Molecular functions of nuclear actin in transcription

Actin is not only a major cytoskeletal component in all eukaryotic cells but also a nuclear protein that plays a role in gene transcription. We put together data from in vitro and in vivo experiments that begin to provide insights into the molecular mechanisms by which actin functions in transcription. Recent studies performed in vitro have suggested that actin, in direct contact with the transcription apparatus, is required in an early step of transcription that is common to all three eukaryotic RNA polymerases. In addition, there is evidence from in vivo studies that actin is involved in the transcription elongation of class II genes. In this case, actin is bound to a specific subset of premessenger RNA binding proteins, and the actin–messenger RNP complex may constitute a molecular platform for recruitment of histone-modifying enzymes. We discuss a general model for actin in RNA polymerase II transcription whereby actin works as a conformational switch in conjunction with specific adaptors to facilitate the remodeling of large macromolecular assemblies at the promoter and along the active gene

Sorting mRNA Molecules for Cytoplasmic Transport and Localization

In eukaryotic cells, gene expression is highly regulated at many layers. Nascent RNA molecules are assembled into ribonucleoprotein complexes that are then released into the nucleoplasmic milieu and transferred to the nuclear pore complex for nuclear export. RNAs are then either translated or transported to the cellular periphery. Emerging evidence indicates that RNA-binding proteins play an essential role throughout RNA biogenesis, from the gene to polyribosomes. However, the sorting mechanisms that regulate whether an RNA molecule is immediately translated or sent to specialized locations for translation are unclear. This question is highly relevant during development and differentiation when cells acquire a specific identity. Here, we focus on the RNA-binding properties of heterogeneous nuclear ribonucleoproteins (hnRNPs) and how these mechanisms are believed to play an essential role in RNA trafficking in polarized cells. Further, by focusing on the specific hnRNP protein CBF-A/hnRNPab and its naturally occurring isoforms, we propose a model on how hnRNP proteins are capable of regulating gene expression both spatially and temporally throughout the RNA biogenesis pathway, impacting both healthy and diseased cells

Analysis of Global Transcriptome Change in Mouse Embryonic Fibroblasts After dsDNA and dsRNA Viral Mimic Stimulation

The activation of innate immunity by viral nucleic acids present in the cytoplasm plays an essential role in controlling viral infection in both immune and non-immune cells. The dsDNA and dsRNA viral mimics can stimulate the cytosolic nucleic acids sensors and activate the antiviral innate immunity. In this study, taking advantage of dsDNA and dsRNA viral mimics, we investigated the global transcriptome changes after the antiviral immunity activation in mouse embryonic fibroblasts. Results from our data identified a positive feedback up-regulation of sensors (e.g., Tlr2, Tlr3, Ddx58, cGAS), transducers (e.g., Traf2, Tbk1) and transcription factors (e.g., Irf7, Jun, Stat1, Stat2) in multiple pathways involved in detecting viral or microbial infections upon viral mimic stimulation. A group of genes involved in DNA damage response and DNA repair such as Parp9, Dtx3l, Rad52 were also up-regulated, implying the involvement of these genes in antiviral immunity. Molecular function analysis further showed that groups of helicase genes (e.g., Dhx58, Helz2), nuclease genes (e.g., Dnase1l3, Rsph10b), methyltransferase genes (e.g., histone methyltransferase Prdm9, Setdb2; RNA methyltransferase Mettl3, Mttl14), and protein ubiquitin-ligase genes (e.g., Trim genes and Rnf genes) were up-regulated upon antiviral immunity activation. In contrast, viral mimic stimulation down-regulated genes involved in a broad range of general biological processes (e.g., cell division, metabolism), cellular components (e.g., mitochondria and ribosome), and molecular functions (e.g., cell-cell adhesion, microtubule binding). In summary, our study provides valuable information about the global transcriptome changes upon antiviral immunity activation. The identification of novel groups of genes up-regulated upon antiviral immunity activation serves as useful resource for mining new antiviral sensors and effectors

Central fatigue and nycthemeral change of serum tryptophan and serotonin in the athletic horse

