DOCpep3-Spindlin1 engagement is compatible with histone readout, an extended SPINDOC fragment containing the K/R-rich region attenuates histone or TCF4 binding by Spindlin1 due to introduced competition. This inhibitory effect is more pronounced for weaker binding targets but not for strong ones such as H3 "K4me3-K9me3" bivalent mark. Further ChIP-seq and RT-qPCR indicated that SPINDOC could promote genomic relocation of Spindlin1, thus modulate downstream gene transcription. Collectively, we revealed multivalent engagement between SPINDOC and Spindlin1, in which a hydrophobic motif acts as the primary binding site for stable SPINDOC-Spindlin1 association, while K/R-rich region modulates the target selectivity of Spindlin1 via competitive inhibition, therefore attenuating the transcriptional co-activator activity of Spindlin1., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: M.T.B. is a cofounder of EpiCypher. Other authors have no competing financial interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Spinal muscular atrophy (SMA) is the leading genetic cause of death in young children, arising from homozygous deletion or mutation of the survival motor neuron 1 (SMN1) gene. SMN protein expressed from a paralogous gene, SMN2, is the primary genetic modifier of SMA; small changes in overall SMN levels cause dramatic changes in disease severity. Thus, deeper insight into mechanisms that regulate SMN protein stability should lead to better therapeutic outcomes. Here, we show that SMA patient-derived missense mutations in the Drosophila SMN Tudor domain exhibit a pronounced temperature sensitivity that affects organismal viability, larval locomotor function and adult longevity. These disease-related phenotypes are domain specific and result from decreased SMN stability at elevated temperature. This system was utilized to manipulate SMN levels during various stages of Drosophila development. Owing to a large maternal contribution of mRNA and protein, Smn is not expressed zygotically during embryogenesis. Interestingly, we find that only baseline levels of SMN are required during larval stages, whereas high levels of the protein are required during pupation. This previously uncharacterized period of elevated SMN expression, during which the majority of adult tissues are formed and differentiated, could be an important and translationally relevant developmental stage in which to study SMN function. Taken together, these findings illustrate a novel in vivo role for the SMN Tudor domain in maintaining SMN homeostasis and highlight the necessity for high SMN levels at crucial developmental time points that are conserved from Drosophila to humans.

For nearly half of the proteome of an important pathogen, Pseudomonas aeruginosa, the function has not yet been recognised. Here, we characterise one such mysterious protein PA2504, originally isolated by us as a sole partner of the RppH RNA hydrolase involved in transcription regulation of multiple genes. This study aims at elucidating details of PA2504 function and discussing its implications for bacterial biology. We show that PA2504 forms homodimers and is evenly distributed in the cytoplasm of bacterial cells. Molecular modelling identified the presence of a Tudor-like domain in PA2504. Transcriptomic analysis of a ΔPA2504 mutant showed that 42 transcripts, mainly coding for proteins involved in sulphur metabolism, were affected by the lack of PA2504. In vivo crosslinking of cellular proteins in the exponential and stationary phase of growth revealed several polypeptides that bound to PA2504 exclusively in the stationary phase. Mass spectrometry analysis identified them as the 30S ribosomal protein S4, the translation elongation factor TufA, and the global response regulator GacA. These results indicate that PA2504 may function as a tether for several important cellular factors.

Epigenetic dysregulation has been reported in multiple cancers including leukemias. Nonetheless, the roles of the epigenetic reader Tudor domains in leukemia progression and therapy remain unexplored. Here, we conducted a Tudor domain-focused CRISPR screen and identified SGF29, a component of SAGA/ATAC acetyltransferase complexes, as a crucial factor for H3K9 acetylation, ribosomal gene expression, and leukemogenesis. To facilitate drug development, we integrated the CRISPR tiling scan with compound docking and molecular dynamics simulation, presenting a generally applicable strategy called CRISPR-Scan Assisted Drug Discovery (CRISPR-SADD). Using this approach, we identified a lead inhibitor that selectively targets SGF29's Tudor domain and demonstrates efficacy against leukemia. Furthermore, we propose that the structural genetics approach used in our study can be widely applied to diverse fields for de novo drug discovery.

