Transcription Complex Research Articles

Overexpression of SlGRAS38 in tomato roots accelerates arbuscular mycorrhiza formation

Recent research has highlighted the role of GRAS family transcription factors (TFs) in arbuscular mycorrhizal (AM) formation, particularly in tomato plants. This study conducted a functional analysis of the SCL32 GRAS transcription factor SlGRAS38 during arbuscular mycorrhizal formation in tomatoes, confirming its positive regulatory effect on mycorrhiza formation. Experiments reveal that overexpression of SlGRAS38 accelerates mycorrhizal colonization and enhances nutrient uptake, flowering, and fruit yield. Transcriptomic data indicate that SlGRAS38 may function as a new transcriptional regulator during mycorrhization. SlGRAS38 has previously been described as a transcriptional activator in ripening fruits, and here we demonstrate its regulatory role in mycorrhizal roots, suggesting potential cross-talk between these physiological processes. However, the exact mechanisms remain to be fully understood. Further research is needed to explore the interplay between SlGRAS38's roles in mycorrhization and fruit ripening, and to confirm its interaction within transcriptional complexes critical for arbuscule formation. This study underscores the importance of SlGRAS38 in AM development and opens avenues for future investigations into its multifaceted functions.

Probenecid Inhibits Extracellular Signal-Regulated Kinase and c-Jun N-Terminal Kinase Mitogen-Activated Protein Kinase Pathways in Regulating Respiratory Syncytial Virus Response.

We have previously shown that probenecid inhibits RSV, influenza virus, and SARS-CoV-2 replication in vitro in preclinical animal models and in humans. In a Phase two randomized, placebo-controlled, single-blind, dose range-finding study using probenecid to treat non-hospitalized patients with symptomatic, mild-to-moderate COVID-19, we previously showed that a 1000 mg twice daily treatment for 5 days reduced the median time to viral clearance from 11 to 7 days, and a 500 mg twice daily treatment for 5 days reduced the time to viral clearance from 11 to 9 days more than the placebo. In this study, we sought to determine the mechanism of action of the probenecid inhibition of RSV replication in human respiratory epithelial (A549) cells. We show that probenecid inhibits the RSV-induced phosphorylation of JNKs and ERKs and the downstream phosphorylation of c-jun, a component of the AP-1 transcription complex needed for virus replication. The inhibition of JNKs by probenecid reversed the repression of transcription factor HNF-4. The probenecid inhibition of JNK and ERK phosphorylation involves the MAPK pathway that precludes virus replication.

A multilineage screen identifies actionable synthetic lethal interactions in human cancers.

Cancers are driven by alterations in diverse genes, creating dependencies that can be therapeutically targeted. However, many genetic dependencies have proven inconsistent across tumors. Here we describe SCHEMATIC, a strategy to identify a core network of highly penetrant, actionable genetic interactions. First, fundamental cellular processes are perturbed by systematic combinatorial knockouts across tumor lineages, identifying 1,805 synthetic lethal interactions (95% unreported). Interactions are then analyzed by hierarchical pooling, revealing that half segregate reliably by tissue type or biomarker status (51%) and a substantial minority are penetrant across lineages (34%). Interactions converge on 49 multigene systems, including MAPK signaling and BAF transcriptional regulatory complexes, which become essential on disruption of polymerases. Some 266 interactions translate to robust biomarkers of drug sensitivity, including frequent genetic alterations in the KDM5C/6A histone demethylases, which sensitize to inhibition of TIPARP (PARP7). SCHEMATIC offers a context-aware, data-driven approach to match genetic alterations to targeted therapies.

Structural proteomics defines a sequential priming mechanism for the progesterone receptor.