BACKGROUND: The serotonergic system is associated with numerous brain functions, including the resetting of the mammalian circadian clock. The synthesis and metabolism of 5-HT in the brain increases in response to exercise and is correlated with high levels of blood-borne tryptophan (TRP). The present investigation was aimed at testing the existence of a daily rhythm of TRP and 5-HT in the blood of athletic horses. METHODS: Blood samples from 5 Thoroughbred mares were collected at 4-hour intervals for 48 hours (starting at 08:00 hours on day 1 and finishing at 4:00 on day 2) via an intravenous cannula inserted into the jugular vein. Tryptophan and serotonin concentrations were assessed by HPLC. Data analysis was conducted by one-way repeated measures analysis of variance (ANOVA) and by the single cosinor method. RESULTS: ANOVA showed a highly significant influence of time both on tryptophan and on serotonin, in all horses, on either day, with p values < 0.0001. Cosinor analysis identified the periodic parameters and their acrophases (expressed in hours) during the 2 days of monitoring. Both parameters studied showed evening acrophases. CONCLUSION: The results showed that serotonin and tryptophan blood levels undergo nycthemeral variation with typical evening acrophases. These results enhance the understanding of the athlete horse's chronoperformance and facilitate the establishment of training programs that take into account the nycthemeral pattern of aminoacids deeply involved in the onset of central fatigue

Toxoplasma gondii in raw milk from Sicily

Toxoplasma gondii tachyzoites shed in milk of infected animals are a potential source of human infection often underestimated. Furthermore, limited risk assessment studies have been carried out to evaluate the transmission of toxoplasmosis by milk consumption. In this frame the availability of few and often not validated analytical methods can be a major shortcoming when investigating the extent of milk contamination and the parasite viability. To address the issue, 1381 samples of raw milk from unknown sero-status cows, donkeys, sheep and goats were examined for the detection of T. gondii DNA by a real time PCR method targeting the 529bp repeating element. The protozoan DNA was detected in 67 samples of cow’s milk (4,93% and 35% individual and herd prevalence respectively) and in one sample of donkey milk. Allegedly, on farm cats cohabitation as well as extensive management in the cattle farms that tested positive, can be accounted for environmental loading and transmission of T. gondii oocysts that are the unique sources of infection for milk-producing animals. Food safety-wise, results are noteworthy since recently raw cow’s milk consumption has become increasingly popular with the spread of automatic raw milk vending machines. Albeit heating treatment before consumption is mandatory for consumers, neglect of this requirement cannot be ruled out and could lead the way for foodborne infection. This is particularly true in view of evidences that T. gondii tachyzoites in experimentally spiked cow’s milk samples could be able to survive in gastric fluids for long enough (1 h) before reaching the intestine and infect the host

Results of five years monitoring for Toxoplasma gondii infection in animals by the official Italian Zoonoses Informative System (SINZOO)

In order to drive effective public health preventive measures for human toxoplasmosis a scrupulous epidemiological monitoring of animal toxoplasmosis is essential. BACKGROUND AND AIM:T. gondii is the fourth most important parasite in the world and second out of 14 foodborne pathogens in the USA and in Europe. Meat-borne transmission of T. gondii causes most of food-borne infections in Europe (EFSA-European Food Safety Authority). SINZOO is part of the Veterinary Informative System of the Italian Ministry of Health. It collects and transmit data to EFSA, published in the annual EFSA/ECDC summary reports on zoonoses. The aim of this study was to evaluate the effectiveness of SINZOO for epidemiological surveillance of toxoplasmosis in Italy. METHODS:Among animal species tested in Italy between 2015 and 2019 the ones most commonly reared for human consumption (sheep, cattle, pig, goats) were selected, moreover wild boars, wild ruminants, cats. RESULTS:Infection rates ranged from 0.73% in wild boars to 45.72% in sheep. Total number of tested animals ranged from 37 pigs in 2015 to 3449 sheep in 2018. Besides a relevant incidence among wild boars in 2018 (45%) and 2019 (32%), higher infection rates were more often reported among sheep and pigs. Between 2018 and 2019 67% of the overall analyses were carried out in one region (Sardinia), mostly on one species (sheep) and emerged from targeted research or clinical investigation. In fact in 2019 83.45% of analyses were performed following clinical suspicions while only 8.43% came from official controls, highlighting toxoplasmosis underestimation by the national veterinary health system. CONCLUSIONS:Despite EFSA recognizes the relevance of toxoplasmosis, this is not included among zoonoses under mandatory notification, making animal epidemiological surveillance rather scarce and uneven. Data reported to SINZOO suggest that T. gondii is still a relevant hazard to monitor by meat inspection and in-farm survey, for effective epidemiological evaluations and appropriate public health interventions. This issue characterizes Italy and Europe, highlighting that toxoplasmosis monitoring should be made mandatory and with uniform rules

