Disruption of a ∼23-24 nucleotide small RNA pathway elevates DNA damage responses in Tetrahymena thermophila

Endogenous RNA interference (RNAi) pathways regulate a wide range of cellular processes in diverse eukaryotes, yet in the ciliated eukaryote, Tetrahymena thermophila, the cellular purpose of RNAi pathways that generate ∼23-24 nucleotide (nt) small (s)RNAs has remained unknown. Here, we investigated the phenotypic and gene expression impacts on vegetatively growing cells when genes involved in ∼23-24 nt sRNA biogenesis are disrupted. We observed slower proliferation and increased expression of genes involved in DNA metabolism and chromosome organization and maintenance in sRNA biogenesis mutants RSP1Δ, RDN2Δ, and RDF2Δ. In addition, RSP1Δ and RDN2Δ cells frequently exhibited enlarged chromatin extrusion bodies, which are non-nuclear, DNA-containing structures that may be akin to mammalian micronuclei. Expression of homologous recombination factor Rad51 was specifically elevated in RSP1Δ and RDN2Δ strains, with Rad51 and double-stranded DNA break (DSB) marker γ-H2A.X localized to discrete macronuclear foci. In addition, an increase in Rad51 and γ-H2A.X foci were also found in knockouts of TWI8, a macronucleus-localized PIWI protein. Together, our findings suggest that an evolutionarily conserved role for RNAi pathways in maintaining genome integrity may be extended even to the early branching eukaryotic lineage that gave rise to Tetrahymena thermophila.

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Spectraplakins: Master orchestrators of cytoskeletal dynamics

The Journal of Cell Biology ◽

10.1083/jcb.201112034 ◽

2012 ◽

Vol 197 (4) ◽

pp. 465-475 ◽

Cited By ~ 110

Author(s):

Kathleen C. Suozzi ◽

Xiaoyang Wu ◽

Elaine Fuchs

Keyword(s):

Differential Splice ◽

Vast Array ◽

Cellular Processes ◽

Tissue Integrity ◽

Multiple Promoters ◽

Wide Range ◽

Evolutionarily Conserved ◽

Cytoskeletal Dynamics ◽

Cytoskeletal Networks ◽

Splice Forms

The dynamics of different cytoskeletal networks are coordinated to bring about many fundamental cellular processes, from neuronal pathfinding to cell division. Increasing evidence points to the importance of spectraplakins in integrating cytoskeletal networks. Spectraplakins are evolutionarily conserved giant cytoskeletal cross-linkers, which belong to the spectrin superfamily. Their genes consist of multiple promoters and many exons, yielding a vast array of differential splice forms with distinct functions. Spectraplakins are also unique in their ability to associate with all three elements of the cytoskeleton: F-actin, microtubules, and intermediate filaments. Recent studies have begun to unveil their role in a wide range of processes, from cell migration to tissue integrity.

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Tight nuclear tethering of cGAS is essential for preventing autoreactivity

eLife ◽

10.7554/elife.47491 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 36

Author(s):

Hannah E Volkman ◽

Stephanie Cambier ◽

Elizabeth E Gray ◽

Daniel B Stetson

Keyword(s):

Resting State ◽

Nuclear Protein ◽

Nuclear Dna ◽

Cell Cycle Phase ◽

Innate Immune ◽

Cycle Phase ◽

Base Pairs ◽

Double Stranded Dna ◽

Evolutionarily Conserved ◽

Activation Status

cGAS is an intracellular innate immune sensor that detects double-stranded DNA. The presence of billions of base pairs of genomic DNA in all nucleated cells raises the question of how cGAS is not constitutively activated. A widely accepted explanation for this is the sequestration of cGAS in the cytosol, which is thought to prevent cGAS from accessing nuclear DNA. Here, we demonstrate that endogenous cGAS is predominantly a nuclear protein, regardless of cell cycle phase or cGAS activation status. We show that nuclear cGAS is tethered tightly by a salt-resistant interaction. This tight tethering is independent of the domains required for cGAS activation, and it requires intact nuclear chromatin. We identify the evolutionarily conserved tethering surface on cGAS and we show that mutation of single amino acids within this surface renders cGAS massively and constitutively active against self-DNA. Thus, tight nuclear tethering maintains the resting state of cGAS and prevents autoreactivity.