The progesterone receptor (PR) is a steroid-responsive nuclear receptor with two isoforms: PR-A and PR-B. Disruption of PR-A:PR-B signaling is associated with breast cancer through interactions with oncogenic co-regulatory proteins (CoRs). However, molecular details of isoform-specific PR-CoR interactions remain poorly understood. Using structural mass spectrometry, we investigate the sequential binding mechanism of purified full-length PR and intact CoRs, steroid receptor coactivator 3 (SRC3) and p300, as complexes on target DNA. Our findings reveal selective CoR NR-box binding by PR and unique interaction surfaces between PR and CoRs during complex assembly, providing a structural basis for CoR sequential binding on PR. Antagonist-bound PR showed persistent CoR interactions, challenging the classical model of nuclear receptor activation and repression. Collectively, we offer a peptide-level perspective on the organization of the PR transcriptional complex and infer the mechanisms behind the interactions of these proteins, both in active and inactive conformations.

The Mediator complex subunit MoMed15 plays an important role in conferring sensitivity to isoprothiolane by modulating xenobiotic metabolism in M. oryzae.

Rice blast caused by Magnaporthe oryzae is one of the most economically important rice diseases. Fungicides such as isoprothiolane (IPT) have been used extensively for rice blast control, but resistance to IPT in M. oryzae is an emerging threat. In this study, molecular mechanisms of resistance in IPT-resistant mutants were identified. Through whole-genome sequencing and genetic transformation, we identified the gene MoMed15, encoding a transcriptional glutamine-rich co-activator Mediator complex subunit, in which mutations or deletion resulted in moderate IPT resistance. Further research found that MoMed15 physically interacted with the IPT resistance regulatory factor MoIRR to simultaneously regulate both MoIRR expression and the expression of multiple xenobiotic-metabolizing enzymes in response to IPT stress. We hypothesize that some xenobiotic-metabolizing enzymes enhance IPT toxicity by modifying the IPT structure. Variation of MoMed15 affected the recruitment of the transcriptional Mediator complex and decreased the expression of these xenobiotic-metabolizing enzymes, resulting in moderate IPT resistance. We also found that MoPGR1, encoding a protein that activates cytochrome P450 enzymes, was essential to confer IPT sensitivity, and its expression was directly regulated by MoIRR.IMPORTANCEIsoprothiolane (IPT) has been used extensively for the management of rice blast disease and IPT-resistant subpopulations have emerged in Chinese rice fields. The emergence of resistant pathogen populations has led to a steep increase in fungicide use, increasing pesticide risk for the applicator and the environment. The molecular mechanisms of IPT resistance in M. oryzae remain elusive. In this study, we demonstrated that transcriptional co-activator MoMed15 interacts with IPT resistance regulator MoIRR to recruit the Mediator complex, which promotes the expression of xenobiotic-metabolizing enzymes, leading to exacerbated IPT toxicity. The MoMed15 could be used for IPT resistance detection in rice fields.

PATH-65. MONITORING MENINGIOMA CELL CYCLE ACTIVITY THROUGH DREAM-MMB ACTIVITY SWITCHING

Abstract Meningiomas account for over 40% of all primary intracranial brain tumors, with approximately one-fifth of these tumors recurring. Our lab has previously used molecular techniques to identify three subgroups: MenG A, B, and C, which have been independently validated. Interestingly, only MenG C tumors harbor recurring patients, making it an aggressive subtype of meningioma. One hallmark of MenG C tumors is the overexpression of a distinct set of genes that harbor in their promoter the CDE-CHR DNA-binding element, used by a transcriptional complex that in dormant cells functions as a repressor (DREAM). Before phosphorylation, this complex functions as an activator (MBB) in cycling cells. Upon a phosphorylation-dependent switch, the dual-specificity tyrosine kinase DYRK1A phosphorylates S28 on LIN52, which itself is dimerized with LIN9 of the core complex. Phosphorylation shifts the complex to DREAM, while lack thereof allows the core complex to interact with MYBL2 (now MBB) and activate the same genes repressed in quiescent cells. We seek to quantify this switch activity using an in vivo luciferase sensor using NanoBiT technology. Our biosensor is based on a split nanoluciferase, where one half is fused to the LIN52/LIN9 dimer and the other is fused to MYBL2. When MYBL2 binds to the LIN52/LIN9 dimer, a functional nanoluciferase is formed, and its activity can be measured using a luciferase-specific substrate. As proof of principle, we have increased cell proliferation by increasing FBS concentration in cultured cells as well as by blocking S28 phosphorylation using the DYRK1A-specific inhibitor Harmine. We screened all possible combinations (alternating NanoBiT fragments) for the highest luciferase activity after stimulation. In the future, this biosensor can be used to monitor cell cycle activity in vitro as well as in vivo using low-cost Luciferase reporter assays to better understand the regulation of specific genes in MenG C tumors.

