scholarly journals The diversity, structure and function of heritable adaptive immunity sequences in the Aedes aegypti genome

2017 ◽  
Author(s):  
Zachary J. Whitfield ◽  
Patrick T. Dolan ◽  
Mark Kunitomi ◽  
Michel Tassetto ◽  
Matthew G. Seetin ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Single Molecule ◽  
Large Scale ◽  
Insect Immunity ◽  
Genomic Context ◽  
Long Read ◽  
Aegypti Genome ◽  
Endogenous Viral Elements ◽  
And Function

AbstractThe Aedes aegypti mosquito is a major vector for arboviruses including dengue, chikungunya and Zika virus. Combating the spread of these viruses requires a more complete understanding of the mosquito immune system. Recent studies have implicated genomic endogenous viral elements (EVEs) derived from non-retroviral RNA viruses in insect immunity. Because these elements are inserted into repetitive regions of the mosquito genome, their large-scale structure and organization with respect to other genomic elements has been difficult to resolve with short-read sequencing. To better define the origin, diversity and biological role of EVEs, we employed single-molecule, real-time sequencing technology to generate a high quality, long-read assembly of the Ae. aegypti-derived Aag2 cell line genome. We leverage the quality and contiguity of this assembly to characterize the diversity and genomic context of EVEs in the genome of this important model system. We find that EVEs in the Aag2 genome are acquired through recombination by LTR retrotransposons, and organize into larger loci (>50kbp) characterized by high LTR density. These EVE containing loci are associated with increased transcription factor binding sight density and increased production of anti-genomic piRNAs. We also detected piRNA processing corresponding to on-going viral infection. This global view of EVEs and piRNA responses demonstrates the ubiquity and diversity of these heritable elements that define small-RNA mediated antiviral immunity in mosquitoes.

scholarly journals Expectations and blind spots for structural variation detection from short-read alignment and long-read assembly

2020 ◽  
Author(s):  
Xuefang Zhao ◽  
Ryan L. Collins ◽  
Wan-Ping Lee ◽  
Alexandra M. Weber ◽  
Yukyung Jun ◽  
...  
Keyword(s):  
Single Molecule ◽  
Large Scale ◽  
Structural Variation ◽  
Human Genetics ◽  
Clinical Diagnostics ◽  
Added Value ◽  
Mendelian Disease ◽  
Segmental Duplications ◽  
Genomic Context ◽  
Long Read

AbstractVirtually all genome sequencing efforts in national biobanks, complex and Mendelian disease programs, and emerging clinical diagnostic approaches utilize short-reads (srWGS), which present constraints for genome-wide discovery of structural variants (SVs). Alternative long-read single molecule technologies (lrWGS) offer significant advantages for genome assembly and SV detection, while these technologies are currently cost prohibitive for large-scale disease studies and clinical diagnostics (∼5-12X higher cost than comparable coverage srWGS). Moreover, only dozens of such genomes are currently publicly accessible by comparison to millions of srWGS genomes that have been commissioned for international initiatives. Given this ubiquitous reliance on srWGS in human genetics and genomics, we sought to characterize and quantify the properties of SVs accessible to both srWGS and lrWGS to establish benchmarks and expectations in ongoing medical and population genetic studies, and to project the added value of SVs uniquely accessible to each technology. In analyses of three trios with matched srWGS and lrWGS from the Human Genome Structural Variation Consortium (HGSVC), srWGS captured ∼11,000 SVs per genome using reference-based algorithms, while haplotype-resolved assembly from lrWGS identified ∼25,000 SVs per genome. Detection power and precision for SV discovery varied dramatically by genomic context and variant class: 9.7% of the current GRCh38 reference is defined by segmental duplications (SD) and simple repeats (SR), yet 91.4% of deletions that were specifically discovered by lrWGS localized to these regions. Across the remaining 90.3% of the human reference, we observed extremely high concordance (93.8%) for deletions discovered by srWGS and lrWGS after error correction using the raw lrWGS reads. Conversely, lrWGS was superior for detection of insertions across all genomic contexts. Given that the non-SD/SR sequences span 90.3% of the GRCh38 reference, and encompass 95.9% of coding exons in currently annotated disease associated genes, improved sensitivity from lrWGS to discover novel and interpretable pathogenic deletions not already accessible to srWGS is likely to be incremental. However, these analyses highlight the added value of assembly-based lrWGS to create new catalogues of functional insertions and transposable elements, as well as disease associated repeat expansions in genomic regions previously recalcitrant to routine assessment.