Objective To investigate the effect of staphylococcal nuclease and tudor domain containing 1(SND1) on the biological function of osteosarcoma cells and decipher the mechanism of SND1 in regulating ferroptosis in osteosarcoma cells via SLC7A11. Methods Human osteoblasts hFOB1.19 and osteosarcoma cell lines Saos-2,U2OS,HOS,and 143B were cultured,in which the expression level of SND1 was determined.Small interfering RNA was employed to knock down the expression of SND1(si-SND1) in the osteosarcoma cell line HOS and 143B.The CCK8 assay kit,colony formation assay,and Transwell assay were employed to examine the effect of SND1 expression on the biological function of osteosarcoma cells.Furthermore,we altered the expression of SND1 and SLC7A11 in osteosarcoma cells to investigate the effect of SND1 on osteosarcoma ferroptosis via SLC7A11. Results The mRNA and protein levels of SND1 in Saos-2,U2OS,HOS,and 143B cells were higher than those in hFOB1.19 cells(all P <0.01).Compared with the control group,transfection with si-SND1 down-regulated the expression level of SND1 in HOS and 143B cells(all P <0.01),decreased the viability of HOS and 143B cells,reduced the number of colony formation,and inhibited cell invasion and migration(all P <0.001).The ferroptosis inducer Erastin promoted the apoptosis of HOS and 143B cells,while the ferroptosis inhibitor Ferrostatin-1 improved the viability of HOS and 143B cells(all P <0.001).After SND-1 knockdown,Erastin reduced the viability of HOS and 143B cells,while Ferrostatin-1 restored the cell viability(all P <0.001).After treatment with Erastin in the si-SND1 group,the levels of iron and malondialdehyde were elevated,and the level of glutathione was lowered(all P <0.001).The results of in vivo experiments showed that SND1 knockdown inhibited the mass of the transplanted tumor in 143B tumor-bearing nude mice( P <0.001).Knocking down the expression of SND1 resulted in down-regulated SLC7A11 expression(all P <0.001) and increased ferroptosis in HOS and 143B cells( P <0.001, P =0.020). Conclusions SND1 presents up-regulated expression in osteosarcoma cells.It may inhibit ferroptosis by up-regulating the expression of SLC7A11,thereby improving the viability of osteosarcoma cells.

Muscle-invasive and metastatic bladder cancer indicates extra worse prognosis. Accumulating evidence roots for the prominent role of circular RNAs(circRNAs) in bladder cancer, while the mechanisms linking circRNAs and bladder cancer metastasis remain limitedly investigated. Here, we identified a significantly upregulated circRNA candidate, hsa&#95;circ&#95;0001583, from online datasets. Validated by qRT-PCR, PCR, sanger sequencing, actinomycin D and RNase R digestion experiments, hsa&#95;circ&#95;0001583 was proved to be a genuine circular RNA with higher expression levels in bladder cancer tissue. Through gain and loss of function experiments, hsa&#95;circ&#95;0001583 exhibited potent migration and invasion powers both in vitro and in vivo. The staphylococcal nuclease and Tudor domain containing 1 (SND1) was identified as an authentic binding partner for hsa&#95;circ&#95;0001583 through RNA pulldown and RIP experiments. Elevated levels of hsa&#95;circ&#95;0001583 could bind more to SND1 and protect the latter from degradation. Rescue experiments demonstrated that such interaction-induced increased in SND1 levels in bladder cancer cells enabled the protein to pump its endonuclease activity, leading to the degradation of tumor-suppressing MicroRNAs (miRNAs) including miR-126-3p, the suppressor of Disintegrin And Metalloproteinase Domain-Containing Protein 9 (ADAM9), ultimately driving cells into a highly migrative and invasive state. In summary, our study is the first to highlight the upregulation of hsa&#95;circ&#95;0001583 in bladder cancer and its role in downregulating miR-126-3p by binding to and stabilizing the SND1 protein, thereby promoting bladder cancer cell migration and invasion. This study adds hsa&#95;circ&#95;0001583 to the pool of bladder cancer metastasis biomarkers and therapeutic targets., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