EPCO-17. CARM1 METHYLATES GLIOBLASTOMA TUMOR SUPPRESSOR QUAKING TO REGULATE ITS TRANSCRIPTIONAL COACTIVATOR FUNCTION

Abstract Glioblastoma (GBM) represents the most prevalent and deadly primary brain tumor in adults, and it remains highly refractory to current therapeutic interventions. Significant intratumor heterogeneity drives rapid invasion, immune escape, and therapeutic resistance. Therapeutic paradigms based on deconvoluting key molecular mechanisms driving gliomagenesis are urgently needed. Quaking (Qki, encoded by Qk) is frequently altered in GBM patients; Qk mutations or deletions occur in 35% percent of cases, and the Qki protein is absent in 50-60% of cases. Our lab has characterized Qki as important tumor suppressor in GBM, where deletion of Qk enables pre-malignant neural stem cells to expand outside their niche and develop into tumors that recapitulate human GBM. We discovered that Qki functions as a transcriptional coactivator of lipid metabolism genes, whereby Qki loss disrupts lipid homeostasis and compromises the integrity of various membrane structures. Several downstream processes governed by Qki have been delineated; however, upstream regulators of Qki have yet to be identified. Recurrent cancer mutations to specific Qki arginine sites implicate the dysregulation of arginine methylation, a common post-translational modification catalyzed by protein arginine methyltransferases (PRMTs). Leveraging in vitro methylation assays, we demonstrate that Qki is selectively methylated by the PRMT coactivator-associated arginine methyltransferase 1 (CARM1), specifically in the Quaking regulatory domain where cancer mutations are reported. We observe that Qki interacts with known CARM1 substrates, functioning in the transcriptional complex assembled by CARM1. Through mutagenesis studies, we identified arginine 242 (R242) and arginine 256 (R256) as specific CARM1-mediated methylation sites on Qki. Importantly, mutations to these sites compromise Qki’s activity as a transcriptional coactivator. Moreover, brain-specific deletion of CARM1 in vivo results in neurological phenotypes reminiscent of Qki deletion. Altogether, we provide evidence that CARM1 represents a novel tumor suppressor in GBM that functions through the arginine methylation of Qki.

PNSC5325 prevents acute respiratory distress syndrome by alleviating inflammation and inhibiting extracellular matrix degradation of alveolar macrophages

BackgroundAcute respiratory distress syndrome (ARDS) is characterized by severe inflammation and significant extracellular matrix (ECM) degradation in the lungs. Our prior research identified the CtBP2-p300-NF-κB (C-terminal-binding protein 2-histone acetyltransferase p300-nuclear factor kappa B) transcriptional complex as critical in ARDS by activating pro-inflammatory cytokine genes. MethodsAn ARDS mouse model was established using intratracheal instillation of lipopolysaccharide (LPS). Small molecules that inhibit the CtBP2-p300 interaction were identified through AlphaScreen. RNA sequencing (RNA-Seq) was conducted to determine differential gene expression. Immunoprecipitation and co-immunoprecipitation analyzed protein interactions. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunoblotting detected gene and protein expression. Histological staining evaluated tissue damage. ResultsThrough AlphaScreen, two natural compounds, PNSC2477 and PNSC5325, were identified for their ability to inhibit the CtBP2-p300 interaction. While PNSC2477 demonstrated toxicity and was deemed unsuitable for further research, PNSC5325 exhibited minimal toxicity. PNSC5325 effectively inhibited the CtBP2-p300 interaction and reduced pro-inflammatory cytokine gene expression. RNA-Seq analysis of PNSC5325-treated cells indicated significant suppression of pro-inflammatory cytokine genes and matrix metalloproteinases (MMPs). Further molecular studies revealed that the CtBP2-p300 complex, in conjunction with activator protein 1 (AP1), activates MMP expression. PNSC5325 simultaneously suppressed both pro-inflammatory cytokines and MMPs by targeting the CtBP2-p300 complex. In LPS-injected mice, PNSC5325 administration significantly reduced ARDS incidence by inhibiting inflammatory and MMP genes. ConclusionThese findings suggest that PNSC5325 protects against ARDS by inhibiting key inflammatory and ECM degradation pathways, highlighting its potential as a novel therapeutic agent for ARDS and paving the way for further clinical investigations.

