scholarly journals Disentangling sRNA-Seq data to study RNA communication between species

2019 ◽  
Author(s):  
JR Bermúdez-Barrientos ◽  
O Ramírez-Sánchez ◽  
FWN Chow ◽  
AH Buck ◽  
C Abreu-Goodger
Keyword(s):  
Small Rnas ◽  
Animal Species ◽  
De Novo ◽  
Extracellular Vesicle ◽  
Host Cells ◽  
Symbiotic System ◽  
Defense Strategies ◽  
Bioinformatic Tools ◽  
Genomic Regions ◽  
Diverse Plant

ABSTRACTMany organisms exchange small RNAs during their interactions, and these RNAs can target or bolster defense strategies in host-pathogen systems. Current sRNA-Seq technology can determine the small RNAs present in any symbiotic system, but there are very few bioinformatic tools available to interpret the results. We show that one of the biggest challenges comes from sequences that map equally well to the genomes of both interacting organisms. This arises due to the small size of the sRNA compared to large genomes, and because many of the produced sRNAs come from genomic regions that encode highly conserved miRNAs, rRNAs or tRNAs. Here we present strategies to disentangle sRNA-Seq data from samples of communicating organisms, developed using diverse plant and animal species that are known to exchange RNA with their parasites. We show that sequence assembly, both de novo and genome-guided, can be used for sRNA-Seq data, greatly reducing the ambiguity of mapping reads. Even confidently mapped sequences can be misleading, so we further demonstrate the use of differential expression strategies to determine the true parasitic sRNAs within host cells. Finally, we validate our methods on new experiments designed to probe the nature of the extracellular vesicle sRNAs from the parasitic nematode H. bakeri that get into mouse epithelial cells.

scholarly journals Disentangling sRNA-Seq data to study RNA communication between species

2019 ◽  
Vol 48 (4) ◽  
pp. e21-e21
Author(s):  
José Roberto Bermúdez-Barrientos ◽  
Obed Ramírez-Sánchez ◽  
Franklin Wang-Ngai Chow ◽  
Amy H Buck ◽  
Cei Abreu-Goodger
Keyword(s):  
De Novo ◽  
Extracellular Vesicle ◽  
Host Cells ◽  
Intestinal Epithelial ◽  
Defense Strategies ◽  
Bioinformatic Tools ◽  
Heligmosomoides Bakeri ◽  
Genomic Regions ◽  
To Receive ◽  
Diverse Plant

Abstract Many organisms exchange small RNAs (sRNAs) during their interactions, that can target or bolster defense strategies in host–pathogen systems. Current sRNA-Seq technology can determine the sRNAs present in any symbiotic system, but there are very few bioinformatic tools available to interpret the results. We show that one of the biggest challenges comes from sequences that map equally well to the genomes of both interacting organisms. This arises due to the small size of the sRNAs compared to large genomes, and because a large portion of sequenced sRNAs come from genomic regions that encode highly conserved miRNAs, rRNAs or tRNAs. Here, we present strategies to disentangle sRNA-Seq data from samples of communicating organisms, developed using diverse plant and animal species that are known to receive or exchange RNA with their symbionts. We show that sequence assembly, both de novo and genome-guided, can be used for these sRNA-Seq data, greatly reducing the ambiguity of mapping reads. Even confidently mapped sequences can be misleading, so we further demonstrate the use of differential expression strategies to determine true parasite-derived sRNAs within host cells. We validate our methods on new experiments designed to probe the nature of the extracellular vesicle sRNAs from the parasitic nematode Heligmosomoides bakeri that get into mouse intestinal epithelial cells.

scholarly journals Benchmarking transposable element annotation methods for creation of a streamlined, comprehensive pipeline

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Shujun Ou ◽  
Weija Su ◽  
Yi Liao ◽  
Kapeel Chougule ◽  
Jireh R. A. Agda ◽  
...  
Keyword(s):  
Transposable Elements ◽  
Animal Species ◽  
Performance Metrics ◽  
De Novo ◽  
Terminal Inverted Repeat ◽  
Miniature Inverted Transposable Elements ◽  
Sensitivity Specificity ◽  
Genomic Regions ◽  
Assembly Algorithms ◽  
Eukaryotic Genomes