The pancreatic β cell synthesizes, packages, and secretes insulin in response to glucose-stimulation to maintain blood glucose homeostasis. Under diabetic conditions, a subset of β cells fail and lose expression of key transcription factors (TFs) required for insulin secretion. Among these TFs is Pancreatic and duodenal homeobox 1 (PDX1), which recruits a unique subset of transcriptional coregulators to modulate its activity. Here we describe a novel interacting partner of PDX1, the Staphylococcal Nuclease and Tudor domain-containing protein (SND1), which has been shown to facilitate protein-protein interactions and transcriptional control through diverse mechanisms in a variety of tissues. PDX1:SND1 interactions were confirmed in rodent β cell lines, mouse islets, and human islets. Utilizing CRISPR-Cas9 gene editing technology, we deleted Snd1 from the mouse β cell lines, which revealed numerous differentially expressed genes linked to insulin secretion and cell proliferation, including limited expression of Glp1r . We observed Snd1 deficient β cell lines had reduced cell expansion rates, GLP1R protein levels, and limited cAMP accumulation under stimulatory conditions, and further show that acute ablation of Snd1 impaired insulin secretion in rodent and human β cell lines. Lastly, we discovered that PDX1:SND1 interactions were profoundly reduced in human β cells from donors with type 2 diabetes (T2D). These observations suggest the PDX1:SND1 complex formation is critical for controlling a subset of genes important for β cell function and is targeted in diabetes pathogenesis.

Piwi-interacting RNAs (piRNAs) direct PIWI proteins to transposons to silence them, thereby preserving genome integrity and fertility. The piRNA population can be expanded in the ping-pong amplification loop. Within this process, piRNA-associated PIWI proteins (piRISC) enter a membraneless organelle called nuage to cleave their target RNA, which is stimulated by Gtsf proteins. The resulting cleavage product gets loaded into an empty PIWI protein to form a new piRISC complex. However, for piRNA amplification to occur, the new RNA substrates, Gtsf-piRISC, and empty PIWI proteins have to be in physical proximity. In this study, we show that in silkworm cells, the Gtsf1 homolog BmGtsf1L binds to piRNA-loaded BmAgo3 and localizes to granules positive for BmAgo3 and BmVreteno. Biochemical assays further revealed that conserved residues within the unstructured tail of BmGtsf1L directly interact with BmVreteno. Using a combination of AlphaFold modeling, atomistic molecular dynamics simulations, and in vitro assays, we identified a novel binding interface on the BmVreteno-eTudor domain, which is required for BmGtsf1L binding. Our study reveals that a single eTudor domain within BmVreteno provides two binding interfaces and thereby interconnects piRNA-loaded BmAgo3 and BmGtsf1L., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Staphylococcal nuclease Tudor domain containing 1 (SND1) protein is an oncogene that 'reads' methylarginine marks through its Tudor domain. Specifically, it recognizes methylation marks deposited by protein arginine methyltransferase 5 (PRMT5), which is also known to promote tumorigenesis. Although SND1 can drive hepatocellular carcinoma (HCC), it is unclear whether the SND1 Tudor domain is needed to promote HCC. We sought to identify the biological role of the SND1 Tudor domain in normal and tumorigenic settings by developing two genetically engineered SND1 mouse models, an Snd1 knockout (Snd1 KO) and an Snd1 Tudor domain-mutated (Snd1 KI) mouse, whose mutant SND1 can no longer recognize PRMT5-catalyzed methylarginine marks. Quantitative PCR analysis of normal, KO, and KI liver samples revealed a role for the SND1 Tudor domain in regulating the expression of genes encoding major acute phase proteins, which could provide mechanistic insight into SND1 function in a tumor setting. Prior studies indicated that ectopic overexpression of SND1 in the mouse liver dramatically accelerates the development of diethylnitrosamine (DEN)-induced HCC. Thus, we tested the combined effects of DEN and SND1 loss or mutation on the development of HCC. We found that both Snd1 KO and Snd1 KI mice were partially protected against malignant tumor development following exposure to DEN. These results support the development of small molecule inhibitors that target the SND1 Tudor domain or the use of upstream PRMT5 inhibitors, as novel treatments for HCC., (© 2023 The Author(s).)

