Ewing Sarcoma Related protein 1 recognizes R-loops by binding DNA forks.

EWSR1 (Ewing Sarcoma Related protein 1) is an RNA binding protein that is ubiquitously expressed across cell lines and involved in multiple parts of RNA processing, such as transcription, splicing, and mRNA transport. EWSR1 has also been implicated in cellular mechanisms to control formation of R-loops, a three-stranded nucleic acid structure consisting of a DNA:RNA hybrid and a displaced single-stranded DNA strand. Unscheduled R-loops result in genomic and transcription stress. Loss of function of EWSR1 functions commonly found in Ewing Sarcoma correlates with high abundance of R-loops. In this study, we investigated the mechanism for EWSR1 to recognize an R-loop structure specifically. Using electrophoretic mobility shift assays (EMSA), we detected the high affinity binding of EWSR1 to substrates representing components found in R-loops. EWSR1 specificity could be isolated to the DNA fork region, which transitions between double- and single-stranded DNA. Our data suggests that the Zinc-finger domain (ZnF) with flanking arginine and glycine rich (RGG) domains provide high affinity binding, while the RNA recognition motif (RRM) with its RGG domains offer improved specificity. This model offers a rational for EWSR1 specificity to encompass a wide range in contexts due to the DNA forks always found with R-loops.

Ewing Sarcoma Related protein 1 recognizes R-loops by binding DNA forks.

EWSR1 (Ewing Sarcoma Related protein 1) is an RNA binding protein that is ubiquitously expressed across cell lines and involved in multiple parts of RNA processing, such as transcription, splicing, and mRNA transport. EWSR1 has also been implicated in cellular mechanisms to control formation of R-loops, a three-stranded nucleic acid structure consisting of a DNA:RNA hybrid and a displaced single-stranded DNA strand. Unscheduled R-loops result in genomic and transcription stress. Loss of function of EWSR1 functions commonly found in Ewing Sarcoma correlates with high abundance of R-loops. In this study, we investigated the mechanism for EWSR1 to recognize an R-loop structure specifically. Using electrophoretic mobility shift assays (EMSA), we detected the high affinity binding of EWSR1 to substrates representing components found in R-loops. EWSR1 specificity could be isolated to the DNA fork region, which transitions between double- and single-stranded DNA. Our data suggests that the Zinc-finger domain (ZnF) with flanking arginine and glycine rich (RGG) domains provide high affinity binding, while the RNA recognition motif (RRM) with its RGG domains offer improved specificity. This model offers a rational for EWSR1 specificity to encompass a wide range in contexts due to the DNA forks always found with R-loops.

Structure of phosphorylated-like RssB, the adaptor delivering σs to the ClpXP proteolytic machinery, reveals an interface switch for activation.

In enterobacteria such as Escherichia coli, the general stress response is mediated by σs, the stationary phase dissociable promoter specificity subunit of RNA polymerase. σs is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which is thought to be stimulated by the phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of full-length RssB bound to a beryllofluoride phosphomimic. Compared to the structure of RssB bound to the IraD anti-adaptor, our new RssB structure with bound beryllofluoride reveals conformational differences and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the interdomain segmented helical linker. These are accompanied by masking of the α4-ß5-α5 (4-5-5) "signaling" face of the RssB receiver domain by its C-terminal domain. Critically, using hydrogen-deuterium exchange mass spectrometry, we identify σs-binding determinants on the 4-5-5 face, implying that this surface needs to be unmasked to effect an interdomain interface switch and enable full σs engagement and hand-off to ClpXP. In activated receiver domains, the 4-5-5 face is often the locus of intermolecular interactions, but its masking by intramolecular contacts upon phosphorylation is unusual, emphasizing that RssB is a response regulator that undergoes atypical regulation.

Folate and Choline: Does It Take Two to Tango in Early Programming of Disease?