Abstract Background Sequencing technology and assembly algorithms have matured to the point that high-quality de novo assembly is possible for large, repetitive genomes. Current assemblies traverse transposable elements (TEs) and provide an opportunity for comprehensive annotation of TEs. Numerous methods exist for annotation of each class of TEs, but their relative performances have not been systematically compared. Moreover, a comprehensive pipeline is needed to produce a non-redundant library of TEs for species lacking this resource to generate whole-genome TE annotations. Results We benchmark existing programs based on a carefully curated library of rice TEs. We evaluate the performance of methods annotating long terminal repeat (LTR) retrotransposons, terminal inverted repeat (TIR) transposons, short TIR transposons known as miniature inverted transposable elements (MITEs), and Helitrons. Performance metrics include sensitivity, specificity, accuracy, precision, FDR, and F1. Using the most robust programs, we create a comprehensive pipeline called Extensive de-novo TE Annotator (EDTA) that produces a filtered non-redundant TE library for annotation of structurally intact and fragmented elements. EDTA also deconvolutes nested TE insertions frequently found in highly repetitive genomic regions. Using other model species with curated TE libraries (maize and Drosophila), EDTA is shown to be robust across both plant and animal species. Conclusions The benchmarking results and pipeline developed here will greatly facilitate TE annotation in eukaryotic genomes. These annotations will promote a much more in-depth understanding of the diversity and evolution of TEs at both intra- and inter-species levels. EDTA is open-source and freely available: https://github.com/oushujun/EDTA.

scholarly journals Rapid evolution of piRNA-mediated silencing of an invading transposable element was driven by abundant de novo mutations

2019 ◽  
Author(s):  
Shuo Zhang ◽  
Erin S. Kelleher
Keyword(s):  
Transposable Element ◽  
Small Rnas ◽  
Evolutionary Dynamics ◽  
De Novo ◽  
Rapid Evolution ◽  
De Novo Mutations ◽  
Element Insertion ◽  
Pirna Cluster ◽  
Genomic Regions ◽  
First Time

ABSTRACTThe regulation of transposable element (TE) activity by small RNAs is a ubiquitous feature of germlines. However, despite the obvious benefits to the host in terms of ensuring the production of viable gametes and maintaining the integrity of the genomes they carry, it remains controversial whether TE regulation evolves adaptively. We examined the emergence and evolutionary dynamics of repressor alleles after P-elements invaded the Drosophila melanogaster genome in the mid 20th century. In many animals including Drosophila, repressor alleles are produced by transpositional insertions into piRNA clusters, genomic regions encoding the Piwi-interacting RNAs (piRNAs) that regulate TEs. We discovered that ∼94% of recently collected isofemale lines in the Drosophila Genetic Reference Panel (DGRP) contain at least one P-element insertion in a piRNA cluster, indicating that repressor alleles are produced by de novo insertion at an exceptional rate. Furthermore, in our sample of ∼200 genomes, we uncovered no fewer than 80 unique P-element insertion alleles in at least 15 different piRNA clusters. Finally, we observe no footprint of positive selection on P-element insertions in piRNA clusters, suggesting that the rapid evolution of piRNA-mediated repression in D. melanogaster was driven primarily by mutation. Our results reveal for the first time how the unique genetic architecture of piRNA production, in which numerous piRNA clusters can encode regulatory small RNAs upon transpositional insertion, facilitates the non-adaptive rapid evolution of repression.

scholarly journals RNA-seq, bioinformatic identification of potential MicroRNA-Like Small RNAs in the edible mushroom Agaricus bisporus and experimental approach for their validation

2021 ◽  
Author(s):  
Francisco R Marin ◽  
Alberto Davalos ◽  
Dylan Kiltschewskij-Brown ◽  
Maria C Crespo ◽  
Murray J Cairns ◽  
...  
Keyword(s):  
Small Rnas ◽  
Agaricus Bisporus ◽  
Metabolic Regulation ◽  
Experimental Approach ◽  
De Novo ◽  
Edible Mushroom ◽  
Rna Seq ◽  
Mushroom Species ◽  
Bioinformatic Tools ◽  
Edible Parts

