scholarly journals Structured RNA Contaminants in Bacterial Ribo-Seq

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Brayon J. Fremin ◽  
Ami S. Bhatt
Keyword(s):  
Rna Structure ◽  
Large Scale ◽  
Partial Information ◽  
Ribosome Profiling ◽  
Micrococcal Nuclease ◽  
Data Sets ◽  
Rna Structures ◽  
Content Type ◽  
Sequencing Library

ABSTRACT Ribosome profiling (Ribo-Seq) is a powerful method to study translation in bacteria. However, Ribo-Seq signal can be observed across RNAs that one would not expect to be bound by ribosomes. For example, Escherichia coli Ribo-Seq libraries also capture reads from most noncoding RNAs (ncRNAs). While some of these ncRNAs may overlap coding regions, this alone does not explain the majority of observed signal across ncRNAs. These fragments of ncRNAs in Ribo-Seq data pass all size selection steps of the Ribo-Seq protocol and survive hours of micrococcal nuclease (MNase) treatment. In this work, we specifically focus on Ribo-Seq signal across ncRNAs and provide evidence to suggest that RNA structure, as opposed to ribosome binding, protects them from degradation and allows them to persist in the Ribo-Seq sequencing library preparation. By inspecting these “contaminant reads” in bacterial Ribo-Seq, we show that data previously disregarded in bacterial Ribo-Seq experiments may, in fact, be used to gain partial information regarding the in vivo secondary structure of ncRNAs. IMPORTANCE Structured ncRNAs are pivotal mediators of bioregulation in bacteria, and their functions are often reliant on their specific structures. Here, we first inspect Ribo-Seq reads across noncoding regions, identifying contaminant reads in these libraries. We observe that contaminant reads in bacterial Ribo-Seq experiments that are often disregarded, in fact, strongly overlap with structured regions of ncRNAs. We then perform several bioinformatic analyses to determine why these contaminant reads may persist in Ribo-Seq libraries. Finally, we highlight some structured RNA contaminants in Ribo-Seq and support the hypothesis that structures in the RNA protect them from MNase digestion. We conclude that researchers should be cautious when interpreting Ribo-Seq signal as coding without considering signal distribution. These findings also may enable us to partially resolve RNA structures, identify novel structured RNAs, and elucidate RNA structure-function relationships in bacteria at a large scale and in vivo through the reanalysis of existing Ribo-Seq data sets.

scholarly journals Repurposing Ribo-Seq to provide insights into structured RNAs

2020 ◽  
Author(s):  
Brayon J. Fremin ◽  
Ami S. Bhatt
Keyword(s):  
Secondary Structure ◽  
Rna Structure ◽  
Large Scale ◽  
Structural Information ◽  
Ribosome Profiling ◽  
Rna Structures ◽  
Size Selection ◽  
Bacterial Ribosome ◽  
Powerful Approach

AbstractRibosome profiling (Ribo-Seq) is a powerful method to study translation in bacteria. However, this method can enrich RNAs that are not bound by ribosomes, but rather, are protected from degradation in another way. For example, Escherichia coli Ribo-Seq libraries also capture reads from most non-coding RNAs (ncRNAs). These fragments of ncRNAs pass all size selection steps of the Ribo-Seq protocol and survive hours of MNase treatment, presumably without protection from the ribosome or other macromolecules or proteins. Since bacterial ribosome profiling does not directly isolate ribosomes, but instead uses broad size range cutoffs to fractionate actively translated RNAs, it is understandable that some ncRNAs are retained after size selection. However, how these ‘contaminants’ survive MNase treatment is unclear. Through analyzing metaRibo-Seq reads across ssrS, a well established structured RNA in E. coli, and structured direct repeats from Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) arrays in Ruminococcus lactaris, we observed that these RNAs are protected from MNase treatment by virtue of their secondary structure. Therefore, large volumes of data previously discarded as contaminants in bacterial Ribo-Seq experiments can, in fact, be used to gain information regarding the in vivo secondary structure of ncRNAs, providing unique insight into their native functional structures.ImportanceWe observe that ‘contaminant’ signals in bacterial Ribo-Seq experiments that are often disregarded and discarded, in fact, strongly overlap with structured regions of ncRNAs. Structured ncRNAs are pivotal mediators of bioregulation in bacteria and their functions are often reliant on their specific structures. We present an approach to access important RNA structural information through merely repurposing ‘contaminant’ signals in bacterial Ribo-Seq experiments. This powerful approach enables us to partially resolve RNA structures, identify novel structured RNAs, and elucidate RNA structure-function relationships in bacteria at a large-scale and in vivo.