SND1 and MTDH are known to promote cancer and therapy resistance, but their mechanisms and interactions with other oncogenes remain unclear. Here, we show that oncoprotein ERG interacts with SND1/MTDH complex through SND1's Tudor domain. ERG, an ETS-domain transcription factor, is overexpressed in many prostate cancers. Knocking down SND1 in human prostate epithelial cells, especially those overexpressing ERG, negatively impacts cell proliferation. Transcriptional analysis shows substantial overlap in genes regulated by ERG and SND1. Mechanistically, we show that ERG promotes nuclear localization of SND1/MTDH. Forced nuclear localization of SND1 prominently increases its growth promoting function irrespective of ERG expression. In mice, prostate-specific Snd1 deletion reduces cancer growth and tumor burden in a prostate cancer model (PB-Cre/Pten flox/flox /ERG mice), Moreover, we find a significant overlap between prostate transcriptional signatures of ERG and SND1. These findings highlight SND1's crucial role in prostate tumorigenesis, suggesting SND1 as a potential therapeutic target in prostate cancer., (© 2023. The Author(s).)

Methyl-lysine reader p53 binding protein 1 (53BP1) is a central mediator of DNA break repair and is associated with various human diseases, including cancer. Thus, high-quality 53BP1 chemical probes can aid in further understanding the role of 53BP1 in genome repair pathways. Herein, we utilized focused DNA-encoded library screening to identify the novel hit compound UNC8531, which binds the 53BP1 tandem Tudor domain (TTD) with an IC 50 of 0.47 ± 0.09 μM in a TR-FRET assay and K d values of 0.85 ± 0.17 and 0.79 ± 0.52 μM in ITC and SPR, respectively. UNC8531 was cocrystallized with the 53BP1 TTD to guide further optimization efforts, leading to UNC9512. NanoBRET and 53BP1-dependent foci formation experiments confirmed cellular target engagement. These results show that UNC9512 is a best-in-class small molecule 53BP1 antagonist that can aid further studies investigating the role of 53BP1 in DNA repair, gene editing, and oncogenesis.

Tudor domain-containing protein 3 (TDRD3) is involved in regulating transcription and translation, promoting breast cancer progression, and modulating neurodevelopment and mental health, making it a promising therapeutic target for associated diseases. The Tudor domain of TDRD3 is essential for its biological functions, and targeting this domain with potent and selective chemical probes may modulate its engagement with chromatin and related functions. Here we reported a study of TDRD3 antagonist following on our earlier work on the development of the SMN antagonist, Compound 1, and demonstrated that TDRD3 can bind effectively to Compound 2, a triple-ring analog of Compound 1. Our structural analysis suggested that the triple-ring compound bound better to TDRD3 due to its smaller side chain at Y566 compared to W102 in SMN. We also revealed that adding a small hydrophobic group to the N-methyl site of Compound 1 can improve binding. These findings provide a path for identifying antagonists for single canonical Tudor domain-containing proteins such as TDRD3 and SMN., Competing Interests: Declaration of competing interest The remaining authors declare no competing interests. Data availability. The coordinates and structure factors of TDRD3-Cmpd 2 have been deposited in the Protein Data Bank (PDB) with an accession code 8JTN., (Copyright © 2023 Elsevier B.V. All rights reserved.)