Although genomes from many edible mushrooms are sequenced, studies on fungal miRNAs are scarce. Most of the bioinformatic tools are designed for plants or animals but fungal miRNAs processing and expression share similarities and differences with both kingdoms. Moreover, since mushroom species such as Agaricus bisporus (white button mushroom) are frequently consumed as food, controversial discussions are still evaluating whether their miRNAs might or might not be assimilated, perhaps within extracellular vesicles (i.e exosomes). Therefore, the A. bisporus RNA-seq was studied in order to identify potential de novo miRNA-like small RNAs (milRNAs) that might allow their later detection in the diet. Results pointed to 1 already known and 37 de novo milRNAss. Three milRNAss were selected for RT-qPCR experiments. Precursors and mature milRNAs were found in the edible parts (caps and stipes) validating the predictions carried out in silico. When their potential gene targets were investigated, results pointed that mostly were involved in primary and secondary metabolic regulation. However, when human transcriptome is used as target, the results suggest that they might interfere with important biological processes related with cancer, infectious process and neurodegenerative diseases.

scholarly journals Publisher Correction: Germline de novo mutation clusters arise during oocyte aging in genomic regions with high double-strand-break incidence

2021 ◽  
Author(s):  
Jakob M. Goldmann ◽  
Vladimir B. Seplyarskiy ◽  
Wendy S. W. Wong ◽  
Thierry Vilboux ◽  
Pieter B. Neerincx ◽  
...  
Keyword(s):  
De Novo ◽  
Strand Break ◽  
Double Strand Break ◽  
De Novo Mutation ◽  
Oocyte Aging ◽  
Genomic Regions

scholarly journals Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

2021 ◽  
pp. eabd6990
Author(s):  
Sang Il Kim ◽  
Jinsung Noh ◽  
Sujeong Kim ◽  
Younggeun Choi ◽  
Duck Kyun Yoo ◽  
...  
Keyword(s):  
B Cells ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Binding Domain ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

STEM-18. STROMAL EXTRACELLULAR VESICLES DRIVE GLIOMA STEM CELL REPROGRAMMING

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi24-vi25
Author(s):  
Lata Adnani ◽  
Brian Meehan ◽  
Jordan Kassouf ◽  
Cristiana Spinelli ◽  
Nadim Tawil ◽  
...  
Keyword(s):  
Molecular Subtype ◽  
Extracellular Vesicle ◽  
Host Cells ◽  
Tumour Mass ◽  
Membrane Structures ◽  
And Function ◽  
Rate Limiting ◽  
The Brain

Abstract Glioblastoma multiforme (GBM) represents the most frequent and lethal form of brain tumors originating from glioma stem cells (GSCs). GBM remains lethal because the rate limiting patho-mechanisms remain poorly understood. In this regard, few limitations involve the diversity 'between' cellular states and the molecular/cellular complexity 'within' the tumour mass. Using cell based- and mouse- models, we explored extracellular vesicle (EV) mediated interactions between cancer and stromal cells impacting phenotypes of GSCs as a function of their molecular subtype. EVs are spherical membrane structures that cells release to expel different molecular cargo (lipids, proteins, RNA, DNA), which can be transported across a distance in the brain and taken up by various ‘recipient’ cells resulting in reprogramming of the recipient cell's content and function. In vivo, GSCs altered their pattern of NOTCH signalling and molecular phenotype as a function of proximity to non-transformed host cells in the brain. In vitro stromal EVs altered GSC sphere forming capacity, proteome and expression of mesenchymal markers. Thus, EV mediated tumour-stromal interactions could represent a biological switch and a novel targeting point in the biology of GBM.

scholarly journals Type III Secretion Decreases Bacterial and Host Survival following Phagocytosis of Yersinia pseudotuberculosis by Macrophages

2008 ◽  
Vol 76 (9) ◽  
pp. 4299-4310 ◽  
Author(s):  
Yue Zhang ◽  
James Murtha ◽  
Margaret A. Roberts ◽  
Richard M. Siegel ◽  
James B. Bliska
Keyword(s):  
Cell Death ◽  
Type Iii Secretion ◽  
Yersinia Pseudotuberculosis ◽  
De Novo ◽  
Host Cells ◽  
Host Responses ◽  
Intracellular Bacteria ◽  
Wild Type ◽  
Macrophage Response ◽  
Type Iii