BACKGROUND: The early life period marks a critical time during which the health trajectory of offspring can be shaped by external influences including maternal nutrition. Folate and choline are water-soluble micronutrients important for fetal development and involved in one-carbon metabolism. Intakes above and below the recommendations commonly occur for both of these nutrients including over-consumption of synthetic folic acid due to widespread vitamin supplement uses and discretionary fortification practices, whereas choline is under-consumed by a majority of the populations including pregnant women. Despite these intake patterns, their long-term impact on offspring health is largely unknown. Moreover, limited attention has been on the combined effects of folate and choline despite being metabolically interrelated as methyl nutrients. This review summarizes evidence from animal models and human studies investigating the role of inadequate or supplemental maternal intakes of folic acid, choline and combined effects of folic acid, and choline as modulators of health and disease in offspring. With the recent rise in the prevalence of obesity and metabolic diseases, our primary measures of interest were metabolic outcomes. SUMMARY: Studies examining the role of maternal intakes of folic acid and/or choline in metabolic phenotypes of offspring have mostly been conducted in animal models with a limited number of reports that consider folate and choline together. An interdependent relationship has been demonstrated between folate and choline in studies where a deficiency in one leads to metabolic aberrations in another. Both deficient and excess maternal intakes of folic acid (in varying doses) have been shown to increase risk of obesity and characteristics of the metabolic syndrome in offspring but these findings were restricted to animal studies. Potential metabolic benefits of choline have been suggested in the presence of obesogenic environment but human data were sparse. An imbalanced intake of high folic acid and inadequate choline in the gestational diet created adverse consequences consistent with the obesogenic phenotypes whereas narrowing this imbalance with high choline blocked these effects. Mechanisms by which maternal folate and/or choline influence offspring outcomes may involve epigenetic modification of gene expression with DNA methylation that can be altered globally and gene-specifically. However, the effects of epigenetic programming were inconsistent as compensatory changes in metabolic products may occur and other contributors including the gut microbiota may provide additional insights into the mechanisms. KEY MESSAGES: Maternal intakes of folic acid and/or choline can impact offspring's long-term health, with metabolic consequences that may arise from imbalances between folate and choline. However, there is a paucity of mechanistic understanding as various contributors influence programming effects including those beyond epigenetics. As folate and choline are metabolically interrelated, future studies need to consider both nutrients to better elucidate metabolic programming of health and disease.

Binding of the nuclear ribonucleoprotein family member FUS to RNA prevents R-loop RNA:DNA hybrid structures.

The protein FUS (FUSed in sarcoma) is a metazoan RNA-binding protein that influences RNA production by all three nuclear polymerases. FUS also binds nascent transcripts, RNA processing factors, RNA polymerases, and transcription machinery. Here, we explored the role of FUS binding interactions for activity during transcription. In vitro run-off transcription assays revealed FUS-enhanced RNA produced by a non-eukaryote polymerase. The activity also reduced the formation of R-loops between RNA products and their DNA template. Analysis by domain mutation and deletion indicated RNA-binding was required for activity. We interpret that FUS binds and sequesters nascent transcripts to prevent R-loops from forming with nearby DNA. DRIP-seq analysis showed that a knockdown of FUS increased R-loop enrichment near expressed genes. Prevention of R-loops by FUS binding to nascent transcripts has the potential to affect transcription by any RNA polymerase, highlighting the broad impact FUS can have on RNA metabolism in cells and disease.

Modification of the serotonergic systems and phenotypes by gestational micronutrients.

Micronutrients consumed in excess or imbalanced amounts during pregnancy may increase the risk of metabolic diseases in offspring, but the mechanisms underlying these effects are unknown. Serotonin (5-hydroxytryptamine, 5-HT), a multifunctional indoleamine in the brain and the gut, may have key roles in regulating metabolism. We investigated the effects of gestational micronutrient intakes on the central and peripheral serotonergic systems as modulators of the offspring's metabolic phenotypes. Pregnant Wistar rats were fed an AIN-93G diet with 1-fold recommended vitamins (RV), high 10-fold multivitamins (HV), high 10-fold folic acid with recommended choline (HFolRC), or high 10-fold folic acid with no choline (HFolNC). Male and female offspring were weaned to a high-fat RV diet for 12 weeks. We assessed the central function using the 5-HT2C receptor agonist, 1-(3-chlorophenyl)piperazine (mCPP), and found that male offspring from the HV- or HFolRC-fed dams were less responsive (P < 0.05) whereas female HFolRC offspring were more responsive to mCPP (P < 0.01) at 6 weeks post-weaning. Male and female offspring from the HV and HFolNC groups, and male HFolRC offspring had greater food intake (males P < 0.001; females P < 0.001) and weight gain (males P < 0.0001; females P < 0.0001), elevated colon 5-HT (males P < 0.01; females P < 0.001) and fasting glucose concentrations (males P < 0.01; females P < 0.01), as well as body composition toward obesity (males P < 0.01; females P < 0.01) at 12 weeks post-weaning. Colon 5-HT was correlated with fasting glucose concentrations (males R2=0.78, P < 0.0001; females R2=0.71, P < 0.0001). Overall, the serotonergic systems are sensitive to the composition of gestational micronutrients, with alterations consistent with metabolic disturbances in offspring.