HnRNPA2B1 deacetylation by SIRT6 restrains local transcription and safeguards genome stability.

Repair of double strand breaks (DSBs) by RNA-binding proteins (RBPs) is vital for ensuring genome integrity. DSB repair is accompanied by local transcriptional repression in the vicinity of transcriptionally active genes, but the mechanism by which RBPs regulate transcriptional regulation is unclear. Here, we demonstrated that RBP hnRNPA2B1 functions as a RNA polymerase-associated factor that stabilizes the transcription complex under physiological conditions. Following a DSB, hnRNPA2B1 is released from damaged chromatin, reducing the efficiency of RNAPII complex assembly, leading to local transcriptional repression. Mechanistically, SIRT6 deacetylates hnRNPA2B1 at K113/173 residues, enforcing its rapid detachment from DSBs. This process disrupts the integrity of the RNAPII complex on active chromatin, which is a pre-requisite for transient but complete repression of local transcription. Functionally, the overexpression of an acetylation mimic stabilizes the transcription complex and facilitates the functioning of the transcription machinery. hnRNPA2B1 acetylation status was negatively correlated with SIRT6 expression, and acetylation mimic enhanced radio-sensitivity in vivo. Our findings demonstrate that hnRNPA2B1 is crucial for transcriptional repression. We have uncovered the missing link between DSB repair and transcriptional regulation in genome stability maintenance, highlighting the potential of hnRNPA2B1 as a therapeutic target.

Classifying the molecular functions of transcription factors beyond activation and repression.

Notch signaling is a highly conserved pathway activated by dynamic cellular interactions that initiates a molecular cascade that ultimately drives changes in gene expression. The Notch transcriptional complex (NTC) regulates genes that influence development and homeostasis. In this issue of Genes & Development, Rogers and colleagues (doi:10.1101/gad.352108.124) leverage a rapid Notch activation system combined with molecular genomic profiling to reveal that the NTC regulates the release of paused RNA polymerase to activate direct target genes. They also highlight the role of the SWI/SNF chromatin remodeling complex in establishing and maintaining chromatin accessibility to potentiate the activation of NTC target genes.

BioID proximity mapping reveals novel SAP18 interactions in the prespliceosomal complex

SAP18 protein was originally discovered in association with the SIN3 transcriptional repressor complex. Subsequent biochemical fractionation studies identified SAP18 as a component of another distinct trimeric complex termed as the apoptosis- and splicing-associated protein (ASAP) complex. The existence of SAP18 in distinct complexes highlights its dual role in transcriptional and splicing regulation. In our study, we aim to define the in vivo interactome of SAP18 using proximity-dependent biotin identification (BioID). Mass spectrometry analysis of streptavidin-purified biotinylated proteins revealed new SIN3-associated interactors, including RBBP4 and SAP30BP. Notably, we identified 72 spliceosomal proteins as highly enriched interactors. Additionally, a complementary immunoprecipitation assay validated novel interactions of SAP18 with the prespliceosomal components SNRNP70, SNRPA, SF3B1, U2AF1, and the SR protein SRSF1. Mutational analysis using a C-terminal SAP18 double point mutant, which is known to be deficient in ASAP-interaction, demonstrated a debilitated interaction with the prespliceosomal proteins. Altogether, our results present a refined understanding of the SAP18 interactome, uncovering its association with the prespliceosome in conjugation with ASAP components.

Seq-Scope: repurposing Illumina sequencing flow cells for high-resolution spatial transcriptomics.