scholarly journals Blakeslea trispora Photoreceptors: Identification and Functional Analysis

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Wei Luo ◽  
Chao Xue ◽  
Yuzheng Zhao ◽  
Huili Zhang ◽  
Zhiming Rao ◽  
...  
Keyword(s):  
Large Scale ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Flavin Mononucleotide ◽  
Blakeslea Trispora ◽  
Scale Production ◽  
Bioinformatics Analyses ◽  
Content Type ◽  
Flavin Binding

ABSTRACT Blakeslea trispora is an industrial fungal species used for large-scale production of carotenoids. However, B. trispora light-regulated physiological processes, such as carotenoid biosynthesis and phototropism, are not fully understood. In this study, we isolated and characterized three photoreceptor genes, btwc-1a, btwc-1b, and btwc-1c, in B. trispora. Bioinformatics analyses of these genes and their protein sequences revealed that the functional domains (PAS/LOV [Per-ARNT-Sim/light-oxygen-voltage] domain and zinc finger structure) of the proteins have significant homology to those of other fungal blue-light regulator proteins expressed by Mucor circinelloides and Neurospora crassa. The photoreceptor proteins were synthesized by heterologous expression in Escherichia coli. The chromogenic groups consisting of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were detected to accompany BTWC-1 proteins by using high-performance liquid chromatography (HPLC) and fluorescence spectrometry, demonstrating that the proteins may be photosensitive. The absorbance changes of the purified BTWC-1 proteins seen under dark and light conditions indicated that they were light responsive and underwent a characteristic photocycle by light induction. Site-directed mutagenesis of the cysteine residual (Cys) in BTWC-1 did not affect the normal expression of the protein in E. coli but did lead to the loss of photocycle response, indicating that Cys represents a flavin-binding domain for photon detection. We then analyzed the functions of BTWC-1 proteins by complementing btwc-1a, btwc-1b, and btwc-1c into the counterpart knockout strains of M. circinelloides for each mcwc-1 gene. Transformation of the btwc-1a complement into mcwc-1a knockout strains restored the positive phototropism, while the addition of btwc-1c complement remedied the deficiency of carotene biosynthesis in the mcwc-1c knockout strains under conditions of illumination. These results indicate that btwc-1a and btwc-1c are involved in phototropism and light-inducible carotenogenesis. Thus, btwc-1 genes share a conserved flavin-binding domain and act as photoreceptors for control of different light transduction pathways in B. trispora. IMPORTANCE Studies have confirmed that light-regulated carotenogenesis is prevalent in filamentous fungi, especially in mucorales. However, few investigations have been done to understand photoinduced synthesis of carotenoids and related mechanisms in B. trispora, a well-known industrial microbial strains. In the present study, three photoreceptor genes in B. trispora were cloned, expressed, and characterized by bioinformatics and photoreception analyses, and then in vivo functional analyses of these genes were constructed in M. circinelloides. The results of this study will lead to a better understanding of photoreception and light-regulated carotenoid synthesis and other physiological responses in B. trispora.

scholarly journals Active ribosome profiling with RiboLace

2017 ◽  
Author(s):  
Massimiliano Clamer ◽  
Toma Tebaldi ◽  
Fabio Lauria ◽  
Paola Bernabò ◽  
Rodolfo F. Gómez-Biagi ◽  
...  
Keyword(s):  
Large Scale ◽  
Positional Information ◽  
Ribosome Profiling ◽  
Input Material ◽  
Nuclease Digestion ◽  
Rna Fragments ◽  
Nucleotide Resolution ◽  
Active Ribosome

Ribosome profiling, or Ribo-Seq, is based around large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information concerning ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-Seq approaches lack the ability to distinguish between fragments protected by ribosomes in active translation or by inactive ribosomes. To overcome these significant limitation, we developed RiboLace: a novel method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied bothin vitroandin vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.

scholarly journals GPRC5b Modulates Inflammatory Response in Glomerular Diseases via NF-κB Pathway

