The RNA-binding protein Snd1/Tudor-SN regulates hypoxia-responsive gene expression.

Snd1 is an evolutionarily conserved RNA-binding protein implicated in several regulatory processes in gene expression including activation of transcription, mRNA splicing, and microRNA decay. Here, we have investigated the outcome of Snd1 gene deletion in the mouse. The knockout mice are viable showing no gross abnormalities apart from decreased fertility, organ and body size, and decreased number of myeloid cells concomitant with decreased expression of granule protein genes. Deletion of Snd1 affected the expression of relatively small number of genes in spleen and liver. However, mRNA expression changes in the knockout mouse liver showed high similarity to expression profile in adaptation to hypoxia. MicroRNA expression in liver showed upregulation of the hypoxia-induced microRNAs miR-96 and -182. Similar to Snd1 deletion, mimics of miR-96/182 enhanced hypoxia-responsive reporter activity. To further elucidate the function of SND1, BioID biotin proximity ligation assay was performed in HEK-293T cells to identify interacting proteins. Over 50% of the identified interactors were RNA-binding proteins, including stress granule proteins. Taken together, our results show that in normal growth conditions, Snd1 is not a critical factor for mRNA transcription in the mouse, and describe a function for Snd1 in hypoxia adaptation through negatively regulating hypoxia-related miRNAs and hypoxia-induced transcription consistent with a role as stress response regulator.

Inhibition of RNA Binding in SND1 Increases the Levels of miR-1-3p and Sensitizes Cancer Cells to Navitoclax.

SND1 is an RNA-binding protein overexpressed in large variety of cancers. SND1 has been proposed to enhance stress tolerance in cancer cells, but the molecular mechanisms are still poorly understood. We analyzed the expression of 372 miRNAs in the colon carcinoma cell line and show that SND1 silencing increases the expression levels of several tumor suppressor miRNAs. Furthermore, SND1 knockdown showed synergetic effects with cancer drugs through MEK-ERK and Bcl-2 family-related apoptotic pathways. To explore whether the SND1-mediated RNA binding/degradation is responsible for the observed effect, we developed a screening assay to identify small molecules that inhibit the RNA-binding function of SND1. The screen identified P2X purinoreceptor antagonists as the most potent inhibitors. Validation confirmed that the best hit, suramin, inhibits the RNA binding ability of SND1. The binding characteristics and mode of suramin to SND1 were characterized biophysically and by molecular docking that identified positively charged binding cavities in Staphylococcus nuclease domains. Importantly, suramin-mediated inhibition of RNA binding increased the expression of miR-1-3p, and enhanced sensitivity of cancer cells to Bcl-2 inhibitor navitoclax treatment. Taken together, we demonstrate as proof-of-concept a mechanism and an inhibitor compound for SND1 regulation of the survival of cancer cells through tumor suppressor miRNAs.

An Activating STAT3 Mutation Causes Neonatal Diabetes through Premature Induction of Pancreatic Differentiation.

Activating germline mutations in STAT3 were recently identified as a cause of neonatal diabetes mellitus associated with beta-cell autoimmunity. We have investigated the effect of an activating mutation, STAT3K392R, on pancreatic development using induced pluripotent stem cells (iPSCs) derived from a patient with neonatal diabetes and pancreatic hypoplasia. Early pancreatic endoderm differentiated similarly from STAT3K392R and healthy-control cells, but in later stages, NEUROG3 expression was upregulated prematurely in STAT3K392R cells together with insulin (INS) and glucagon (GCG). RNA sequencing (RNA-seq) showed robust NEUROG3 downstream targets upregulation. STAT3 mutation correction with CRISPR/Cas9 reversed completely the disease phenotype. STAT3K392R-activating properties were not explained fully by altered DNA-binding affinity or increased phosphorylation. Instead, reporter assays demonstrated NEUROG3 promoter activation by STAT3 in pancreatic cells. Furthermore, proteomic and immunocytochemical analyses revealed increased nuclear translocation of STAT3K392R. Collectively, our results demonstrate that the STAT3K392R mutation causes premature endocrine differentiation through direct induction of NEUROG3 expression.

Poly(A)(+) mRNA-binding protein Tudor-SN regulates stress granules aggregation dynamics.