Spatial transcriptomics technologies aim to advance gene expression studies by profiling the entire transcriptome with intact spatial information from a single histological slide. However, the application of spatial transcriptomics is limited by low resolution, limited transcript coverage, complex procedures, poor scalability and high costs of initial setup and/or individual experiments. Seq-Scope repurposes the Illumina sequencing platform for high-resolution, high-content spatial transcriptome analysis, overcoming these limitations. It offers submicrometer resolution, high capture efficiency, rapid turnaround time and precise annotation of histopathology at a much lower cost than commercial alternatives. This protocol details the implementation of Seq-Scope with an Illumina NovaSeq 6000 sequencing flow cell, allowing the profiling of multiple tissue sections in an area of 7 mm × 7 mm or larger. We describe the preparation of a fresh-frozen tissue section for both histological imaging and sequencing library preparation and provide a streamlined computational pipeline with comprehensive instructions to integrate histological and transcriptomic data for high-resolution spatial analysis. This includes the use of conventional software tools for single-cell and spatial analysis, as well as our recently developed segmentation-free method for analyzing spatial data at submicrometer resolution. Aside from array production and sequencing, which can be done in batches, tissue processing, library preparation and running the computational pipeline can be completed within 3 days by researchers with experience in molecular biology, histology and basic Unix skills. Given its adaptability across various biological tissues, Seq-Scope establishes itself as an invaluable tool for researchers in molecular biology and histology.

The HSP90/R2TP Quaternary Chaperone Scaffolds Assembly of the TSC Complex

The R2TP chaperone is composed of the RUVBL1/RUVBL2 AAA+ ATPases and two adapter proteins, RPAP3 and PIH1D1. Together with HSP90, it functions in the assembly of macromolecular complexes that are often involved in cell proliferation. Here, proteomic experiments using the isolated PIH domain reveals additional R2TP partners, including the Tuberous Sclerosis Complex (TSC) and many transcriptional complexes. The TSC is a key regulator of mTORC1 and is composed of TSC1, TSC2 and TBC1D7. We show a direct interaction of TSC1 with the PIH phospho-binding domain of PIH1D1, which is, surprisingly, phosphorylation independent. Via the use of mutants and KO cell lines, we observe that TSC2 makes independent interactions with HSP90 and the TPR domains of RPAP3. Moreover, inactivation of PIH1D1 or the RUVBL1/2 ATPase activity inhibits the association of TSC1 with TSC2. Taken together, these data suggest a model in which the R2TP recruits TSC1 via PIH1D1 and TSC2 via RPAP3 and HSP90, and use the chaperone-like activities of RUVBL1/2 to stimulate their assembly.

MiRNA29a-3p negatively regulates ISL1–Integrin β1 axis to suppress gastric cancer progression

Insulin gene enhancer protein 1 (ISL1) belongs to the LIM homeodomain transcription factor family, which is closely related to the development of several cancers. We previously found that abnormally high ISL1 expression is involved in gastric cancer (GC) metastasis. However, the specific role of ISL1 and its regulatory mechanisms in GC metastasis warrant elucidation. In this study, we found that ISL1 is highly expressed in GC tissues and positively correlated with GC development, promoting cell migration and invasion in vivo and in vitro. Moreover, miRNA29a-3p can target ISL1 and thus inhibit GC cell migration. Furthermore, ISL1 upregulates ITGB1 by binding to its enhancer; nevertheless, ISL1–ITGB1 axis expression can be regulated using miRNA29a-3p. In GC cell nuclei, ISL1 and annexin A2 (ANXA2) form a transcriptional activator complex at the ITGB1 enhancer, thus promoting ITGB1 expression. In GC cell cytoplasm, the ISL1–ANXA2 complex synergistically activates matrix metalloproteinases, thus promoting cell migration. In conclusion, ISL1 is a potential therapeutic target for GC.

Distinct deregulation trends of transcriptional protein complexes in aging naive T cells.