scholarly journals TRPV channel OCR-2 is distributed along C. elegans chemosensory cilia by diffusion in a local interplay with intraflagellar transport

2020 ◽  
Author(s):  
Jaap van Krugten ◽  
Noémie Danné ◽  
Erwin J.G. Peterman
Keyword(s):  
Single Molecule ◽  
Transmembrane Proteins ◽  
Intraflagellar Transport ◽  
Single Molecule Tracking ◽  
C Elegans ◽  
Normal Diffusion ◽  
Cellular Signal ◽  
Underlying Mechanisms ◽  
And Function

AbstractSensing and reacting to the environment is essential for survival and procreation of most organisms. Caenorhabditis elegans senses soluble chemicals with transmembrane proteins (TPs) in the cilia of its chemosensory neurons. Development, maintenance and function of these cilia relies on intraflagellar transport (IFT), in which motor proteins transport cargo, including sensory TPs, back and forth along the ciliary axoneme. Here we use live fluorescence imaging to show that IFT machinery and the sensory TP OCR-2 reversibly redistribute along the cilium after exposure to repellant chemicals. To elucidate the underlying mechanisms, we performed single-molecule tracking experiments and found that OCR-2 distribution depends on an intricate interplay between IFT-driven transport, normal diffusion and subdiffusion that depends on the specific location in the cilium. These insights in the role of IFT on the dynamics of cellular signal transduction contribute to a deeper understanding of the regulation of sensory TPs and chemosensing.

scholarly journals Energetic dependencies dictate folding mechanism in a complex protein

2019 ◽  
Vol 116 (51) ◽  
pp. 25641-25648 ◽  
Author(s):  
Kaixian Liu ◽  
Xiuqi Chen ◽  
Christian M. Kaiser
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Large Scale ◽  
Elongation Factor ◽  
Multidomain Protein ◽  
Domain Interactions ◽  
The Stability ◽  
Domain Iii ◽  
And Function ◽  
Terminal Domains

Large proteins with multiple domains are thought to fold cotranslationally to minimize interdomain misfolding. Once folded, domains interact with each other through the formation of extensive interfaces that are important for protein stability and function. However, multidomain protein folding and the energetics of domain interactions remain poorly understood. In elongation factor G (EF-G), a highly conserved protein composed of 5 domains, the 2 N-terminal domains form a stably structured unit cotranslationally. Using single-molecule optical tweezers, we have defined the steps leading to fully folded EF-G. We find that the central domain III of EF-G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N-terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C-terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C-terminal neighbors disrupts cotranslational folding and imposes a posttranslational mechanism on the folding of the C-terminal part of EF-G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large-scale conformational transitions that are part of the EF-G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF-G function, complicates the folding of this large multidomain protein.

scholarly journals Non-retroviral endogenous viral element limits cognate virus replication in Aedes aegypti ovaries

2020 ◽  
Author(s):  
Yasutsugu Suzuki ◽  
Artem Baidaliuk ◽  
Pascal Miesen ◽  
Lionel Frangeul ◽  
Anna B. Crist ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Virus Replication ◽  
Insect Virus ◽  
Functional Link ◽  
Naturally Occurring ◽  
Specific Virus ◽  
Virus Interaction ◽  
Endogenous Viral Elements ◽  
Viral Sequences

