scholarly journals Single molecule structure sequencing reveals RNA structural dependencies, breathing and ensembles

2020 ◽  
Author(s):  
Teshome Tilahun Bizuayehu ◽  
Kornel Labun ◽  
Kirill Jefimov ◽  
Eivind Valen
Keyword(s):  
Single Molecule ◽  
High Throughput Sequencing ◽  
Structural Heterogeneity ◽  
Structural Features ◽  
Limiting Factor ◽  
Tertiary Structures ◽  
Rna Molecules ◽  
Molecule Structure ◽  
Chemical Probing ◽  
And Function

ABSTRACTRNA molecules can form secondary and tertiary structures that determine their localization and function1. Using enzymatic or chemical probing together with high-throughput sequencing, secondary structure can be mapped across the entire transcriptome2. A limiting factor, however, is that only population averages can be obtained, since each read is an independent measurement. In addition, no information about structural heterogeneity across molecules or dependencies within each molecule is accessible. Here, we present Single Molecule Structure sequencing (SMS-seq) that combines structural probing with native RNA sequencing to provide non-amplified, structural profiles of individual molecules. Each RNA is probed at numerous bases enabling the discovery of dependencies and heterogeneity of structural features. In addition to revealing known structural features of mRNAs, SMS-seq shows compartmentalization of structural dependencies across CDSs and 3’UTRs. Finally, we show that SMS-seq can capture structural breathing, tertiary interactions and dynamics of riboswitch ligand binding.

scholarly journals Lead-seq: transcriptome-wide structure probing in vivo using lead(II) ions

2020 ◽  
Vol 48 (12) ◽  
pp. e71-e71 ◽  
Author(s):  
Christian Twittenhoff ◽  
Vivian B Brandenburg ◽  
Francesco Righetti ◽  
Aaron M Nuss ◽  
Axel Mosig ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Yersinia Pseudotuberculosis ◽  
Structural Information ◽  
Dimethyl Sulfate ◽  
Bacterial Pathogen ◽  
Structural Features ◽  
Global Scale ◽  
Rna Molecules ◽  
Different Temperatures

Abstract The dynamic conformation of RNA molecules within living cells is key to their function. Recent advances in probing the RNA structurome in vivo, including the use of SHAPE (Selective 2′-Hydroxyl Acylation analyzed by Primer Extension) or kethoxal reagents or DMS (dimethyl sulfate), provided unprecedented insights into the architecture of RNA molecules in the living cell. Here, we report the establishment of lead probing in a global RNA structuromics approach. In order to elucidate the transcriptome-wide RNA landscape in the enteric pathogen Yersinia pseudotuberculosis, we combined lead(II) acetate-mediated cleavage of single-stranded RNA regions with high-throughput sequencing. This new approach, termed ‘Lead-seq’, provides structural information independent of base identity. We show that the method recapitulates secondary structures of tRNAs, RNase P RNA, tmRNA, 16S rRNA and the rpsT 5′-untranslated region, and that it reveals global structural features of mRNAs. The application of Lead-seq to Y. pseudotuberculosis cells grown at two different temperatures unveiled the first temperature-responsive in vivo RNA structurome of a bacterial pathogen. The translation of candidate genes derived from this approach was confirmed to be temperature regulated. Overall, this study establishes Lead-seq as complementary approach to interrogate intracellular RNA structures on a global scale.

scholarly journals CoDNaS-RNA: a database of Conformational Diversity in the Native State of RNA

2020 ◽  
Author(s):  
Martín González Buitrón ◽  
Ronaldo Romario Tunque Cahui ◽  
Emilio García Ríos ◽  
Layla Hirsh ◽  
María Silvina Fornasari ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Features ◽  
Rna Structures ◽  
Rna Molecules ◽  
Functional Annotations ◽  
Public Resource ◽  
Conformational Diversity ◽  
Dynamics And Function ◽  
And Function ◽  
The Relationship

AbstractConformational changes in RNA native ensembles are central to fulfill many of their biological roles. Systematic knowledge of the extent and possible modulators of this conformational diversity is desirable to better understand the relationship between RNA dynamics and function.We have developed CoDNaS-RNA as the first database of conformational diversity in RNA molecules. Known RNA structures are retrieved and clustered to identify alternative conformers of each molecule. Pairwise structural comparisons within each cluster allows to measure the variability of the molecule. Additional data on structural features, molecular interactions and functional annotations are provided. CoDNaS-RNA is implemented as a public resource that can be of much interest for computational and bench scientists alike.AvailabilityCoDNaS-RNA is freely accessible at http://ufq.unq.edu.ar/[email protected]

scholarly journals Single-molecule and Single-cell Approaches in Molecular Bioengineering