2019 ◽  
Vol 30 (9) ◽  
pp. 1573-1586 ◽  
Author(s):  
Sonia Zambrano ◽  
Katja Möller-Hackbarth ◽  
Xidan Li ◽  
Patricia Q. Rodriguez ◽  
Emmanuelle Charrin ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Knockout Mice ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Inflammatory Cells ◽  
Data Sets ◽  
Glomerular Diseases ◽  
Filtration Barrier ◽  
Inflammatory Processes

BackgroundInflammatory processes play an important role in the pathogenesis of glomerulopathies. Finding novel ways to suppress glomerular inflammation may offer a new way to stop disease progression. However, the molecular mechanisms that initiate and drive inflammation in the glomerulus are still poorly understood.MethodsWe performed large-scale gene expression profiling of glomerulus-associated G protein–coupled receptors (GPCRs) to identify new potential therapeutic targets for glomerulopathies. The expression of Gprc5b in disease was analyzed using quantitative PCR and immunofluorescence, and by analyzing published microarray data sets. In vivo studies were carried out in a podocyte-specific Gprc5b knockout mouse line. Mechanistic studies were performed in cultured human podocytes.ResultsWe identified an orphan GPCR, Gprc5b, as a novel gene highly enriched in podocytes that was significantly upregulated in common human glomerulopathies, including diabetic nephropathy, IgA nephropathy, and lupus nephritis. Similar upregulation of Gprc5b was detected in LPS-induced nephropathy in mice. Studies in podocyte-specific Gprc5b knockout mice showed that Gprc5b was not essential for normal development of the glomerular filtration barrier. However, knockout mice were partially protected from LPS-induced proteinuria and recruitment of inflammatory cells. Mechanistically, RNA sequencing in Gprc5b knockouts mice and experiments in cultured human podocytes showed that Gpr5cb regulated inflammatory response in podocytes via NF-κB signaling.ConclusionsGPRC5b is a novel podocyte-specific receptor that regulates inflammatory response in the glomerulus by modulating the NF-κB signaling pathway. Upregulation of Gprc5b in human glomerulopathies suggests that it may play a role in their pathogenesis.

An efficient algorithm for planar drawing of RNA structures with pseudoknots of any type

2016 ◽  
Vol 14 (03) ◽  
pp. 1650009 ◽  
Author(s):  
Yanga Byun ◽  
Kyungsook Han
Keyword(s):  
Rna Structure ◽  
Large Scale ◽  
Structural Elements ◽  
Rna Structures ◽  
Structural Element ◽  
Rna Pseudoknots ◽  
Number Of Stems ◽  
Rna Pseudoknot ◽  
Aesthetic Structure ◽  
Planar Drawing

An RNA pseudoknot is a tertiary structural element in which bases of a loop pair with complementary bases are outside the loop. A drawing of RNA secondary structures is a tree, but a drawing of RNA pseudoknots is a graph that has an inner cycle within a pseudoknot and possibly outer cycles formed between the pseudoknot and other structural elements. Visualizing a large-scale RNA structure with pseudoknots as a planar drawing is challenging because a planar drawing of an RNA structure requires both pseudoknots and an entire structure enclosing the pseudoknots to be embedded into a plane without overlapping or crossing. This paper presents an efficient heuristic algorithm for visualizing a pseudoknotted RNA structure as a planar drawing. The algorithm consists of several parts for finding crossing stems and page mapping the stems, for the layout of stem-loops and pseudoknots, and for overlap detection between structural elements and resolving it. Unlike previous algorithms, our algorithm generates a planar drawing for a large RNA structure with pseudoknots of any type and provides a bracket view of the structure. It generates a compact and aesthetic structure graph for a large pseudoknotted RNA structure in O([Formula: see text]) time, where n is the number of stems of the RNA structure.

Size matters

2014 ◽  
Vol 24 (3) ◽  
pp. 224-237 ◽  
Author(s):  
Valerie Johnson ◽  
Sonia Ranade ◽  
David Thomas
Keyword(s):  
Large Scale ◽  
Cost Savings ◽  
Large Data ◽  
Data Sets ◽  
Content Type ◽  
Large Scale Data ◽  
Archival Record ◽  
Original Record ◽  
Definition Of ◽  
Practical Implications