The impact of aging on T cell subsets, specifically CD4+ and CD8+ T cells, leading to immune system dysfunction, has been the focus of scientific investigation due to its potential to reverse age-associated deterioration. Transcriptomic and epigenomic studies have identified the primary regulators in T cell aging. However, comprehending the underlying dynamic mechanisms requires studying these proteins with their interactors. Here, we integrated single-cell RNA sequencing data of naive CD4+ and CD8+ T cells obtained from three different age groups with protein-protein and domain-domain interaction networks to predict and compare the transcriptional protein complexes and identify their capacity to explain age-associated variances. Our novel approach revealed significant effects of aging on the repertoire of complexes, which remains unchanged in naive CD4+ T cells, while in naive CD8+ T cells, it diminishes. In both cell types, there was major deregulation of complexes with the same composition, involving a range of transcription factors. This aging-associated deregulation is characterized by a specific set of protein complexes in naive CD4+ T cells, but this pattern is not observed in naive CD8+ T cells. SMAD3 and BCL11A complexes emerge as key markers in defining a trajectory in aging naive CD4+ T cells. These complexes can accurately distinguish between three different age groups, indicating their potential as targets. The direct link between SMAD3 and FOS complexes whose regulatory role has been previously implicated in aging and MBD3 as the novel key link between SMAD3 and BCL11A complexes implicate a coordinated mechanism in age-associated deregulation.

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On the Cover: lncRNAs mechanisms of action: As a scaffold aids in the assembly of RNA protein complexes, thereby facilitating or inhibiting transcription. (B) As a signal molecule recruits chromatin modification enzymes such as deacetylases, acetylases, and histone methylases in response to varying stimuli to suppress locus genes. (C) LncRNAs sponge miRNAs to repress miRNA‐mediated gene regulation. (D) As a guide from transcription complexes to control the expression of downstream genes. (E) Decoy lncRNAs prevent the binding of regulatory factors to the genome and terminate its regulation. (F) Upon binding to RBPs, and changing their cellular localization which in turn alters the expression of RBPs target genes by Ebrahimnezhad et al.; Article first published: 1 August 2024; https://doi.org/10.1002/iub.2888.image

Tumor suppressor NME1/NM23-H1 modulates DNA binding of NF-κB RelA.

The dimeric NF-κB family of transcription factors activates transcription by binding sequence-specifically to DNA response elements known as κB sites, located within the promoters and enhancers of their target genes. While most NF-κB remain inactive in the cytoplasm of unstimulated cells, a small amount of RelA, one of its members, persists in the nucleus, ensuring low-level expression of genes essential for homeostasis. Several cofactors have been identified that aid in DNA binding of RelA. In this study, we identify NME1 (nucleoside diphosphate kinase 1) as a cofactor that enhances RelA's ability to bind κB sites within the promoters of a subset of its target genes, promoting their expression under both unstimulated and stimulated conditions. Depletion of NME1 influences activation or repression of several genes that are unresponsive to TNFα, despite containing κB sites in their promoters but not in clusters. This suggests that clustering of kB sites may be necessary for RelA-dependent transcription complex assembly. NME1 appears to act as a cofactor for other transcription factors to regulate these genes. NME1 does not directly contact κB DNA but interacts with RelA, with this interaction being further strengthened in the presence of κB DNA. Notably, NME1 alone has a marginal effect in enhancing RelA's DNA binding, suggesting that NME1 likely cooperate with other cofactors to regulate DNA binding and transcription through RelA. These observations underscore the intricate assembly of transcription complexes centered on NF-κB.

Characterization and phylogenetic analysis of vomeronasal receptors in the female muskrat (Ondatra zibethicus)