2020 ◽  
Vol 74 (9) ◽  
pp. 704-709
Author(s):  
Michael A. Nash
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
High Throughput Screening ◽  
Protein Sequences ◽  
Structural Features ◽  
Functional Protein ◽  
Molecular Systems ◽  
Measurement Tools ◽  
And Function ◽  
Control Protein

Protein sequences inhabit a discrete set in macromolecular space with incredible capacity to treat human disease. Despite our ability to program and manipulate protein sequences, the vast majority of protein development efforts are still done heuristically without a unified set of guiding principles. This article highlights work in understanding biophysical stability and function of proteins, developing new biophysical measurement tools and building high-throughput screening platforms to explore functional protein sequences. We highlight two primary areas. First, molecular biomechanics is a subfield concerned with the response of proteins to mechanical forces, and how we can leverage mechanical force to control protein function. The second subfield investigates the use of polymers and hydrogels in protein engineering and directed evolution in pursuit of new molecular systems with therapeutic applications. These two subdisciplines complement each other by shedding light onto sequence and structural features that can be used to impart stability into therapeutic proteins.

scholarly journals Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers

2018 ◽  
Author(s):  
Yu Fu ◽  
Pei-Hsuan Wu ◽  
Timothy Beane ◽  
Phillip D. Zamore ◽  
Zhiping Weng
Keyword(s):  
Single Molecule ◽  
Small Rna ◽  
High Throughput Sequencing ◽  
Starting Material ◽  
Quality Data ◽  
Rna Seq ◽  
High Quality Data ◽  
Pcr Duplicates ◽  
Substantial Bias ◽  
And Function

AbstractRNA-seq and small RNA-seq are powerful, quantitative tools to study gene regulation and function. Common high-throughput sequencing methods rely on polymerase chain reaction (PCR) to expand the starting material, but not every molecule amplifies equally, causing some to be overrepresented. Unique molecular identifiers (UMIs) can be used to distinguish undesirable PCR duplicates derived from a single molecule and identical but biologically meaningful reads from different molecules. We have incorporated UMIs into RNA-seq and small RNA-seq protocols and developed tools to analyze the resulting data. Our UMIs contain stretches of random nucleotides whose lengths sufficiently capture diverse molecule species in both RNA-seq and small RNA-seq libraries generated from mouse testis. Our approach yields high-quality data while allowing unique tagging of all molecules in high-depth libraries. Using simulated and real datasets, we demonstrate that our methods increase the reproducibility of RNA-seq and small RNA-seq data. Notably, we find that the amount of starting material and sequencing depth, but not the number of PCR cycles, determine PCR duplicate frequency. Finally, we show that computational removal of PCR duplicates based only on their mapping coordinates introduces substantial bias into data analysis.

Unfolding Single RNA Molecules with Optical Tweezers

2001 ◽  
Vol 7 (S2) ◽  
pp. 26-27
Author(s):  
Carlos Bustamante ◽  
Jan Liphardt ◽  
Bibiana Onoa ◽  
Steven B. Smith ◽  
Delphine Collin ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Structural Features ◽  
Rna Molecules ◽  
Group I ◽  
Tertiary Folding ◽  
Tertiary Interactions

RNA molecules must fold into specific three-dimensional shapes to perform their structural and catalytic functions. Unlike proteins, RNAs secondary structural features are usually stable enough to form by themselves in solution. The reason is that in RNA, the stabilization energy gained from the formation of secondary structure is substantially larger than the energies involved in tertiary interactions. As a result, the formation of tertiary interactions is expected to alter only slightly the pre-existing secondary structural contacts. Moreover, secondary structure prediction is robust and can be made without taking into consideration tertiary folding. However, bulk studies of the energetics and kinetics of their secondary and tertiary folding are often frustrated by the presence of multiple species and multiple folding pathways in solution. These problems are circumvented in single-molecule studies in which the folding/unfolding trajectories of the individual molecules can be followed. The T. thermophila group I intron ribozyme is organized into several domains whose mechanical unfolding can be investigated independently, and whose tertiary contacts are stabilized by numerous Mg++ ions.We have begun characterization of the ribozyme by analysis of the P5abc domain because:

scholarly journals RNA modifications detection by comparative Nanopore direct RNA sequencing