ABSTRACT Yersinia pseudotuberculosis uses a plasmid (pYV)-encoded type III secretion system (T3SS) to translocate a set of effectors called Yops into infected host cells. YopJ functions to induce apoptosis, and YopT, YopE, and YopH act to antagonize phagocytosis in macrophages. Because Yops do not completely block phagocytosis and Y. pseudotuberculosis can replicate in macrophages, it is important to determine if the T3SS modulates host responses to intracellular bacteria. Isogenic pYV-cured, pYV+ wild-type, and yop mutant Y. pseudotuberculosis strains were allowed to infect bone marrow-derived murine macrophages at a low multiplicity of infection under conditions in which the survival of extracellular bacteria was prevented. Phagocytosis, the intracellular survival of the bacteria, and the apoptosis of the infected macrophages were analyzed. Forty percent of cell-associated wild-type bacteria were intracellular after a 20-min infection, allowing the study of the macrophage response to internalized pYV+ Y. pseudotuberculosis. Interestingly, macrophages restricted survival of pYV+ but not pYV-cured or ΔyopB Y. pseudotuberculosis within phagosomes: only a small fraction of the pYV+ bacteria internalized replicated by 24 h. In addition, ∼20% of macrophages infected with wild-type pYV+ Y. pseudotuberculosis died of apoptosis after 20 h. Analysis of yop mutants expressing catalytically inactive effectors revealed that YopJ was important for apoptosis, while a role for YopE, YopH, and YopT in modulating macrophage responses to intracellular bacteria could not be identified. Apoptosis was reduced in Toll-like receptor 4-deficient macrophages, indicating that cell death required signaling through this receptor. Treatment of macrophages harboring intracellular pYV+ Y. pseudotuberculosis with chloramphenicol reduced apoptosis, indicating that the de novo bacterial protein synthesis was necessary for cell death. Our finding that the presence of a functional T3SS impacts the survival of both bacterium and host following phagocytosis of Y. pseudotuberculosis suggests new roles for the T3SS in Yersinia pathogenesis.

scholarly journals Nurse cell-derived small RNAs define paternal epigenetic inheritance in Arabidopsis

2021 ◽  
Author(s):  
Jincheng Long ◽  
James Walker ◽  
Wenjing She ◽  
Billy Aldridge ◽  
Hongbo Gao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Small Rnas ◽  
Nurse Cell ◽  
De Novo ◽  
Epigenetic Inheritance ◽  
Genome Integrity ◽  
Nurse Cells ◽  
Male Germline ◽  
Methylation Patterns

AbstractThe plant male germline undergoes DNA methylation reprogramming, which methylates genes de novo and thereby alters gene expression and facilitates meiosis. Why reprogramming is limited to the germline and how specific genes are chosen is unknown. Here, we demonstrate that genic methylation in the male germline, from meiocytes to sperm, is established by germline-specific siRNAs transcribed from transposons with imperfect sequence homology. These siRNAs are synthesized by meiocyte nurse cells (tapetum) via activity of the tapetum-specific chromatin remodeler CLASSY3. Remarkably, tapetal siRNAs govern germline methylation throughout the genome, including the inherited methylation patterns in sperm. Finally, we demonstrate that these nurse cell-derived siRNAs (niRNAs) silence germline transposons, thereby safeguarding genome integrity. Our results reveal that tapetal niRNAs are sufficient to reconstitute germline methylation patterns and drive extensive, functional methylation reprogramming analogous to piRNA-mediated reprogramming in animal germlines.

scholarly journals A new form of actin assembly around the Shigella-containing vacuole regulates its intracellular niche

2020 ◽  
Author(s):  
Sonja Kühn ◽  
John Bergqvist ◽  
Laura Barrio ◽  
Stephanie Lebreton ◽  
Chiara Zurzolo ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
De Novo ◽  
Shigella Flexneri ◽  
Host Cells ◽  
Host Recognition ◽  
Actin Assembly ◽  
Actin Structure ◽  
New Form ◽  
Structure And Functions

SUMMARYThe enteroinvasive bacterium Shigella flexneri forces its uptake into non-phagocytic host cells through the translocation of T3SS effectors that subvert the actin cytoskeleton. Here, we report de novo actin polymerization after cellular entry around the bacterial containing vacuole (BCV) leading to the formation of a dynamic actin cocoon. This cocoon is thicker than any described cellular actin structure and functions as a gatekeeper for the cytosolic access of the pathogen. Host Cdc42, Toca-1, N-WASP, WIP, the Arp2/3 complex, cortactin, coronin, and cofilin are recruited to the actin cocoon. They are subverted by T3SS effectors, such as IpgD, IpgB1, and IcsB. IcsB immobilizes components of the actin polymerization machinery at the BCV. This represents a novel microbial subversion strategy through localized entrapment of host actin regulators causing massive actin assembly. We propose that the cocoon protects Shigella’s niche from canonical maturation or host recognition.

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