PEPPI: Whole-proteome Protein-protein Interaction Prediction through Structure and Sequence Similarity, Functional Association, and Machine Learning.

Proteome-wide identification of protein-protein interactions is a formidable task which has yet to be sufficiently addressed by experimental methodologies. Many computational methods have been developed to predict proteome-wide interaction networks, but few leverage both the sensitivity of structural information and the wide availability of sequence data. We present PEPPI, a pipeline which integrates structural similarity, sequence similarity, functional association data, and machine learning-based classification through a naïve Bayesian classifier model to accurately predict protein-protein interactions at a proteomic scale. Through benchmarking against a set of 798 ground truth interactions and an equal number of non-interactions, we have found that PEPPI attains 4.5% higher AUROC than the best of other state-of-the-art methods. As a proteomic-scale application, PEPPI was applied to model the interactions which occur between SARS-CoV-2 and human host cells during coronavirus infection, where 403 high-confidence interactions were identified with predictions covering 73% of a gold standard dataset from PSICQUIC and demonstrating significant complementarity with the most recent high-throughput experiments. PEPPI is available both as a webserver and in a standalone version and should be a powerful and generally applicable tool for computational screening of protein-protein interactions.

Traumatic injury compromises nucleocytoplasmic transport and leads to TDP-43 pathology.

Traumatic brain injury (TBI) is a predisposing factor for many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and chronic traumatic encephalopathy (CTE). Although defects in nucleocytoplasmic transport (NCT) is reported ALS and other neurodegenerative diseases, whether defects in NCT occur in TBI remains unknown. We performed proteomic analysis on Drosophila exposed to repeated TBI and identified resultant alterations in several novel molecular pathways. TBI upregulated nuclear pore complex (NPC) and nucleocytoplasmic transport (NCT) proteins as well as alter nucleoporin stability. Traumatic injury disrupted RanGAP1 and NPC protein distribution in flies and a rat model and led to coaggregation of NPC components and TDP-43. In addition, trauma-mediated NCT defects and lethality are rescued by nuclear export inhibitors. Importantly, genetic upregulation of nucleoporins in vivo and in vitro triggered TDP-43 cytoplasmic mislocalization, aggregation, and altered solubility and reduced motor function and lifespan of animals. We also found NUP62 pathology and elevated NUP62 concentrations in postmortem brain tissues of patients with mild or severe CTE as well as co-localization of NUP62 and TDP-43 in CTE. These findings indicate that TBI leads to NCT defects, which potentially mediate the TDP-43 pathology in CTE.

Fusion protein EWS-FLI1 is incorporated into a protein granule in cells.

Ewing sarcoma is driven by fusion proteins containing a low complexity (LC) domain that is intrinsically disordered and a powerful transcriptional regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing Sarcoma RNA-binding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend Leukemia Virus Integration 1). EWS-FLI1 can bind RNA polymerase II (RNA Pol II) and self-assemble through its low-complexity (LC) domain. The ability of RNA-binding proteins like EWSR1 to self-assemble or phase separate in cells has raised questions about the contribution of this process to EWS-FLI1 activity. We examined EWSR1 and EWS-FLI1 activity in Ewing sarcoma cells by siRNA-mediated knockdown and RNA-seq analysis. More transcripts were affected by the EWSR1 knockdown than expected and these included many EWS-FLI1 regulated genes. We reevaluated physical interactions between EWS-FLI1, EWSR1, and RNA Pol II, and employed a cross-linking based strategy to investigate protein assemblies associated with the proteins. The LC domain of EWS-FLI1 was required for the assemblies observed to form in cells. These results offer new insights into a protein assembly that may enable EWS-FLI1 to bind its wide network of protein partners and contribute to regulation of gene expression in Ewing sarcoma.

Isolating and Analyzing Protein Containing Granules from Cells.

Recent advancements in detection methods have made protein condensates, also called granules, a major area of study, but tools to characterize these assemblies need continued development to keep up with evolving paradigms. We have optimized a protocol to separate condensates from cells using chemical cross-linking followed by size-exclusion chromatography (SEC). After SEC fractionation, the samples can be characterized by a variety of approaches including enzyme-linked immunosorbent assay, dynamic light scattering, electron microscopy, and mass spectrometry. The protocol described here has been optimized for cultured mammalian cells and E. coli expressing recombinant proteins. Since the lysates are fractionated by size, this protocol can be modified to study other large protein assemblies, including the nuclear pore complex, and for other tissues or organisms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: SEC separation of cross-linked mammalian cell lysates Alternate Protocol: Preparation of non-crosslinked mammalian cells Basic Protocol 2: SEC separation of E. coli lysate Support Protocol 1: Detecting protein of interest by ELISA Support Protocol 2: TCA precipitation of SEC fractions.