Vomeronasal receptors (VRs) play a crucial role in recognizing pheromones, which are essential for social chemical communication. The male muskrat (Ondatra zibethicus) secretes musk, which contains pheromones as a reproductive signal, and the female can recognize it through the VNO to mediate social communication behavior. This study aimed to identify the genomic information of VRs (OzVRs) in the female muskrat and elucidate their physicochemical properties and evolutionary relationship. Six predominantly expressed OzVR genes were identified using the RACE technique, and a comprehensive analysis was conducted on their gene structure, subcellular distribution, functional predictions, and mRNA levels, revealed that all OzVRs were transmembrane proteins. Phylogenetic analysis clustered OzVR genes into two clades (V1Rs: OzV1R21, OzV1R81, OzV1R105; V2Rs: OzV2R33, OzV2R44, OzV2R60). Physiochemically, OzV1Rs were basic proteins, while OzV2Rs exhibited weakly acidic character. Among them, OzV1R81 and OzV2R44 were identified as hydrophobicitystable proteins, with the remainder categorized as hydrophobicity-unstable proteins. Promoters analysis revealed the involvement of transcription factors and complexes, including Ahr::Arnt, Runx1, Arnt, and TFAP2A, in regulating the expression of the OzVR genes. Conserved domain and motif analyses demonstrated a high conservation of the VRs superfamily in rodents, with many conserved domains linked to pheromone binding. Functional predictions confirmed that OzVRs were associated with pheromones detection. Finally, the expression patterns of OzVR genes in different tissues and seasons indicated that OzVRs have the highest level of expression in the vomeronasal organ, and OzV1Rs notably higher in the breeding season than that in the non-breeding season, however the expression levels of OzV2Rs were higher in the non-breeding season. This study provided insights into the phylogenetic relationships, gene structure, physicochemical properties, promoter binding sites, functions and gene expression patterns of OzVRs, offering a theoretical reference for further examination of VR gene functions and a foundation for understanding chemical signaling mechanisms in the muskrat.

Simulations reveal unique roles for the FXR hinge in the FXR-RXR nuclear receptor heterodimer.

Nuclear receptors are multidomain transcription factors whose full-length quaternary architecture is poorly described and understood. Most nuclear receptors bind DNA as heterodimers or homodimers, which could encompass a variety of arrangements of the individual domains. Only a handful of experimental structures currently exist describing these architectures. Given that domain interactions and protein-DNA interactions within transcriptional complexes are tightly linked to function, understanding the arrangement of nuclear receptor domains on DNA is of utmost importance. Here, we employ modeling and molecular dynamics (MD) simulations to describe the structure of the full-length farnesoid X receptor (FXR) and retinoid X receptor alpha (RXR) heterodimer bound to DNA. Combining over 100 microseconds of atomistic MD simulations with enhanced sampling simulations, we characterize the dynamic behavior of eight FXR-RXR-DNA complexes, showing that these complexes support a range of quaternary architectures. Our simulations reveal critical roles for the hinge in the DNA-bound dimer in facilitating interdomain allostery, mediating DNA binding and driving the dynamic flexibility of the complex. These roles have been hard to appreciate previously due to experimental limitations in studying the flexible hinge. These studies provide a much-needed framework that will enable the field to obtain a complete understanding of nuclear receptor quaternary architectures.

Cellular NS1-BP protein interacts with the mRNA export receptor NXF1 to mediate nuclear export of influenza virus M mRNAs

Influenza A viruses have eight genomic RNAs that are transcribed in the host cell nucleus. Two of the viral mRNAs undergo alternative splicing. The M1 mRNA encodes the matrix protein 1 (M1) and is also spliced into M2 mRNA, which encodes the proton channel matrix protein 2 (M2). Our previous studies have shown that the cellular NS1-binding protein (NS1-BP) interacts with the viral non-structural protein 1 (NS1) and M1 mRNA to promote M1 to M2 splicing. Another pool of NS1 protein binds the mRNA export receptor NXF1 (nuclear RNA export factor-1), leading to nuclear retention of cellular mRNAs. Here we show a series of biochemical and cell biological findings that suggest a model for nuclear export of M1 and M2 mRNAs despite the mRNA nuclear export inhibition imposed by the viral NS1 protein. NS1-BP competes with NS1 for NXF1 binding, allowing the recruitment of NXF1 to the M mRNAs after splicing. NXF1 then binds GANP (Germinal-center Associated Nuclear Protein), a member of the TRanscription and EXport complex (TREX)-2. Although both NS1 and NS1-BP remain in complex with GANP-NXF1, they dissociate once this complex docks at the nuclear pore complex (NPC), and the M mRNAs are translocated to the cytoplasm. Since this mRNA nuclear export pathway is key for expression of M1 and M2 proteins that function in viral intracellular trafficking and budding, these viral-host interactions are critical for influenza virus replication.