Influenza polymerase uses unique mechanisms to synthesize capped and polyadenylated mRNAs from the genomic viral RNA (vRNA) template, which is packaged inside ribonucleoprotein particles (vRNPs). ...Here, we visualize by cryoelectron microscopy the conformational dynamics of the polymerase during the complete transcription cycle from pre-initiation to termination, focusing on the template trajectory. After exiting the active site cavity, the template 3′ extremity rebinds into a specific site on the polymerase surface. Here, it remains sequestered during all subsequent transcription steps, forcing the template to loop out as it further translocates. At termination, the strained connection between the bound template 5′ end and the active site results in polyadenylation by stuttering at uridine 17. Upon product dissociation, further conformational changes release the trapped template, allowing recycling back into the pre-initiation state. Influenza polymerase thus performs transcription while tightly binding to and protecting both template ends, allowing efficient production of multiple mRNAs from a single vRNP.
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•Cryo-EM snapshots of the transcription elongation, termination, and recycling states•After being copied, the template 3′ end rebinds the polymerase in a secondary site•Mechanism of viral mRNA poly(A) tail formation by stuttering elucidated•Efficient reformation of the promoter allows multiple transcripts from one RNP
Influenza polymerase transcribes the negative sense viral RNA genome into mRNA in the nucleus of infected cells. This work by Cusack and colleagues reports high-resolution cryo-EM structures of the polymerase at various stages of transcription providing a molecular basis for the complete transcription cycle, which should enable improved inhibitor design.
Segmented negative-strand RNA bunyaviruses encode a multi-functional polymerase that performs genome replication and transcription. Here, we establish conditions for in vitro activity of La Crosse ...virus polymerase and visualize its conformational dynamics by cryo-electron microscopy, unveiling the precise molecular mechanics underlying its essential activities. We find that replication initiation is coupled to distal duplex promoter formation, endonuclease movement, prime-and-realign loop extension and closure of the polymerase core that direct the template towards the active site. Transcription initiation depends on C-terminal region closure and endonuclease movements that prompt primer cleavage prior to primer entry in the active site. Product realignment after priming, observed in replication and transcription, is triggered by the prime-and-realign loop. Switch to elongation results in polymerase reorganization and core region opening to facilitate template-product duplex formation in the active site cavity. The uncovered detailed mechanics should be helpful for the future design of antivirals counteracting bunyaviral life threatening pathogens.
Cancer-initiating cell (CIC) exosomes (CIC-TEX) are suggested reprogramming Non-CIC. Mode of message transfer and engagement of CIC-markers being disputed, we elaborated the impact of CD44v6 and ...Tspan8 on the response of Non-CIC.
Non-metastasizing CD44v6- and Tspan8-knockdown (kd) pancreatic cancer cells served as Non-CIC. CIC-TEX coculture-induced changes were evaluated by deep-sequencing and functional assays. Tumor progression was surveyed during in vivo CIC-TEX treatment.
Deep-sequencing of CIC-TEX-cocultured CD44v6kd-Non-CIC revealed pronounced mRNA changes in signaling, transport, transcription and translation; altered miRNA affected metabolism, signaling and transcription. CIC-TEX coculture-induced changes in Tspan8kd-Non-CIC mostly relied on CIC-TEX-Tspan8 being required for targeting. CIC-TEX transfer supported apoptosis resistance and significantly promoted epithelial mesenchymal transition, migration, invasion and (lymph)angiogenesis of the kd Non-CIC in vitro and in vivo, deep-sequencing allowing individual mRNA and miRNA assignment to altered functions. Importantly, CIC-TEX act as a hub, initiated by CD44v6-dependent RTK, GPCR and integrin activation and involving CD44v6-assisted transcription and RNA processing. Accordingly, a kinase inhibitor hampered CIC-TEX-fostered tumor progression, which was backed by an anti-Tspan8 blockade of CIC-TEX binding.
This in depth report on the in vitro and in vivo impact of CIC-TEX on CD44v6kd and Tspan8kd Non-CIC unravels hub CIC-TEX activity, highlighting a prominent contribution of the CIC-markers CD44v6 to signaling cascade activation, transcription, translation and miRNA processing in Non-CIC and of Tspan8 to CIC-TEX targeting. Blocking CIC-TEX binding/uptake and uptake-initiated target cell activation significantly mitigated the deleterious CIC-TEX impact on CD44v6kd and Tspan8kd Non-CIC.