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Naphthalimide-Containing BP100 Leads to Higher Model Membranes Interactions and Antimicrobial Activity

Biomolecules ◽

10.3390/biom11040542 ◽

2021 ◽

Vol 11 (4) ◽

pp. 542

Author(s):

Gustavo Penteado Battesini Carretero ◽

Greice Kelle Viegas Saraiva ◽

Magali Aparecida Rodrigues ◽

Sumika Kiyota ◽

Marcelo Porto Bemquerer ◽

...

Keyword(s):

Anticancer Agents ◽

Biological Activities ◽

Biological Effects ◽

Membrane Surface ◽

Helical Structure ◽

Helical Conformation ◽

Property A ◽

Cellular Processes ◽

Double Stranded Dna ◽

Low Toxicity

In a large variety of organisms, antimicrobial peptides (AMPs) are primary defenses against pathogens. BP100 (KKLFKKILKYL-NH2), a short, synthetic, cationic AMP, is active against bacteria and displays low toxicity towards eukaryotic cells. BP100 acquires a α-helical conformation upon interaction with membranes and increases membrane permeability. Despite the volume of information available, the action mechanism of BP100, the selectivity of its biological effects, and possible applications are far from consensual. Our group synthesized a fluorescent BP100 analogue containing naphthalimide linked to its N-terminal end, NAPHT-BP100 (Naphthalimide-AAKKLFKKILKYL-NH2). The fluorescence properties of naphthalimides, especially their spectral sensitivity to microenvironment changes, are well established, and their biological activities against transformed cells and bacteria are known. Naphthalimide derived compounds are known to interact with DNA disturbing related processes as replication and transcription, and used as anticancer agents due to this property. A wide variety of techniques were used to demonstrate that NAPHT-BP100 bound to and permeabilized zwitterionic POPC and negatively charged POPC:POPG liposomes and, upon interaction, acquired a α-helical structure. Membrane surface high peptide/lipid ratios triggered complete permeabilization of the liposomes in a detergent-like manner. Membrane disruption was driven by charge neutralization, lipid aggregation, and bilayer destabilization. NAPHT-BP100 also interacted with double-stranded DNA, indicating that this peptide could also affect other cellular processes besides causing membrane destabilization. NAPHT-BP100 showed increased antibacterial and hemolytic activities, compared to BP100, and may constitute an efficient antimicrobial agent for dermatological use. By conjugating BP100 and naphthalimide DNA binding properties, NAPHT-BP100 bound to a large extent to the bacterial membrane and could more efficiently destabilize it. We also speculate that peptide could enter the bacteria cell and interact with its DNA in the cytoplasm.

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Intracellular Ionic Strength Sensing Using NanoLuc

International Journal of Molecular Sciences ◽

10.3390/ijms22020677 ◽

2021 ◽

Vol 22 (2) ◽

pp. 677

Author(s):

Tausif Altamash ◽

Wesam Ahmed ◽

Saad Rasool ◽

Kabir H. Biswas

Keyword(s):

Thermal Stability ◽

Phase Separation ◽

Drug Discovery ◽

Ionic Strength ◽

Cellular Signaling ◽

Live Cells ◽

Protein Activity ◽

Cellular Survival ◽

Cellular Processes ◽

Wide Range

Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.

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SCAPP: an algorithm for improved plasmid assembly in metagenomes

Microbiome ◽

10.1186/s40168-021-01068-z ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

David Pellow ◽

Alvah Zorea ◽

Maraike Probst ◽

Ori Furman ◽

Arik Segal ◽

...

Keyword(s):

Bacterial Species ◽

Bacterial Genome ◽

Biological Knowledge ◽

Assessment Procedure ◽

Metagenomic Sequencing ◽

Sequencing Data ◽

Human Gut ◽

Double Stranded Dna ◽

Wide Range ◽

Python Package

Abstract Background Metagenomic sequencing has led to the identification and assembly of many new bacterial genome sequences. These bacteria often contain plasmids: usually small, circular double-stranded DNA molecules that may transfer across bacterial species and confer antibiotic resistance. These plasmids are generally less studied and understood than their bacterial hosts. Part of the reason for this is insufficient computational tools enabling the analysis of plasmids in metagenomic samples. Results We developed SCAPP (Sequence Contents-Aware Plasmid Peeler)—an algorithm and tool to assemble plasmid sequences from metagenomic sequencing. SCAPP builds on some key ideas from the Recycler algorithm while improving plasmid assemblies by integrating biological knowledge about plasmids. We compared the performance of SCAPP to Recycler and metaplasmidSPAdes on simulated metagenomes, real human gut microbiome samples, and a human gut plasmidome dataset that we generated. We also created plasmidome and metagenome data from the same cow rumen sample and used the parallel sequencing data to create a novel assessment procedure. Overall, SCAPP outperformed Recycler and metaplasmidSPAdes across this wide range of datasets. Conclusions SCAPP is an easy to use Python package that enables the assembly of full plasmid sequences from metagenomic samples. It outperformed existing metagenomic plasmid assemblers in most cases and assembled novel and clinically relevant plasmids in samples we generated such as a human gut plasmidome. SCAPP is open-source software available from: https://github.com/Shamir-Lab/SCAPP.