Phospho-dependent signaling during the general stress response by the atypical response regulator and ClpXP adaptor RssB.

In the model organism Escherichia coli and related species, the general stress response relies on tight regulation of the intracellular levels of the promoter specificity subunit RpoS. RpoS turnover is exclusively dependent on RssB, a two-domain response regulator that functions as an adaptor that delivers RpoS to ClpXP for proteolysis. Here, we report crystal structures of the receiver domain of RssB both in its unphosphorylated form and bound to the phosphomimic BeF3- . Surprisingly, we find only modest differences between these two structures, suggesting that truncating RssB may partially activate the receiver domain to a "meta-active" state. Our structural and sequence analysis points to RssB proteins not conforming to either the Y-T coupling scheme for signaling seen in prototypical response regulators, such as CheY, or to the signaling model of the less understood FATGUY proteins.

Effect of a brief scenario-tailored educational program on parents' risk knowledge, perceptions, and decisions to administer prescribed opioids: a randomized controlled trial.

ABSTRACT: This randomized, controlled trial evaluated whether a brief educational program (ie, Scenario-Tailored Opioid Messaging Program [STOMP]) would improve parental opioid risk knowledge, perceptions, and analgesic efficacy; ensure safe opioid use decisions; and impact prescription opioid use after surgery. Parent-child dyads (n = 604) who were prescribed an opioid for short-term use were randomized to routine instruction (Control) or routine plus STOMP administered preoperatively. Baseline and follow-up surveys assessed parents' awareness and perceived seriousness of adverse opioid effects, and their analgesic efficacy. Parents' decisions to give an opioid in hypothetical scenarios and total opioid doses they gave to children at home were assessed at follow-up. Scenario-Tailored Opioid Messaging Program parents gained enhanced perceptions of opioid-related risks over time, whereas Controls did not; however, risk perceptions did not differ between groups except for addiction risk. Scenario-Tailored Opioid Messaging Program parents exhibited marginally greater self-efficacy compared to Controls (mean difference vs controls = 0.58 [95% confidence interval 0.08-1.09], P = 0.023). Scenario-Tailored Opioid Messaging Program parents had a 53% lower odds of giving an opioid in an excessive sedation scenario (odds ratio 0.47 [95% confidence interval 0.28-0.78], P = 0.003), but otherwise made similar scenario-based opioid decisions. Scenario-Tailored Opioid Messaging Program was not associated with total opioid doses administered at home. Instead, parents' analgesic efficacy and pain-relief preferences explained 7%, whereas child and surgical factors explained 22% of the variance in opioid doses. Scenario-tailored education enhanced parents' opioid risk knowledge, perceptions, and scenario-based decision-making. Although this may inform later situation-specific decision-making, our research did not demonstrate an impact on total opioid dosing, which was primarily driven by surgical and child-related factors.

Discriminating changes in protein structure using tyrosine conjugation.

Chemical modification of proteins has been crucial in engineering protein-based therapies, targeted biopharmaceutics, molecular probes, and biomaterials. Here, we explore the use of a conjugation-based approach to sense alternative conformational states in proteins. Tyrosine has both hydrophobic and hydrophilic qualities, thus allowing it to be positioned at protein surfaces, or binding interfaces, or to be buried within a protein. Tyrosine can be conjugated with 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD). We hypothesized that individual protein conformations could be distinguished by labeling tyrosine residues in the protein with PTAD. We conjugated tyrosine residues in a well-folded protein, bovine serum albumin (BSA), and quantified labeled tyrosine with liquid chromatography with tandem mass spectrometry. We applied this approach to alternative conformations of BSA produced in the presence of urea. The amount of PTAD labeling was found to relate to the depth of each tyrosine relative to the protein surface. This study demonstrates a new use of tyrosine conjugation using PTAD as an analytic tool able to distinguish the conformational states of a protein.

Changes to the TDP-43 and FUS Interactomes Induced by DNA Damage.