Stress granules (SGs) and processing bodies (PBs) comprise the main types of cytoplasmic RNA foci during stress. Our previous data indicate that knockdown of human Tudor staphylococcal nuclease (Tudor-SN) affects the aggregation of SGs. However, the precise molecular mechanism has not been determined fully. In the present study, we demonstrate that Tudor-SN binds and colocalizes with many core components of SGs, such as poly(A)(+) mRNA binding protein 1, T-cell internal antigen-1-related protein and poly(A)(+) mRNA, and SG/PB sharing proteins Argonaute 1/2, but not PB core proteins, such as decapping enzyme 1 a/b, confirming that Tudor-SN is an SG-specific protein. We also demonstrate that the Tudor-SN granule actively communicates with the nuclear and cytosolic pool under stress conditions. Tudor-SN can regulate the aggregation dynamics of poly(A)(+) mRNA-containing SGs and selectively stabilize the SG-associated mRNA during cellular stress.

Tudor-SN, a novel coactivator of peroxisome proliferator-activated receptor γ protein, is essential for adipogenesis.

Adipogenesis, in which mesenchymal precursor cells differentiate into mature adipocytes, is a well orchestrated process. In the present study we identified Tudor-SN as a novel co-activator of the transcription factor peroxisome proliferator-activated receptor γ (PPARγ). We provide the first evidence that Tudor-SN and PPARγ exist in the same complex. Both are up-regulated by the early factor C/EBPß during adipogenesis and significantly influence the regulation of PPARγ target genes in both 3T3-L1 pre-adipocyte and mouse embryonic fibroblasts (MEF) upon exposure to a mixture of hormonal mixture. Moreover, aP2-PPARγ response element (PPRE) interacts with both PPARγ and Tudor-SN, and the gene transcriptional activation of PPRE-luc is enhanced by ectopic expression of Tudor-SN. Deletion of Tudor-SN protein (MEF-KO) affects but does not completely abolish the association of PPARγ and aP2-PPRE. Loss-of-function studies further verified that Tudor-SN is required for adipogenesis, as deletion of Tudor-SN (MEF-KO) impairs dexamethasone, 3-isobutyl-1-methylxanthine, and insulin (DMI)-induced adipocyte differentiation and the expression of PPARγ target genes, such as aP2 and adipsin. Furthermore, H3 acetylation levels were lower in MEF-KO than MEF-WT. Both HDAC1 and HDAC3 are stably associated with PPARγ in MEF-KO, whereas only a small amount of association was observed in MEF-WT after 5 days of treatment during adipogenesis. PPARγ requires various co-activators or co-repressors, which may dynamically associate with and regulate the higher order chromatin remodeling of the promoter region of PPARγ-bound target genes; Tudor-SN is likely one of these co-activators.

Expression analysis of Tudor-SN protein in mouse tissues.

Tudor-SN (SND1, p100) has been shown to function as a transcriptional coactivator as well as a modulator of RNA metabolism and biogenesis and a component in the RNA-induced silencing complex (RISC). Tudor-SN consists of five repeats of staphylococcus nuclease-like domains (SN1-SN5) and, a Tudor domain implicated in binding to methylated ligands. The protein is highly conserved through evolution from fission yeast to mammals and it exists as a single gene without any close homologs. Tudor-SN is found to be overexpressed in several cancers such as colon adenocarcinomas and prostate cancer. The conservation of Tudor-SN along evolution suggests it may have important functions; however, the physiological function of Tudor-SN has not yet been characterized. In this study we analyzed the expression and localization of Tudor-SN in mouse tissues and organs by immunohistochemistry, fluorescent immunostaining, Western blotting and RT-qPCR. Expression analysis indicated that Tudor-SN is widely expressed in most organs with the exception of muscle cells. Up-regulated expression was observed in rapidly dividing cells and progenitor cells such as in spermatogonial cells in testis, in the follicular cells of ovary, in the cells of crypts of Lieberkühn of ileum and basal keratinocytes of skin and hair follicle when compared to more differentiated or terminally differentiated cells in the respective organs. Moreover, Tudor-SN was robustly expressed in T-cells and Tudor-SN was co-expressed with CD3 in T-cells in the Peyer's patch, spleen and lymph node. The wide expression pattern of Tudor-SN and high expression in proliferating and self-differentiating cells suggests that the protein serves functions related to activated state of cells.