SummaryEndogenous viral elements (EVEs) are viral sequences integrated in host genomes. A large number of non-retroviral EVEs was recently detected in Aedes mosquito genomes, leading to the hypothesis that mosquito EVEs may control exogenous infections by closely related viruses. Here, we experimentally investigated the role of an EVE naturally found in Aedes aegypti populations and derived from the widespread insect-specific virus, cell-fusing agent virus (CFAV). Using CRISPR/Cas9 genome editing, we created an Ae. aegypti line lacking the CFAV EVE. Absence of the EVE resulted in increased CFAV replication in ovaries, possibly modulating vertical transmission of the virus. Viral replication was controlled by targeting of viral RNA by EVE-derived piRNAs. Our results provide evidence that antiviral piRNAs are produced in the presence of a naturally occurring EVE and its cognate virus, demonstrating a functional link between non-retroviral EVEs and antiviral immunity in a natural insect-virus interaction.

scholarly journals Actin bundles play different role in shaping scale as compare to bristle in mosquito Aedes aegypti

2020 ◽  
Author(s):  
Sanja Djokic ◽  
Bakhrat Anna ◽  
Ido Zurim ◽  
Nadya Urakova ◽  
Jason L. Rasgon ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Actin Polymerization ◽  
Morphological Changes ◽  
Cylindrical Shape ◽  
Actin Bundles ◽  
Membrane Invagination ◽  
Flat Shape ◽  
Cellular Extensions ◽  
And Function

AbstractInsect epithelial cells contain cellular extensions such as bristles, hairs and scales. It has been suggested that these cellular extensions are homologous structures that differ in morphology and function. These cellular extensions contain actin bundles that dictate their cellular morphology; bristle and hair are cylindrical in shape, while scales are wider and flattened. While the organization, function and identity of the major actin bundling protein in bristles and hairs is known, this information in scales is unknown. In this study, we characterized the development of scales and the role of actin bundles in the mosquito, Aedes aegypti. We show that scales undergo drastic morphological changes during development, from cylindrical shape to flat shape with longer membrane invagination. Scale actin bundle distribution changes during development, from symmetrical organization of actin bundles located throughout the bristle membrane, to asymmetrical organization of the actin bundles. By chemically inhibiting actin polymerization and by knocking-out the forked gene in the mosquito (Ae-Forked; a known actin bundling protein), by CRISPR-Cas9 gene editing, we showed that actin bundles are required for shaping bristle, hair and scale morphology. We demonstrated that actin bundles and Ae-Forked are required for bristle elongation, but not that of scales. In scales, actin bundles are required for width formation. Our results reveal a differential requirement of actin bundles in shaping mosquito scales compared to bristles.

scholarly journals Single-nucleus full-length RNA profiling in plants incorporates isoform information to facilitate cell type identification

2020 ◽  
Author(s):  
Yanping Long ◽  
Zhijian Liu ◽  
Jinbu Jia ◽  
Weipeng Mo ◽  
Liang Fang ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Molecule ◽  
Large Scale ◽  
Alternative Polyadenylation ◽  
Full Length ◽  
Cell Type ◽  
Arabidopsis Root ◽  
Rna Profiling ◽  
Long Read ◽  
Single Nucleus

AbstractThe broad application of large-scale single-cell RNA profiling in plants has been restricted by the prerequisite of protoplasting. We recently found that the Arabidopsis nucleus contains abundant polyadenylated mRNAs, many of which are incompletely spliced. To capture the isoform information, we combined 10x Genomics and Nanopore long-read sequencing to develop a protoplasting-free full-length single-nucleus RNA profiling method in plants. Our results demonstrated using Arabidopsis root that nuclear mRNAs faithfully retain cell identity information, and single-molecule full-length RNA sequencing could further improve cell type identification by revealing splicing status and alternative polyadenylation at single-cell level.