The RNA-binding proteins TDP-43 and FUS are tied as the third leading known genetic cause for amyotrophic lateral sclerosis (ALS), and TDP-43 proteopathies are found in nearly all ALS patients. Both the natural function and contribution to pathology for TDP-43 remain unclear. The intersection of functions between TDP-43 and FUS can focus attention for those natural functions mostly likely to be relevant to disease. Here, we compare the role played by TDP-43 and FUS, maintaining chromatin stability for dividing HEK293T cells. We also determine and compare the interactomes of TDP-43 and FUS, quantitating changes in those before and after DNA damage. Finally, selected interactions with known importance to DNA damage repair were validated by co-immunoprecipitation assays. This study uncovered TDP-43 and FUS binding to several factors important to DNA repair mechanisms that can be replication-dependent, -independent, or both. These results provide further evidence that TDP-43 has an important role in DNA stability and provide new ways that TDP-43 can bind to the machinery that guards DNA integrity in cells.

TDP-43 regulates transcription at protein-coding genes and Alu retrotransposons.

The 43-kDa transactive response DNA-binding protein (TDP-43) is an example of an RNA-binding protein that regulates RNA metabolism at multiple levels from transcription and splicing to translation. Its role in post-transcriptional RNA processing has been a primary focus of recent research, but its role in regulating transcription has been studied for only a few human genes. We characterized the effects of TDP-43 on transcription genome-wide and found that TDP-43 broadly affects transcription of protein-coding and noncoding RNA genes. Among protein-coding genes, the effects of TDP-43 were greatest for genes <30 thousand base pairs in length. Surprisingly, we found that the loss of TDP-43 resulted in increased evidence for transcription activity near repetitive Alu elements found within expressed genes. The highest densities of affected Alu elements were found in the shorter genes, whose transcription was most affected by TDP-43. Thus, in addition to its role in post-transcriptional RNA processing, TDP-43 plays a critical role in maintaining the transcriptional stability of protein-coding genes and transposable DNA elements.

Fused in sarcoma silences HIV gene transcription and maintains viral latency through suppressing AFF4 gene activation.

BACKGROUND: The human immunodeficiency virus (HIV) cell reservoir is currently a main obstacle towards complete eradication of the virus. This infected pool is refractory to anti-viral therapy and harbors integrated proviruses that are transcriptionally repressed but replication competent. As transcription silencing is key for establishing the HIV reservoir, significant efforts have been made to understand the mechanism that regulate HIV gene transcription, and the role of the elongation machinery in promoting this step. However, while the role of the super elongation complex (SEC) in enhancing transcription activation of HIV is well established, the function of SEC in modulating viral latency is less defined and its cell partners are yet to be identified. RESULTS: In this study we identify fused in sarcoma (FUS) as a partner of AFF4 in cells. FUS inhibits the activation of HIV transcription by AFF4 and ELL2, and silences overall HIV gene transcription. Concordantly, depletion of FUS elevates the occupancy of AFF4 and Cdk9 on the viral promoter and activates HIV gene transcription. Live cell imaging demonstrates that FUS co-localizes with AFF4 within nuclear punctuated condensates, which are disrupted upon treating cells with aliphatic alcohol. In HIV infected cells, knockout of FUS delays the gradual entry of HIV into latency, and similarly promotes viral activation in a T cell latency model that is treated with JQ1. Finally, effects of FUS on HIV gene transcription are also exhibited genome wide, where FUS mainly occupies gene promoters at transcription starting sites, while its knockdown leads to an increase in AFF4 and Cdk9 occupancy on gene promoters of FUS affected genes. CONCLUSIONS: Towards eliminating the HIV infected reservoir, understanding the mechanisms by which the virus persists in the face of therapy is important. Our observations show that FUS regulates both HIV and global gene transcription and modulates viral latency, thus can potentially serve as a target for future therapy that sets to reactivate HIV from its latent state.

Sexually experienced, but not naïve, female rats show a conditioned object preference (COP) for mating after a single training trial.

Female rats with mating experience spend more time with the male rat, exhibit shorter contact-return latency to intromission, and display more proceptive behaviors in the male rat's compartment than during the first mating experience. The present study tested 1) whether mating induced conditioned object preference (COP) is possible with a single conditioning trial and 2) whether a preference is induced for an object associated with the first mating encounter or the fifth mating encounter in female rats. Ovariectomized, Long-Evans female rats were primed with estradiol benzoate + progesterone and either exposed to an empty paced mating chamber for 15 min (Naïve) or received a 15 intromission test of paced mating behavior (Experienced) on four separate occasions before undergoing the COP procedure. Experienced, but not Naïve, female rats developed a COP for a single mating bout, indicating that mating is highly rewarding for sexually experienced female rats. The findings raise questions about the effect of sexual experience on reward regions in the brain, the responsiveness of genital tissue, and learning mechanisms.