2019 ◽  
Author(s):  
Adrien Leger ◽  
Paulo P. Amaral ◽  
Luca Pandolfini ◽  
Charlotte Capitanchik ◽  
Federica Capraro ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Single Molecule ◽  
High Throughput Sequencing ◽  
Control Sample ◽  
Analytical Framework ◽  
Rna Modifications ◽  
Rna Molecules ◽  
Naturally Occurring ◽  
Oxford Nanopore ◽  
Experimental Approaches

AbstractRNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. To date, over 150 naturally occurring PTMs have been identified, however the overwhelming majority of their functions remain elusive. In recent years, a small number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing (DRS) technology has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework to evaluate the presence of modifications in DRS data. To do so, we compare an RNA sample of interest against a non-modified control sample. Our strategy does not require a training set and allows the use of replicates to model biological variability. Here, we demonstrate the ability of Nanocompore to detect RNA modifications at single-molecule resolution in human polyA+ RNAs, as well as in targeted non-coding RNAs. Our results correlate well with orthogonal methods, confirm previous observations on the distribution of N6-methyladenosine sites and provide novel insights into the distribution of RNA modifications in the coding and non-coding transcriptomes. The latest version of Nanocompore can be obtained at https://github.com/tleonardi/nanocompore.

kangaroo, a Mobile Element From Volvox carteri, Is a Member of a Newly Recognized Third Class of Retrotransposons

Genetics ◽  
2002 ◽  
Vol 162 (4) ◽  
pp. 1617-1630
Author(s):  
Leonard Duncan ◽  
Kristine Bouckaert ◽  
Fay Yeh ◽  
David L Kirk
Keyword(s):  
Genomic Structure ◽  
Mobile Element ◽  
Direct Repeat ◽  
Structural Features ◽  
Volvox Carteri ◽  
Developmentally Regulated ◽  
Closed Circle ◽  
Integration Mechanisms ◽  
And Function

Abstract Retrotransposons play an important role in the evolution of genomic structure and function. Here we report on the characterization of a novel retrotransposon called kangaroo from the multicellular green alga, Volvox carteri. kangaroo elements are highly mobile and their expression is developmentally regulated. They probably integrate via double-stranded, closed-circle DNA intermediates through the action of an encoded recombinase related to the λ-site-specific integrase. Phylogenetic analysis indicates that kangaroo elements are closely related to other unorthodox retrotransposons including PAT (from a nematode), DIRS-1 (from Dictyostelium), and DrDIRS1 (from zebrafish). PAT and kangaroo both contain split direct repeat (SDR) termini, and here we show that DIRS-1 and DrDIRS1 elements contain terminal features structurally related to SDRs. Thus, these mobile elements appear to define a third class of retrotransposons (the DIRS1 group) that are unified by common structural features, genes, and integration mechanisms, all of which differ from those of LTR and conventional non-LTR retrotransposons.

scholarly journals Tree Diversity Reduces Fungal Endophyte Richness and Diversity in a Large-Scale Temperate Forest Experiment

Diversity ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 234 ◽  
Author(s):  
Eric A. Griffin ◽  
Joshua G. Harrison ◽  
Melissa K. McCormick ◽  
Karin T. Burghardt ◽  
John D. Parker
Keyword(s):  
Phylogenetic Diversity ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Spatial Scales ◽  
Fungal Endophytes ◽  
Fungal Endophyte ◽  
Tree Diversity ◽  
Trophic Levels ◽  
Community Diversity ◽  
And Function

Although decades of research have typically demonstrated a positive correlation between biodiversity of primary producers and associated trophic levels, the ecological drivers of this association are poorly understood. Recent evidence suggests that the plant microbiome, or the fungi and bacteria found on and inside plant hosts, may be cryptic yet important drivers of important processes, including primary production and trophic interactions. Here, using high-throughput sequencing, we characterized foliar fungal community diversity, composition, and function from 15 broadleaved tree species (N = 545) in a recently established, large-scale temperate tree diversity experiment using over 17,000 seedlings. Specifically, we tested whether increases in tree richness and phylogenetic diversity would increase fungal endophyte diversity (the “Diversity Begets Diversity” hypothesis), as well as alter community composition (the “Tree Diversity–Endophyte Community” hypothesis) and function (the “Tree Diversity–Endophyte Function” hypothesis) at different spatial scales. We demonstrated that increasing tree richness and phylogenetic diversity decreased fungal species and functional guild richness and diversity, including pathogens, saprotrophs, and parasites, within the first three years of a forest diversity experiment. These patterns were consistent at the neighborhood and tree plot scale. Our results suggest that fungal endophytes, unlike other trophic levels (e.g., herbivores as well as epiphytic bacteria), respond negatively to increasing plant diversity.

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