Purpose – This paper aims to focus on a highly significant yet under-recognised concern: the huge growth in the volume of digital archival information and the implications of this shift for information professionals. Design/methodology/approach – Though data loss and format obsolescence are often considered to be the major threats to digital records, the problem of scale remains under-acknowledged. This paper discusses this issue, and the challenges it brings using a case study of a set of Second World War service records. Findings – TNA’s research has shown that it is possible to digitise large volumes of records to replace paper originals using rigorous procedures. Consequent benefits included being able to link across large data sets so that further records could be released. Practical implications – The authors will discuss whether the technical capability, plus space and cost savings will result in increased pressure to retain, and what this means in creating a feedback-loop of volume. Social implications – The work also has implications in terms of new definitions of the “original” archival record. There has been much debate on challenges to the definition of the archival record in the shift from paper to born-digital. The authors will discuss where this leaves the digitised “original” record. Originality/value – Large volumes of digitised and born-digital records are starting to arrive in records and archive stores, and the implications for retention are far wider than simply digital preservation. By sharing novel research into the practical implications of large-scale data retention, this paper showcases potential issues and some approaches to their management.

scholarly journals Genome-wide mapping of SARS-CoV-2 RNA structures identifies therapeutically-relevant elements

2020 ◽  
Vol 48 (22) ◽  
pp. 12436-12452 ◽  
Author(s):  
Ilaria Manfredonia ◽  
Chandran Nithin ◽  
Almudena Ponce-Salvatierra ◽  
Pritha Ghosh ◽  
Tomasz K Wirecki ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Structure ◽  
Therapeutic Strategies ◽  
Rna Structures ◽  
Genome Wide ◽  
High Sequence Conservation ◽  
Infected Cells ◽  
Rna Elements

Abstract SARS-CoV-2 is a betacoronavirus with a linear single-stranded, positive-sense RNA genome, whose outbreak caused the ongoing COVID-19 pandemic. The ability of coronaviruses to rapidly evolve, adapt, and cross species barriers makes the development of effective and durable therapeutic strategies a challenging and urgent need. As for other RNA viruses, genomic RNA structures are expected to play crucial roles in several steps of the coronavirus replication cycle. Despite this, only a handful of functionally-conserved coronavirus structural RNA elements have been identified to date. Here, we performed RNA structure probing to obtain single-base resolution secondary structure maps of the full SARS-CoV-2 coronavirus genome both in vitro and in living infected cells. Probing data recapitulate the previously described coronavirus RNA elements (5′ UTR and s2m), and reveal new structures. Of these, ∼10.2% show significant covariation among SARS-CoV-2 and other coronaviruses, hinting at their functionally-conserved role. Secondary structure-restrained 3D modeling of these segments further allowed for the identification of putative druggable pockets. In addition, we identify a set of single-stranded segments in vivo, showing high sequence conservation, suitable for the development of antisense oligonucleotide therapeutics. Collectively, our work lays the foundation for the development of innovative RNA-targeted therapeutic strategies to fight SARS-related infections.

scholarly journals Lost and Found: Re-searching and Re-scoring Proteomics Data Aids Genome Annotation and Improves Proteome Coverage

mSystems ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Patrick Willems ◽  
Igor Fijalkowski ◽  
Petra Van Damme
Keyword(s):  
Genome Annotation ◽  
Deinococcus Radiodurans ◽  
Gene Annotation ◽  
Bacterial Genome ◽  
Prokaryotic Genome ◽  
Ribosome Profiling ◽  
Great Promise ◽  
Data Sets ◽  
Proteome Coverage ◽  
Content Type

ABSTRACT Prokaryotic genome annotation is heavily dependent on automated gene annotation pipelines that are prone to propagate errors and underestimate genome complexity. We describe an optimized proteogenomic workflow that uses ribosome profiling (ribo-seq) and proteomic data for Salmonella enterica serovar Typhimurium to identify unannotated proteins or alternative protein forms. This data analysis encompasses the searching of cofragmenting peptides and postprocessing with extended peptide-to-spectrum quality features, including comparison to predicted fragment ion intensities. When this strategy is applied, an enhanced proteome depth is achieved, as well as greater confidence for unannotated peptide hits. We demonstrate the general applicability of our pipeline by reanalyzing public Deinococcus radiodurans data sets. Taken together, our results show that systematic reanalysis using available prokaryotic (proteome) data sets holds great promise to assist in experimentally based genome annotation. IMPORTANCE Delineation of open reading frames (ORFs) causes persistent inconsistencies in prokaryote genome annotation. We demonstrate that by advanced (re)analysis of omics data, a higher proteome coverage and sensitive detection of unannotated ORFs can be achieved, which can be exploited for conditional bacterial genome (re)annotation, which is especially relevant in view of annotating the wealth of sequenced prokaryotic genomes obtained in recent years.