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Role of Transposable Elements in Gene Regulation in the Human Genome

Life ◽

10.3390/life11020118 ◽

2021 ◽

Vol 11 (2) ◽

pp. 118

Author(s):

Arsala Ali ◽

Kyudong Han ◽

Ping Liang

Keyword(s):

Gene Regulation ◽

Transposable Elements ◽

Regulatory Sequences ◽

Rna Sequences ◽

Expression Of Genes ◽

Wide Range ◽

Lineage Specificity ◽

Regulatory Rnas ◽

Potential Factors ◽

Interspersed Repeats

Transposable elements (TEs), also known as mobile elements (MEs), are interspersed repeats that constitute a major fraction of the genomes of higher organisms. As one of their important functional impacts on gene function and genome evolution, TEs participate in regulating the expression of genes nearby and even far away at transcriptional and post-transcriptional levels. There are two known principal ways by which TEs regulate the expression of genes. First, TEs provide cis-regulatory sequences in the genome with their intrinsic regulatory properties for their own expression, making them potential factors for regulating the expression of the host genes. TE-derived cis-regulatory sites are found in promoter and enhancer elements, providing binding sites for a wide range of trans-acting factors. Second, TEs encode for regulatory RNAs with their sequences showed to be present in a substantial fraction of miRNAs and long non-coding RNAs (lncRNAs), indicating the TE origin of these RNAs. Furthermore, TEs sequences were found to be critical for regulatory functions of these RNAs, including binding to the target mRNA. TEs thus provide crucial regulatory roles by being part of cis-regulatory and regulatory RNA sequences. Moreover, both TE-derived cis-regulatory sequences and TE-derived regulatory RNAs have been implicated in providing evolutionary novelty to gene regulation. These TE-derived regulatory mechanisms also tend to function in a tissue-specific fashion. In this review, we aim to comprehensively cover the studies regarding these two aspects of TE-mediated gene regulation, mainly focusing on the mechanisms, contribution of different types of TEs, differential roles among tissue types, and lineage-specificity, based on data mostly in humans.

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A YoeB toxin cleaves both RNA and DNA

Scientific Reports ◽

10.1038/s41598-021-82950-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Julia McGillick ◽

Jessica R. Ames ◽

Tamiko Murphy ◽

Christina R. Bourne

Keyword(s):

Dna Cleavage ◽

Divalent Cations ◽

Nuclease Activity ◽

Dose Dependence ◽

Cleavage Activity ◽

Double Stranded Dna ◽

Dna Cleavage Activity ◽

Evolutionarily Conserved ◽

Toxin Protein ◽

Rna And Dna

AbstractType II toxin-antitoxin systems contain a toxin protein, which mediates diverse interactions within the bacterial cell when it is not bound by its cognate antitoxin protein. These toxins provide a rich source of evolutionarily-conserved tertiary folds that mediate diverse catalytic reactions. These properties make toxins of interest in biotechnology applications, and studies of the catalytic mechanisms continue to provide surprises. In the current work, our studies on a YoeB family toxin from Agrobacterium tumefaciens have revealed a conserved ribosome-independent non-specific nuclease activity. We have quantified the RNA and DNA cleavage activity, revealing they have essentially equivalent dose-dependence while differing in requirements for divalent cations and pH sensitivity. The DNA cleavage activity is as a nickase for any topology of double-stranded DNA, as well as cleaving single-stranded DNA. AtYoeB is able to bind to double-stranded DNA with mid-micromolar affinity. Comparison of the ribosome-dependent and -independent reactions demonstrates an approximate tenfold efficiency imparted by the ribosome. This demonstrates YoeB toxins can act as non-specific nucleases, cleaving both RNA and DNA, in the absence of being bound within the ribosome.