The ability to perceive geomagnetic fields (GMFs) represents a fascinating biological phenomenon. Studies on transgenic flies have provided evidence that photosensitive Cryptochromes (Cry) are ...involved in the response to magnetic fields (MFs). However, none of the studies tackled the problem of whether the Cry-dependent magnetosensitivity is coupled to the sole MF presence or to the direction of MF vector. In this study, we used gene silencing and a directional MF to show that mammalian-like Cry2 is necessary for a genuine directional response to periodic rotations of the GMF vector in two insect species. Longer wavelengths of light required higher photon fluxes for a detectable behavioral response, and a sharp detection border was present in the cyan/green spectral region. Both observations are consistent with involvement of the FADox, FAD•− and FADH⁻ redox forms of flavin. The response was lost upon covering the eyes, demonstrating that the signal is perceived in the eye region. Immunohistochemical staining detected Cry2 in the hemispherical layer of laminal glia cells underneath the retina. Together, these findings identified the eye-localized Cry2 as an indispensable component and a likely photoreceptor of the directional GMF response. Our study is thus a clear step forward in deciphering the in vivo effects of GMF and supports the interaction of underlying mechanism with the visual system.
Various cellular quality control mechanisms support proteostasis. While, ribosome-associated chaperones prevent the misfolding of nascent chains during translation, importins were shown to prevent ...the aggregation of specific cargoes in a post-translational mechanism prior the import into the nucleoplasm. Here, we hypothesize that importins may already bind ribosome-associated cargo in a co-translational manner. We systematically measure the nascent chain association of all importins in Saccharomyces cerevisiae by selective ribosome profiling. We identify a subset of importins that bind to a wide range of nascent, often uncharacterized cargoes. This includes ribosomal proteins, chromatin remodelers and RNA binding proteins that are aggregation prone in the cytosol. We show that importins act consecutively with other ribosome-associated chaperones. Thus, the nuclear import system is directly intertwined with nascent chain folding and chaperoning.
The treatment of autoimmune diseases still poses a major challenge, frequently relying on non-specific immunosuppressive drugs. Current efforts aim at reestablishing self tolerance using immune cells ...with suppressive activity like the regulatory T cells (Treg) or the myeloid-derived suppressor cells (MDSC). We have demonstrated therapeutic efficacy of MDSC in mouse Alopecia Areata (AA). In the same AA model, we now asked whether MDSC exosomes (MDSC-Exo) can replace MDSC. MDSC-Exo from bone marrow cells (BMC) cultures of healthy donors could substantially facilitate treatment. With knowledge on MDSC-Exo being limited, their suitability needs to be verified in advance. Protein marker profiles suggest comparability of BMC- to
collected inflammatory MDSC/MDSC-Exo in mice with a chronic contact dermatitis, which is a therapeutic option in AA. Proteome analyses substantiated a large overlap of function-relevant molecules in MDSC and MDSC-Exo. Furthermore, MDSC-Exo are taken up by T cells, macrophages, NK, and most avidly by Treg and MDSC-Exo uptake exceeds binding of MDSC themselves. In AA mice, MDSC-Exo preferentially target skin-draining lymph nodes and cells in the vicinity of remnant hair follicles. MDSC-Exo uptake is accompanied by a strong increase in Treg, reduced T helper proliferation, mitigated cytotoxic activity, and a slight increase in lymphocyte apoptosis. Repeated MDSC-Exo application in florid AA prevented progression and sufficed for partial hair regrowth. Deep sequencing of lymphocyte mRNA from these mice revealed a significant increase in immunoregulatory mRNA, including FoxP3 and arginase 1. Downregulated mRNA was preferentially engaged in prohibiting T cell hyperreactivity. Taken together, proteome analysis provided important insights into potential MDSC-Exo activities, these Exo preferentially homing into AA-affected organs. Most importantly, changes in leukocyte mRNA seen after treatment of AA mice with MDSC-Exo sustainably supports the strong impact on the adaptive and the non-adaptive immune system, with Treg expansion being a dominant feature. Thus, MDSC-Exo could potentially serve as therapeutic agents in treating AA and other autoimmune diseases.