Tudor staphylococcal nuclease (Tudor-SN) participates in small ribonucleoprotein (snRNP) assembly via interacting with symmetrically dimethylated Sm proteins.

Human Tudor staphylococcal nuclease (Tudor-SN) is composed of four tandem repeats of staphylococcal nuclease (SN)-like domains, followed by a tudor and SN-like domain (TSN) consisting of a central tudor flanked by two partial SN-like sequences. The crystal structure of the tudor domain displays a conserved aromatic cage, which is predicted to hook methyl groups. Here, we demonstrated that the TSN domain of Tudor-SN binds to symmetrically dimethylarginine (sDMA)-modified SmB/B' and SmD1/D3 core proteins of the spliceosome. We demonstrated that this interaction ability is reduced by the methyltransferase inhibitor 5-deoxy-5-(methylthio)adenosine. Mutagenesis experiments indicated that the conserved amino acids (Phe-715, Tyr-721, Tyr-738, and Tyr-741) in the methyl-binding cage of the TSN domain are required for Tudor-SN-SmB interaction. Furthermore, depletion of Tudor-SN affects the association of Sm protein with snRNAs and, as a result, inhibits the assembly of uridine-rich small ribonucleoprotein mediated by the Sm core complex in vivo. Our results reveal the molecular basis for the involvement of Tudor-SN in regulating small nuclear ribonucleoprotein biogenesis, which provides novel insight related to the biological activity of Tudor-SN.

Not4 enhances JAK/STAT pathway-dependent gene expression in Drosophila and in human cells.

The JAK/STAT pathway is essential for organogenesis, innate immunity, and stress responses in Drosophila melanogaster. The JAK/STAT pathway and its associated regulators have been highly conserved in evolution from flies to humans. We have used a genome-wide RNAi screen in Drosophila S2 cells to identify regulators of the JAK/STAT pathway, and here we report the characterization of Not4 as a positive regulator of the JAK/STAT pathway. Overexpression of Not4 enhanced Stat92E-mediated gene responses in vitro and in vivo in Drosophila. Specifically, Not4 increased Stat92E-mediated reporter gene activation in S2 cells; and in flies, Not4 overexpression resulted in an 8-fold increase in Turandot M (TotM) and in a 4-fold increase in Turandot A (TotA) stress gene activation when compared to wild-type flies. Drosophila Not4 is structurally related to human CNOT4, which was found to regulate interferon-γ- and interleukin-4-induced STAT-mediated gene responses in human HeLa cells. Not4 was found to coimmunoprecipitate with Stat92E but not to affect tyrosine phosphorylation of Stat92E in Drosophila cells. However, Not4 is required for binding of Stat92E to its DNA recognition sequence in the TotM gene promoter. In summary, Not4/CNOT4 is a novel positive regulator of the JAK/STAT pathway in Drosophila and in humans.

PTB-associated splicing factor (PSF) functions as a repressor of STAT6-mediated Ig epsilon gene transcription by recruitment of HDAC1.

Regulation of transcription requires cooperation between sequence-specific transcription factors and numerous coregulatory proteins. In IL-4/IL-13 signaling several coactivators for STAT6 have been identified, but the molecular mechanisms of STAT6-mediated gene transcription are still not fully understood. Here we identified by proteomic approach that the PTB-associated splicing factor (PSF) interacts with STAT6. In intact cells the interaction was observed only after IL-4 stimulation. The IL-4-induced tyrosine phosphorylation of both STAT6 and PSF is a prerequisite for the efficient association of the two proteins. Functional analysis demonstrated that ectopic expression of PSF resulted in inhibition of STAT6-mediated transcriptional activation and mRNA expression of the Igε germline heavy chain gene, whereas knockdown of PSF increased the STAT6-mediated responses. PSF recruited histone deacetylase 1 (HDAC1) to the STAT6 transcription complex, which resulted in reduction of H3 acetylation at the promoter regions of Ig heavy chain germline Igε and inhibition of STAT6-mediated transcription. In addition, the HDACs inhibitor trichostatin A (TSA) enhanced H3 acetylation, and reverted the PSF-mediated transcriptional repression of Igε gene transcription. In summary, these results identify PSF as a repressor of STAT6-mediated transcription that functions through recruitment of HDAC to the STAT6 transcription complex, and delineates a novel regulatory mechanism of IL-4 signaling that may have implications in the pathogenesis of allergic diseases and pharmacological HDAC inhibition in lymphomas.

Tudor-SN interacts with and co-localizes with G3BP in stress granules under stress conditions.

SGs are mRNA containing cytoplasmic structures that are assembled in response to stress. Tudor-SN protein is a ubiquitously expressed protein. Here, Tudor-SN protein was found to physiologically interact with G3BP, which is the marker and effector of SG. The kinetics of the assembly of SGs in the living cells demonstrated that Tudor-SN co-localizes with G3BP and is recruited to the same SGs in response to different stress stimuli. Knockdown of endogenous Tudor-SN did not inhibit the formation of SGs, but retarded the aggregation of small SGs into large SGs. Thus Tudor-SN may not be an initiator as essential as G3BP for the formation of SGs, but affects the aggregation of SGs. These findings identify Tudor-SN as a novel component of SGs.

Monoclonal antibodies against human Tudor-SN.

Tudor-SN is a multifunctional regulator of gene expression that has been shown to function as a transcriptional co-activator, regulator of miRNA processing, mRNA splicing, and stability. Tudor-SN has also been identified as a component in RNA-induced silencing complex. Here we have produced and characterized seven monoclonal antibody (MAb) clones against human Tudor-SN. Antibodies were generated against the fourth staphylococcal nuclease-like domain (SN4) and the Tudor domain of human Tudor-SN. The MAbs recognize the Tudor-SN protein in Western blot analysis and immunoprecipitation, and detect the specific antigen in immunohistochemistry assays. One of the antibody clones also recognizes the Drosophila melanogaster and Danio rerio Tudor-SN. Immunocytochemistry of HeLa cells revealed Tudor-SN localization in nucleolus, suggesting a possible new function for the protein in the compartment. An extensive expression analysis in human tissue arrays shows moderate to high expression of Tudor-SN in a wide range of organs and tissues, especially in epithelial cell types.

Calmodulin inhibition of E2A stops expression of surrogate light chains of the pre-B-cell receptor and CD19.

To create antibody diversity, B lymphocyte development is characterized by the ordered rearrangement of first immunoglobulin (Ig) heavy chain gene segments and then Ig light-chain gene segments. Early in B-cell development, expression of a pre-B-cell receptor (pre-BCR) composed of membrane-bound Ig heavy chain protein associated with surrogate light-chain (SLC) proteins serves as a critical checkpoint that monitors for functional heavy chain rearrangement. Signaling from the pre-BCR induces clonal expansion, but it also turns off transcription of the genes for the SLC proteins lambda5 and VpreB, which limits this proliferation. Here we show that signaling from the pre-BCR rapidly down-regulates lambda5 and VpreB and also the co-receptor CD19 in primary pre-B-cells. We show that calcium (Ca(2+)) signaling is essential for this silencing of the SLC and CD19 genes. The SLC genes are activated by the E2A transcription factor, and we show that E2A is required for pre-BCR-mediated regulation of the genes. E2A mutated in its binding site for the Ca(2+) sensor protein calmodulin, and thus with calmodulin-resistant DNA binding, makes lambda5, VpreB and CD19 expression resistant to the inhibition following pre-BCR activation. Thus, Ca(2+) down-regulates SLC and CD19 gene expression upon pre-BCR activation through inhibition of E2A by Ca(2+)/calmodulin.

Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome.

Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals. Plants lack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases. Although metacaspases are essential for PCD, their natural substrates remain unknown. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our results establish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN.

Calcium regulation of myogenesis by differential calmodulin inhibition of basic helix-loop-helix transcription factors.

