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.
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.
MicroRNAs (miRNAs) are small non-coding RNA molecules, with sizes ranging from 18 to 25 nucleotides, which are key players in gene expression regulation. These molecules play an important role in ...fine-tuning early vertebrate embryo development. However, there are scarce publicly available miRNA datasets from non-mammal embryos, such as the chicken (Gallus gallus), which is a classical model system to study vertebrate embryogenesis. Here, we performed microRNA-sequencing to characterize the early stages of trunk and limb development in the chick embryo. For this, we profiled three chick embryonic tissues, namely, Undetermined Presomitic Mesoderm (PSM_U), Determined Presomitic Mesoderm (PSM_D) and Forelimb Distal Cyclic Domain (DCD). We identified 926 known miRNAs, and 1,141 novel candidate miRNAs, which nearly duplicates the number of Gallus gallus entries in the miRBase database. These data will greatly benefit the avian research community, particularly by highlighting new miRNAs potentially involved in the regulation of early vertebrate embryo development, that can be prioritized for further experimental testing.
During the co-translational assembly of protein complexes, a fully synthesized subunit engages with the nascent chain of a newly synthesized interaction partner. Such events are thought to contribute ...to productive assembly, but their exact physiological relevance remains underexplored. Here, we examine structural motifs contained in nucleoporins for their potential to facilitate co-translational assembly. We experimentally test candidate structural motifs and identify several previously unknown co-translational interactions. We demonstrate by selective ribosome profiling that domain invasion motifs of beta-propellers, coiled-coils, and short linear motifs may act as co-translational assembly domains. Such motifs are often contained in proteins that are members of multiple complexes (moonlighters) and engage with closely related paralogs. Surprisingly, moonlighters and paralogs assemble co-translationally in only some but not all of the relevant biogenesis pathways. Our results highlight the regulatory complexity of assembly pathways.
Mosquito saliva facilitates blood feeding through the anti-haemostatic, anti-inflammatory and immunomodulatory properties of its proteins. However, the potential contribution of non-coding RNAs to ...host manipulation is still poorly understood. We analysed small RNAs from Aedes aegypti saliva and salivary glands and show here that chikungunya virus-infection triggers both the siRNA and piRNA antiviral pathways with limited effects on miRNA expression profiles. Saliva appears enriched in specific miRNA subsets and its miRNA content is well conserved among mosquitoes and ticks, clearly pointing to a non-random sorting and occurrence. Finally, we provide evidence that miRNAs from Ae. aegypti saliva may target human immune and inflammatory pathways, as indicated by prediction analysis and searching for experimentally validated targets of identical human miRNAs. Overall, we believe these observations convincingly support a scenario where both proteins and miRNAs from mosquito saliva are injected into vertebrates during blood feeding and contribute to the complex vector-host-pathogen interactions.
Squamous cell carcinomas (SCCs) in the anogenital and head and neck regions are associated with high-risk types of human papillomaviruses (HR-HPV). Deregulation of miRNA expression is an important ...contributor to carcinogenesis. This study aimed to pinpoint commonly and uniquely deregulated miRNAs in cervical, anal, vulvar, and tonsillar tumors of viral or non-viral etiology, searching for a common set of deregulated miRNAs linked to HPV-induced carcinogenesis. RNA was extracted from tumors and nonmalignant tissues from the same locations. The miRNA expression level was determined by next-generation sequencing. Differential expression of miRNAs was calculated, and the patterns of miRNA deregulation were compared between tumors. The total of deregulated miRNAs varied between tumors of different locations by two orders of magnitude, ranging from 1 to 282. The deregulated miRNA pool was largely tumor-specific. In tumors of the same location, a low proportion of miRNAs were exclusively deregulated and no deregulated miRNA was shared by all four types of HPV-positive tumors. The most significant overlap of deregulated miRNAs was found between tumors which differed in location and HPV status (HPV-positive cervical tumors vs. HPV-negative vulvar tumors). Our results imply that HPV infection does not elicit a conserved miRNA deregulation in SCCs.
The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the primary focus for vaccine development. In this study, we combined ...cryo-electron tomography, subtomogram averaging, and molecular dynamics simulations to structurally analyze S in situ. Compared with the recombinant S, the viral S was more heavily glycosylated and occurred mostly in the closed prefusion conformation. We show that the stalk domain of S contains three hinges, giving the head unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and potentially to the development of safe vaccines.
Aim
This study aims to map intimate partner violence evidence among indigenous women and explore the prevalence, social and systemic factors contributing to this occurrence.
Methods
This is a scoping ...review following the steps recommended by the JBI. We searched the MEDLINE/PubMed®, Web of Science™, Embase, CINAHL and LILACS databases on March 2023. Studies that addressed the intimate partner violence topic among indigenous women and risk factors, without time and language limitations, were included. Detailed information was extracted, standardized by JBI.
Results
Twenty studies of different designs were included, all published in English, between 2004 and 2022. A high intimate partner violence prevalence among indigenous women was identified, associated with a great diversity of risk factors.
Conclusion
The great diversity of identified factors associated with its occurrence shows the complexity of this problem and the vulnerability of indigenous women.
Summary statement
What is already known about this topic?
Intimate partner violence is a serious social and public health problem.
Worldwide, a third of women aged between 15 and 49 who have a partner have already experience or are experiencing intimate partner violence.
One in three women experience intimate partner violence during their lifetime.
What this paper adds?
The study presents intimate partner violence prevalence among indigenous women based on evidence.
The study explores the social and systemic factors that contribute to this prevalence.
The implications of this paper:
Nurses are necessary agents not only in providing care but also in articulating and developing strategies to deal with violence, especially intimate partner violence.
In nurses' work with indigenous health, it is essential to understand the health–disease process in a broader way, including the ethnic–cultural aspects.
The study points out that embracing, understanding and respecting cultural diversity and social support are aspects to be considered in prevention, protection and health promotion strategies for these women.
In January 2021, Germany commenced surveillance of SARS-CoV-2 variants under the Corona Surveillance Act, which ceased in July 2023. The objective was to bolster pandemic control, as specific ...alterations in amino acids, particularly within the spike protein, were linked to heightened transmission and decreased vaccine effectiveness.
Consequently, our team conducted whole genome sequencing using the commercially accessible ARTIC protocol on Illumina's NextSeq500 platform and MiSeq for SARS-CoV-2 positive samples obtained from patients at Heidelberg University Hospital, affiliated hospitals, and the public health office in the Rhine-Neckar/Heidelberg region. Throughout the pandemic, we refined the existing ARTIC V4 protocol as well as our bioinformatics pipeline, the details of which are outlined in this report. This report reflects the protocol for the MiSeq analysis, the protocol for the NextSeq500 can be found in our previous publication.
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