GEMIN5 is essential for core assembly of small nuclear Ribonucleoproteins (snRNPs), the building blocks of spliceosome formation. Loss-of-function mutations in GEMIN5 lead to a neurodevelopmental syndrome among patients presenting with developmental delay, motor dysfunction, and cerebellar atrophy by perturbing SMN complex protein expression and assembly. Currently, molecular determinants of GEMIN5-mediated disease have yet to be explored. Here, we identified SMN as a genetic suppressor of GEMIN5-mediated neurodegeneration in vivo. We discovered that an increase in SMN expression by either SMN gene therapy replacement or the antisense oligonucleotide (ASO), Nusinersen, significantly upregulated the endogenous levels of GEMIN5 in mammalian cells and mutant GEMIN5-derived iPSC neurons. Further, we identified a strong functional association between the expression patterns of SMN and GEMIN5 in patient Spinal Muscular Atrophy (SMA)-derived motor neurons harboring loss-of-function mutations in the SMN gene. Interestingly, SMN binds to the C-terminus of GEMIN5 and requires the Tudor domain for GEMIN5 binding and expression regulation. Finally, we show that SMN upregulation ameliorates defective snRNP biogenesis and alternative splicing defects caused by loss of GEMIN5 in iPSC neurons and in vivo. Collectively, these studies indicate that SMN acts as a regulator of GEMIN5 expression and neuropathologies., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Coactivator-associated arginine methyltransferase 1 (CARM1) is an arginine methyltransferase that posttranslationally modifies proteins that regulate multiple levels of RNA production and processing. Its substrates include histones, transcription factors, coregulators of transcription, and splicing factors. CARM1 is overexpressed in many different cancer types, and often promotes transcription factor programs that are co-opted as drivers of the transformed cell state, a process known as transcription factor addiction. Targeting these oncogenic transcription factor pathways is difficult but could be addressed by removing the activity of the key coactivators on which they rely. CARM1 is ubiquitously expressed, and its KO is less detrimental in embryonic development than deletion of the arginine methyltransferases protein arginine methyltransferase 1 and protein arginine methyltransferase 5, suggesting that therapeutic targeting of CARM1 may be well tolerated. Here, we will summarize the normal in vivo functions of CARM1 that have been gleaned from mouse studies, expand on the transcriptional pathways that are regulated by CARM1, and finally highlight recent studies that have identified oncogenic properties of CARM1 in different biological settings. This review is meant to kindle an interest in the development of human drug therapies targeting CARM1, as there are currently no CARM1 inhibitors available for use in clinical trials., Competing Interests: Conflict of interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Mark T. Bedford is the co-founder of EpiCypher., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

SAGA and ATAC are two distinct chromatin modifying co-activator complexes with distinct enzymatic activities involved in RNA polymerase II (Pol II) transcription regulation. To investigate the mobility of co-activator complexes and general transcription factors in live-cell nuclei, we performed imaging experiments based on photobleaching. SAGA and ATAC, but also two general transcription factors ( TFIID and TFIIB), were highly dynamic, exhibiting mainly transient associations with chromatin, contrary to Pol II, which formed more stable chromatin interactions. Fluorescence correlation spectroscopy analyses revealed that the mobile pool of the two co-activators, as well as that of TFIID and TFIIB, can be subdivided into 'fast' (free) and 'slow' (chromatin-interacting) populations. Inhibiting transcription elongation decreased H3K4 trimethylation and reduced the 'slow' population of SAGA, ATAC, TFIIB and TFIID. In addition, inhibiting histone H3K4 trimethylation also reduced the 'slow' populations of SAGA and ATAC. Thus, our results demonstrate that in the nuclei of live cells the equilibrium between fast and slow population of SAGA or ATAC complexes is regulated by active transcription via changes in the abundance of H3K4me3 on chromatin. [ABSTRACT FROM AUTHOR]

Background and purpose Most spinal muscular atrophy (SMA) patients are homozygous for survival of motor neuron 1 gene ( SMN1 ) deletion. However, some SMA patients carry an intragenic SMN1 mutation. Such patients provide a clue to understanding the function of the SMN protein and the role of each domain of the protein. We previously identified mutations in the Tudor domain and C-terminal region of the SMN protein in three Japanese SMA patients. To clarify the effect of these mutations on protein stability, we conducted expression assays of SMN with mutated domains. Patients and methods Patients A and B carried a mutation in SMN1 exon 3, which encodes a Tudor domain, c.275G>C (p.Trp92Ser). Patient C carried a mutation in SMN1 exon 6, which encodes a YG-box; c.819_820insT (p.Thr274Tyrfs). We constructed plasmid expression vectors containing wild-type and mutant SMN1 cDNAs. After transfection of HeLa cells with the expression plasmids, RNA and protein were isolated and analyzed by reverse-transcription PCR and western blot analysis. Results The abundance of wild-type and mutant SMN1 transcripts in HeLa cells was almost the same. However, western blot analysis showed lower levels of mutant SMN proteins compared with wild-type SMN. In mutant SMN proteins, it is noteworthy that the level of the p.Thr274Tyrfs mutant was much reduced compared with that of the p.Trp92Ser mutant. Conclusions SMN mutations may affect the stability and levels of the protein. [ABSTRACT FROM AUTHOR]