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Designer Oncolytic Adenovirus: Coming of Age

Cancers ◽

10.3390/cancers10060201 ◽

2018 ◽

Vol 10 (6) ◽

pp. 201 ◽

Cited By ~ 35

Author(s):

Alexander Baker ◽

Carmen Aguirre-Hernández ◽

Gunnel Halldén ◽

Alan Parker

Keyword(s):

Checkpoint Inhibitors ◽

Coming Of Age ◽

Oncolytic Adenovirus ◽

Detailed Knowledge ◽

Immunomodulatory Agents ◽

Double Stranded Dna ◽

Wide Range ◽

Anti Cancer ◽

Targeting Ability

The licensing of talimogene laherparepvec (T-Vec) represented a landmark moment for oncolytic virotherapy, since it provided unequivocal evidence for the long-touted potential of genetically modified replicating viruses as anti-cancer agents. Whilst T-Vec is promising as a locally delivered virotherapy, especially in combination with immune-checkpoint inhibitors, the quest continues for a virus capable of specific tumour cell killing via systemic administration. One candidate is oncolytic adenovirus (Ad); it’s double stranded DNA genome is easily manipulated and a wide range of strategies and technologies have been employed to empower the vector with improved pharmacokinetics and tumour targeting ability. As well characterised clinical and experimental agents, we have detailed knowledge of adenoviruses’ mechanisms of pathogenicity, supported by detailed virological studies and in vivo interactions. In this review we highlight the strides made in the engineering of bespoke adenoviral vectors to specifically infect, replicate within, and destroy tumour cells. We discuss how mutations in genes regulating adenoviral replication after cell entry can be used to restrict replication to the tumour, and summarise how detailed knowledge of viral capsid interactions enable rational modification to eliminate native tropisms, and simultaneously promote active uptake by cancerous tissues. We argue that these designer-viruses, exploiting the viruses natural mechanisms and regulated at every level of replication, represent the ideal platforms for local overexpression of therapeutic transgenes such as immunomodulatory agents. Where T-Vec has paved the way, Ad-based vectors now follow. The era of designer oncolytic virotherapies looks decidedly as though it will soon become a reality.

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The AAA+ ATPase p97, a cellular multitool

Biochemical Journal ◽

10.1042/bcj20160783 ◽

2017 ◽

Vol 474 (17) ◽

pp. 2953-2976 ◽

Cited By ~ 42

Author(s):

Lasse Stach ◽

Paul S. Freemont

Keyword(s):

Atp Hydrolysis ◽

Current Status ◽

Cellular Functions ◽

Aaa Atpase ◽

Cellular Processes ◽

Proteotoxic Stress ◽

Binding Domains ◽

Wide Range ◽

Aaa Atpases ◽

Substrate Proteins

The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy.

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Autophagy is activated in the ovarian tissue of polycystic ovary syndrome

Reproduction ◽

10.1530/rep-17-0499 ◽

2018 ◽

Vol 155 (1) ◽

pp. 85-92 ◽

Cited By ~ 16

Author(s):

Da Li ◽

Yue You ◽

Fang-Fang Bi ◽

Tie-Ning Zhang ◽

Jiao Jiao ◽

...

Keyword(s):

Polycystic Ovary Syndrome ◽

Potential Role ◽

Polycystic Ovary ◽

Ovarian Tissue ◽

Ovary Syndrome ◽

Cellular Processes ◽

Transmission Electron ◽

Wide Range ◽

Response To Stress

The importance of autophagy in polycystic ovary syndrome (PCOS)-related metabolic disorders is increasingly being recognized, but few studies have investigated the role of autophagy in PCOS. Here, transmission electron microscopy demonstrated that autophagy was enhanced in the ovarian tissue from both humans and rats with PCOS. Consistent with this, ovarian granulosa cells from PCOS rats showed increases in the autophagy marker protein light chain 3B (LC3B), whereas levels of the autophagy substrate SQSTM1/p62 were decreased. In addition, the ratio of LC3-II/LC3-I was markedly elevated in human PCOS ovarian tissue compared with normal ovarian tissue. Real-time PCR arrays indicated that 7 and 34 autophagy-related genes were down- and up-regulated in human PCOS , Signal-Net, and regression analysis suggested that there are a wide range of interactions among these 41 genes, and a potential network based on EGFR, ERBB2, FOXO1, MAPK1, NFKB1, IGF1, TP53 and MAPK9 may be responsible for autophagy activation in PCOS. Systematic functional analysis of 41 differential autophagy-related genes indicated that these genes are highly involved in specific cellular processes such as response to stress and stimulus, and are linked to four significant pathways, including the insulin, ERBB, mTOR signaling pathways and protein processing in the endoplasmic reticulum. This study provides evidence for a potential role of autophagy disorders in PCOS in which autophagy may be an important molecular event in the pathogenesis of PCOS.

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