The members of the MyoD family of basic helix-loop-helix (bHLH) transcription factors are critical regulators of skeletal muscle differentiation that function as heterodimers with ubiquitously expressed E-protein bHLH transcription factors. These heterodimers must compete successfully with homodimers of E12 and other E-proteins to enable myogenesis. Here, we show that E12 mutants resistant to Ca(2+)-loaded calmodulin (CaM) inhibit MyoD-initiated myogenic conversion of transfected fibroblasts. Ca(2+) channel blockers reduce, and Ca(2+) stimulation increases, transcription by coexpressed MyoD and wild-type E12 but not CaM-resistant mutant E12. Furthermore, CaM-resistant E12 gives lower MyoD binding and higher E12 binding to a MyoD-responsive promoter in vivo and cannot rescue myogenic differentiation that has been inhibited by siRNA against E12 and E47. Our data support the concept that Ca(2+)-loaded CaM enables myogenesis by inhibiting DNA binding of E-protein homodimers, thereby promoting occupancy of myogenic bHLH protein/E-protein heterodimers on promoters of myogenic target genes.

B-cell receptor activation inhibits AID expression through calmodulin inhibition of E-proteins.

Upon encountering antigens, B-lymphocytes can adapt to produce a highly specific and potent antibody response. Somatic hypermutation, which introduces point mutations in the variable regions of antibody genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both somatic hypermutation and CSR. The mutagenic AID enzyme has to be tightly controlled. Here, we show that engagement of the membrane-bound antibodies of the B-cell receptor (BCR), which signals that good antibody affinity has been reached, inhibits AID gene expression and that calcium (Ca(2+)) signaling is essential for this inhibition. Moreover, we show that overexpression of the Ca(2+) sensor protein calmodulin inhibits AID gene expression, and that the transcription factor E2A is required for regulation of the AID gene by the BCR. E2A mutated in the binding site for calmodulin, and thus showing calmodulin-resistant DNA binding, makes AID expression resistant to the inhibition through BCR activation. Thus, BCR activation inhibits AID gene expression through Ca(2+)/calmodulin inhibition of E2A.

The multifunctional human p100 protein 'hooks' methylated ligands.

The human p100 protein is a vital transcription regulator that increases gene transcription by forming a physical bridge between promoter-specific activators and the basal transcription machinery. Here we demonstrate that the tudor and SN (TSN) domain of p100 interacts with U small nuclear ribonucleoprotein (snRNP) complexes, suggesting a role for p100 in the processing of precursor messenger RNA. We determined the crystal structure of the p100 TSN domain to delineate the molecular basis of p100's proposed functions. The interdigitated structure resembles a hook, with a hinge controlling the movement and orientation of the hook. Our studies suggest that a conserved aromatic cage hooks methyl groups of snRNPs and anchors p100 to the spliceosome. These structural insights partly explain the distinct roles of p100 in transcription and splicing.

Calcium/calmodulin inhibition of transcriptional activity of E-proteins by prevention of their binding to DNA.

The Ca2+ sensor protein calmodulin can interact with the DNA binding basic helix-loop-helix (bHLH) domain of E12, E47, and SEF2-1 (E2-2), which belong to the E-protein subclass of bHLH transcription factors. This interaction inhibits the DNA binding of these bHLH proteins in vitro, and an ionophore that increases intracellular Ca2+ can inhibit transcriptional activation by the E-proteins. Here we have attempted to determine if these phenomena reflect a direct calmodulin-dependent inhibition of DNA binding by E-proteins in vivo. We show that calmodulin overexpression inhibits the transcriptional activity of E12, E47, and SEF2-1. We have compared calmodulin effects on DNA binding in vitro and on activation of transcription in vivo using a series of E12 mutants harboring defined alterations within the basic sequence of the bHLH domain that reduce their ability to bind calmodulin to varying degrees. We find a striking direct correlation between the ability of calmodulin to inhibit their DNA binding in vitro and the ability of overexpressed calmodulin or cellular Ca2+ mobilization to inhibit their transcriptional activity in vivo. Furthermore, E12 and overexpressed calmodulin were co-localized in the nucleus, and calmodulin pull-down experiments with cell extracts showed a Ca2+-dependent interaction between calmodulin and E12 but not with a calmodulin inhibition-deficient E12 mutant. Chromatin immunoprecipitation showed that calmodulin overexpression leads to decreased binding of E12 and E47, but not a calmodulin inhibition-deficient E12 mutant, to the DNA recognition sequence in vivo. The data suggest that Ca2+ signaling can inhibit the transcriptional activities of E-proteins through direct binding of Ca2+/calmodulin to the basic sequence of E-proteins, resulting in inhibition of their DNA binding.