Background: Histone posttranslational modifications (PTMs) function to regulate chromatin structure and function in part through the recruitment of effector proteins that harbor specialized "reader" domains. Despite efforts to elucidate reader domain--PTM interactions, the influence of neighboring PTMs and the target specificity of many reader domains is still unclear. The aim of this study was to use a high-throughput histone peptide microarray platform to interrogate 83 known and putative histone reader domains from the chromo and Tudor domain families to identify their interactions and characterize the influence of neighboring PTMs on these interactions. Results: Nearly a quarter of the chromo and Tudor domains screened showed interactions with histone PTMs by peptide microarray, revealing known and several novel methyllysine interactions. Specifically, we found that the CBX/ HP1 chromodomains that recognize H3K9me also recognize H3K23me2/3--a poorly understood histone PTM. We also observed that, in addition to their interaction with H3K4me3, Tudor domains of the Spindlin family also recognized H4K20me3--a previously uncharacterized interaction. Several Tudor domains also showed novel interactions with H3K4me as well. Conclusions: These results provide an important resource for the epigenetics and chromatin community on the interactions of many human chromo and Tudor domains. They also provide the basis for additional studies into the functional significance of the novel interactions that were discovered. [ABSTRACT FROM AUTHOR]

Background: Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication initiation determinant protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING E3 ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation., Methods: The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis., Results: RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter., Conclusion: This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production. Video Abstract., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Increased expression and hyperactivation of the methyltransferase SET domain bifurcated 1 (SETDB1) are commonly observed in cancer and central nervous system disorders. However, there are currently no reported SETDB1-specific methyltransferase inhibitors in the literature, suggesting that this is a challenging target. Here, we disclose that the previously reported small-molecule ligand for SETDB1's triple tudor domain, ( R , R )-59, is unexpectedly able to increase SETDB1 methyltransferase activity both in vitro and in cells. Specifically, ( R , R )-59 promotes in vitro SETDB1-mediated methylation of lysine 64 of the protein kinase Akt1. Treatment with ( R , R )-59 also increased Akt1 threonine 308 phosphorylation and activation, a known consequence of Akt1 methylation, resulting in stimulated cell proliferation in a dose-dependent manner. ( R , R )-59 is the first SETDB1 small-molecule positive activator for the methyltransferase activity of this protein. Mechanism of action studies show that full-length SETDB1 is required for significant in vitro methylation of an Akt1-K64 peptide and that this activity is stimulated by ( R , R )-59 primarily through an increase in catalytic activity rather than a change in S -adenosyl methionine binding.

SMNDC1 is a Tudor domain protein that recognizes di-methylated arginines and controls gene expression as an essential splicing factor. Here, we study the specific contributions of the SMNDC1 Tudor domain to protein-protein interactions, subcellular localization, and molecular function. To perturb the protein function in cells, we develop small molecule inhibitors targeting the dimethylarginine binding pocket of the SMNDC1 Tudor domain. We find that SMNDC1 localizes to phase-separated membraneless organelles that partially overlap with nuclear speckles. This condensation behavior is driven by the unstructured C-terminal region of SMNDC1, depends on RNA interaction and can be recapitulated in vitro. Inhibitors of the protein's Tudor domain drastically alter protein-protein interactions and subcellular localization, causing splicing changes for SMNDC1-dependent genes. These compounds will enable further pharmacological studies on the role of SMNDC1 in the regulation of nuclear condensates, gene regulation and cell identity., (© 2023. The Author(s).)