scholarly journals Chromosome organization by a conserved condensin-ParB system in the actinobacterium Corynebacterium glutamicum

2019 ◽  
Author(s):  
Kati Böhm ◽  
Giacomo Giacomelli ◽  
Andreas Schmidt ◽  
Axel Imhof ◽  
Romain Koszul ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Single Molecule ◽  
Chromosomal Dna ◽  
Chromosome Conformation ◽  
Long Range Interactions ◽  
Domains Of Life ◽  
Dna Partitioning ◽  
And Function ◽  
Single Molecule Localization Microscopy

AbstractHigher-order chromosome folding and segregation is tightly regulated in all domains of life. In bacteria, details on nucleoid organization regulatory mechanisms and function remains poorly characterized, especially in non-model species. Here, we investigate the role of DNA partitioning protein ParB and condensin complexes, two key players in bacterial chromosome structuring, in the actinobacterium Corynebacterium glutamicum. Chromosome conformation capture reveals SMC-mediated long-range interactions around ten centromere-like parS sites clustered at the replication origin (oriC). At least one oriC-proximal parS site is necessary for a reliable chromosome segregation. Using a combination of chromatin immunoprecipitation and photoactivated single molecule localization microscopy evidences the formation of distinct ParB-nucleoprotein subclusters in dependence of parS numbers. We further identified and functionally characterized two condensin paralogs. Whereas SMC/ScpAB complexes are loaded via ParB at parS sites mediating chromosomal inter-arm contacts like in Bacillus subtilis, the MukBEF-like SMC complex MksBEFG does not contribute to chromosomal DNA-folding. Rather, the MksBEFG complex is involved in plasmid maintenance and interacts with the polar oriC-tethering factor DivIVA. These data complement current models of ParB-SMC/ScpAB crosstalk, while showing that some condensin complexes evolved functions uncoupled from chromosome folding.

scholarly journals Biophysical, Biochemical, and Cell Based Approaches Used to Decipher the Role of Carbonic Anhydrases in Cancer and to Evaluate the Potency of Targeted Inhibitors

2018 ◽  
Vol 2018 ◽  
pp. 1-18 ◽  
Author(s):  
Mam Y. Mboge ◽  
Anusha Kota ◽  
Robert McKenna ◽  
Susan C. Frost
Keyword(s):  
Cancer Progression ◽  
Large Scale ◽  
Metastatic Potential ◽  
Migration And Invasion ◽  
Carbonic Anhydrases ◽  
X Ray Crystallography ◽  
Targeted Inhibitors ◽  
Nonspecific Cytotoxicity ◽  
And Function

Carbonic anhydrases (CAs) are thought to be important for regulating pH in the tumor microenvironment. A few of the CA isoforms are upregulated in cancer cells, with only limited expression in normal cells. For these reasons, there is interest in developing inhibitors that target these tumor-associated CA isoforms, with increased efficacy but limited nonspecific cytotoxicity. Here we present some of the biophysical, biochemical, and cell based techniques and approaches that can be used to evaluate the potency of CA targeted inhibitors and decipher the role of CAs in tumorigenesis, cancer progression, and metastatic processes. These techniques include esterase activity assays, stop flow kinetics, and mass inlet mass spectroscopy (MIMS), all of which measure enzymatic activity of purified protein, in the presence or absence of inhibitors. Also discussed is the application of X-ray crystallography and Cryo-EM as well as other structure-based techniques and thermal shift assays to the studies of CA structure and function. Further, large-scale genomic and proteomic analytical methods, as well as cell based techniques like those that measure cell growth, apoptosis, clonogenicity, and cell migration and invasion, are discussed. We conclude by reviewing approaches that test the metastatic potential of CAs and how the aforementioned techniques have contributed to the field of CA cancer research.

scholarly journals Single-molecule real-time sequencing identifies massive full-length cDNAs and alternative-splicing events that facilitate comparative and functional genomics study in the hexaploid crop sweet potato