The CRISPR/Cas9 technique is widely used in experimentation with human cell lines as well as with other model systems, such as mice
, zebrafish
, and the fruit fly
. However, publications describing ...the use of CRISPR/Cas9 for genome editing in non-model organisms, including non-model insects, are scarce. The introduction of this relatively new method presents many problems even for experienced researchers, especially with the lack of procedures to tackle issues concerning the efficiency of mutant generation. Here we present a protocol for efficient genome editing in the non-model insect species
. We collected data from several independent trials that targeted several genes using the CRISPR/Cas9 system and determined that several crucial optimization steps led to a remarkably increased efficiency of mutant production. The main steps are as follows: the timing of embryo injection, the use of the heteroduplex mobility assay as a screening method,
testing of sgRNA efficiency, and G
germline mosaicism screening. The timing and the method of egg injections used here need to be optimized for other species, but other here-described optimization solutions can be applied immediately for genome editing in other insect species.
Tumor cell-derived extracellular vesicles (TEX) expressing tetraspanin Tspan8-alpha4/beta1 support angiogenesis. Tspan8-alpha6/beta4 facilitates lung premetastatic niche establishment. TEX-promoted ...target reprogramming is still being disputed, we explored rat endothelial cell (EC) and lung fibroblast (Fb) mRNA and miRNA profile changes after coculture with TEX. TEX were derived from non-metastatic BSp73AS (AS) or metastatic BSp73ASML (ASML) rat tumor lines transfected with Tspan8 (AS-Tspan8) or Tspan8-shRNA (ASML-Tspan8kd). mRNA was analyzed by deep sequencing and miRNA by array analysis of EC and Fb before and after coculture with TEX. EC and Fb responded more vigorously to AS-Tspan8- than AS-TEX. Though EC and Fb responses differed, both cell lines predominantly responded to membrane receptor activation with upregulation and activation of signaling molecules and transcription factors. Minor TEX-initiated changes in the miRNA profile relied, at least partly, on long noncoding RNA (lncRNA) that also affected chromosome organization and mRNA processing. These analyses uncovered three important points. TEX activate target cell autonomous programs. Responses are initiated by TEX targeting units and are target cell-specific. The strong TEX-promoted lncRNA impact reflects lncRNA shuttling and location-dependent distinct activities. These informations urge for an in depth exploration on the mode of TEX-initiated target cell-specific remodeling including, as a major factor, lncRNA.
Background
Chronic heart failure (CHF) leads to diaphragm myopathy that significantly impairs quality of life and worsens prognosis. In this study, we aimed to assess the efficacy of a recently ...discovered small‐molecule inhibitor of MuRF1 in treating CHF‐induced diaphragm myopathy and loss of contractile function.
Methods
Myocardial infarction was induced in mice by ligation of the left anterior descending coronary artery. Sham‐operated animals (sham) served as controls. One week post‐left anterior descending coronary artery ligation animals were randomized into two groups—one group was fed control rodent chow, whereas the other group was fed a diet containing 0.1% of the compound ID#704946—a recently described MuRF1‐interfering small molecule. Echocardiography confirmed development of CHF after 10 weeks. Functional and molecular analysis of the diaphragm was subsequently performed.
Results
Chronic heart failure induced diaphragm fibre atrophy and contractile dysfunction by ~20%, as well as decreased activity of enzymes involved in mitochondrial energy production (P < 0.05). Treatment with compound ID#704946 in CHF mice had beneficial effects on the diaphragm: contractile function was protected, while mitochondrial enzyme activity and up‐regulation of the MuRF1 and MuRF2 was attenuated after infarct.
Conclusions
Our murine CHF model presented with diaphragm fibre atrophy, impaired contractile function, and reduced mitochondrial enzyme activities. Compound ID#704946 rescued from this partially, possibly by targeting MuRF1/MuRF2. However, at this stage of our study, we refrain to claim specific mechanism(s) and targets of compound ID#704946, because the nature of changes after 12 weeks of feeding is likely to be complex and is not necessarily caused by direct mechanistic effects.
Photon number resolving detectors are the ultimate measurement of quantum optics, which is the reason why developing the technology is getting significant attention in recent years. With this arises ...the question of how to evaluate the performance of the detectors. We suggest that performance of a photon number detector can be evaluated by comparing it to a multiplex of on-off detectors in a practical scenario: conditional preparation of a photon number state. Here, both the quality of the prepared state and the probability of the preparation are limited by the number of on-off detectors in the multiplex, which allows us to set benchmarks that can be achieved or surpassed by the photon number resolving detectors.
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