scholarly journals The Small RNA sr8384 Is a Crucial Regulator of Cell Growth in Solventogenic Clostridia

2020 ◽  
Vol 86 (13) ◽  
Author(s):  
Yunpeng Yang ◽  
Huan Zhang ◽  
Nannan Lang ◽  
Lu Zhang ◽  
Changsheng Chai ◽  
...  
Keyword(s):  
Cell Growth ◽  
Clostridium Acetobutylicum ◽  
Large Scale ◽  
Biological Processes ◽  
Content Type ◽  
Functional Studies ◽  
Biochemical Analyses ◽  
Solventogenic Clostridia

ABSTRACT Small RNAs (sRNAs) are crucial regulatory molecules in organisms and are well-known not only for their roles in the control of diverse crucial biological processes but also for their value in regulation rewiring. However, to date, in Gram-positive anaerobic solventogenic clostridia (a group of important industrial bacteria with exceptional substrate and product diversity), sRNAs remain minimally explored, and thus there is a lack of detailed understanding regarding these important molecules and their use as targets for genetic improvement. Here, we performed large-scale phenotypic screens of a transposon-mediated mutant library of Clostridium acetobutylicum, a typical solventogenic clostridial species, and discovered a novel sRNA (sr8384) that functions as a crucial regulator of cell growth. Comparative transcriptomic data combined with genetic and biochemical analyses revealed that sr8384 acts as a pleiotropic regulator and controls multiple targets that are associated with crucial biological processes through direct or indirect interactions. Notably, the in vivo expression level of sr8384 determined the cell growth rate, thereby affecting the solvent titer and productivity. These findings indicate the importance of the sr8384-mediated regulatory network in C. acetobutylicum. Furthermore, a homolog of sr8384 was discovered and proven to be functional in another important Clostridium species, C. beijerinckii, suggesting the potential broad role of this sRNA in clostridia. Our work showcases a previously unknown potent and complex role of sRNAs in clostridia, providing new opportunities for understanding and engineering these anaerobes. IMPORTANCE The uses of sRNAs as new resources for functional studies and strain modifications are promising strategies in microorganisms. However, these crucial regulatory molecules have hardly been explored in industrially important solventogenic clostridia. Here, we identified sr8384 as a novel determinant sRNA controlling the cell growth of solventogenic Clostridium acetobutylicum. Based on a detailed functional analysis, we further reveal the pleiotropic function of sr8384 and its multiple direct and indirect crucial targets, which represents a valuable source for understanding and optimizing this anaerobe. Of note, manipulation of this sRNA achieves improved cell growth and solvent synthesis. Our findings provide a new perspective for future studies on regulatory sRNAs in clostridia.

scholarly journals Light-Dependent Translation Change of Arabidopsis psbA Correlates with RNA Structure Alterations at the Translation Initiation Region

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 322
Author(s):  
Piotr Gawroński ◽  
Christel Enroth ◽  
Peter Kindgren ◽  
Sebastian Marquardt ◽  
Stanisław Karpiński ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Translation Initiation ◽  
Rna Structure ◽  
Regulatory Protein ◽  
High Light ◽  
Ribosome Profiling ◽  
General Mechanism ◽  
Mrna Secondary Structure ◽  
Translation Initiation Region

mRNA secondary structure influences translation. Proteins that modulate the mRNA secondary structure around the translation initiation region may regulate translation in plastids. To test this hypothesis, we exposed Arabidopsis thaliana to high light, which induces translation of psbA mRNA encoding the D1 subunit of photosystem II. We assayed translation by ribosome profiling and applied two complementary methods to analyze in vivo RNA secondary structure: DMS-MaPseq and SHAPE-seq. We detected increased accessibility of the translation initiation region of psbA after high light treatment, likely contributing to the observed increase in translation by facilitating translation initiation. Furthermore, we identified the footprint of a putative regulatory protein in the 5′ UTR of psbA at a position where occlusion of the nucleotide sequence would cause the structure of the translation initiation region to open up, thereby facilitating ribosome access. Moreover, we show that other plastid genes with weak Shine-Dalgarno sequences (SD) are likely to exhibit psbA-like regulation, while those with strong SDs do not. This supports the idea that changes in mRNA secondary structure might represent a general mechanism for translational regulation of psbA and other plastid genes.

Sign in / Sign up

Export Citation Format

Share Document