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7933 ◽  
Author(s):  
Na Ding ◽  
Huihui Cui ◽  
Ying Miao ◽  
Jun Tang ◽  
Qinghe Cao ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Functional Genomics ◽  
Real Time ◽  
Sweet Potato ◽  
Single Molecule ◽  
Large Scale ◽  
Full Length ◽  
Full Length Cdna ◽  
Cdna Sequences ◽  
Long Read

Background Sweet potato (Ipomoea batatas (L.) Lam.) is one of the most important crops in many developing countries and provides a candidate source of bioenergy. However, neither a complete reference genome nor large-scale full-length cDNA sequences for this outcrossing hexaploid crop are available, which in turn impedes progress in research studies in I. batatas functional genomics and molecular breeding. Methods In this study, we sequenced full-length transcriptomes in I. batatas and its diploid ancestor I. trifida by single-molecule real-time sequencing and Illumina second-generation sequencing technologies. With the generated datasets, we conducted comprehensive intraspecific and interspecific sequence analyses and experimental characterization. Results A total of 53,861/51,184 high-quality long-read transcripts were obtained, which covered about 10,439/10,452 loci in the I. batatas/I. trifida genome. These datasets enabled us to predict open reading frames successfully in 96.83%/96.82% of transcripts and identify 34,963/33,637 full-length cDNA sequences, 1,401/1,457 transcription factors, 25,315/27,090 simple sequence repeats, 1,656/1,389 long non-coding RNAs, and 5,251/8,901 alternative splicing events. Approximately, 32.34%/38.54% of transcripts and 46.22%/51.18% multi-exon transcripts underwent alternative splicing in I. batatas/I. trifida. Moreover, we validated one alternative splicing event in each of 10 genes and identified tuberous-root-specific expressed isoforms from a starch-branching enzyme, an alpha-glucan phosphorylase, a neutral invertase, and several ABC transporters. Overall, the collection and analysis of large-scale long-read transcripts generated in this study will serve as a valuable resource for the I. batatas research community, which may accelerate the progress in its structural, functional, and comparative genomics studies.

scholarly journals Autophosphorylation of the KaiC-like protein ArlH inhibits oligomerisation and interaction with ArlI, the motor ATPase of the archaellum

2021 ◽  
Author(s):  
João Nuno de Sousa Machado ◽  
Leonie Vollmar ◽  
Julia Schimpf ◽  
Paushali Chaudhury ◽  
Rashmi Kumariya ◽  
...  
Keyword(s):  
Single Molecule ◽  
Pyrococcus Furiosus ◽  
Bound State ◽  
Central Component ◽  
Oligomeric State ◽  
Fluorescence Measurements ◽  
Biochemical Assays ◽  
And Function ◽  
Atp Binding Protein

Motile archaea are propelled by the archaellum, whose motor complex consists of the membrane protein ArlJ, the ATPase ArlI, and the ATP-binding protein ArlH. Despite its essential function and the existence of structural and biochemical data on ArlH, the role of ArlH in archaellum assembly and function remains elusive. ArlH is a structural homolog of KaiC, the central component of the cyanobacterial circadian clock. Similar to KaiC, ArlH exhibits autophosphorylation activity, which was observed for both ArlH of the euryarchaeon Pyrococcus furiosus (PfArlH) and the crenarchaeon Sulfolobus acidocaldarius (SaArlH). Using a combination of single molecule fluorescence measurements and biochemical assays, it is shown that autophosphorylation of ArlH is closely linked to the oligomeric state of ArlH bound to ArlI. These experiments also strongly suggest that ArlH is a hexamer in its functional ArlI bound state. Mutagenesis of the putative catalytic residue Glu-57 in SaArlH results in a reduced autophosphorylation activity and abolished archaellation and motility, suggesting that optimum phosphorylation activity of ArlH is essential for both archaellation and motility.

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