Toxoplasma gondii in animals and food: results of four-years monitoring by the official italian zoonoses informative system.

When considering human toxoplasmosis, food remains the main source of infection. Whether is meat from susceptible animals that naturally harbour Toxoplasma gondii tissue cysts or food like fresh vegetables or fishery products (mussels, fish) contaminated by environmentally derived oocysts, information about animal toxoplasmosis prevalence should continue to be under the spotlight. Directive 2003/99/EC of the European Parliament and the Council classifies toxoplasmosis and its causative agent as a class B zoonosis/agent for which monitoring depends on the epidemiological situation. Surveillance systems on animal toxoplasmosis are different across Europe and regulated by national legislation. In Italy, administrative regions are called annually to submit data on animal toxoplasmosis to SINZOO (Zoonoses Informative System) that is part of the Veterinary Informative System managed by the Italian Ministry of Health. Foodborne zoonoses data collected at the national level are then processed and summarized by EFSA to became part of the annual EFSA/ECDC joint European Union summary reports on trends and sources of zoonoses, zoonotic agents and foodborne outbreaks. No foodborne outbreaks of toxoplasmosis have been reported in Italy during 2017, the most recent year for which a report is available. Unfortunately, prevalence data are often the results of fragmented researches or clinical investigations but, nevertheless, they provide baseline data to monitor epidemiological trends for guiding public health preventive measures. Over the past four years (2014-2017), in Italy, sheep, cattle, pig, goats, wild boars and wild ruminants were the main species tested besides pet animals. Prevalence of the infection ranged: 3.49-11.10% in cattle (209 to 2289 animals tested); 11.46-34.55% in pigs (37 to 2051 animals tested); 18.01-45.72% in sheep (912 to 2682 animals tested); 5.98-23.25% in goats (234 to 432 animals tested. Positive among wildlife ranged from 0 to 10.24% for wild boars (26 to 901 animals tested) and from 0.76 to 11.25% for wild ruminants (240 to 394 animals tested). For cats, that are solely responsible for contamination with environmentally-resistant oocysts, the prevalence ranged from 19.48 to 34.77% (77 to 696 animals tested). Although these data cannot support epidemiological analyses, they suggest that pigs and small ruminants are still the major sources of meatborne toxoplasmosis. Cattle are also under a special watch list since beef consumption has been predicted as the most important source of meatborne toxoplasmosis at least in Italy and the Netherlands as resulting from quantitative risk assessment studies. Cattle are often found positive to serological screening although direct detection of T. gondii via biological assay or PCR is uncommon with lack of concordance between antibodies and parasite DNA detection. The informative system also reports very limited information on food such as fresh vegetables or fishery products that can be contaminated by T. gondii oocysts dispersed in the environment although 1 sample of mussels and 3 samples of fish resulted positive by PCR in 2017. These findings suggest that surveillance on animal toxoplasmosis and meat and other food contamination with T. gondii should be enhanced since it is one of the few effective tools to control the parasite diffusion and prevent the transmission of the zoonosis

Glycogen Synthase Kinase (GSK) 3β phosphorylates and protects nuclear myosin 1c from proteasome-mediated degradation to activate rDNA transcription in early G1 cells

Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3β phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3β selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3β-/- mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3β directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3β-mediated phosphorylation of NM1 is required for pol I transcription activation

Actin: its cumbersome pilgrimage through cellular compartments

In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days’ knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin

Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus.

Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes