scholarly journals Allosteric Mechanism of the V. vulnificus Adenine Riboswitch Resolved by Four-dimensional Chemical Mapping

2017 ◽  
Author(s):  
Siqi Tian ◽  
Wipapat Kladwang ◽  
Rhiju Das
Keyword(s):  
Strong Support ◽  
Chemical Mapping ◽  
Rna Molecules ◽  
Conformational Selection ◽  
Allosteric Mechanism ◽  
Ligand Free ◽  
Gene Regulatory ◽  
Nucleotide Resolution ◽  
Regulatory Functions ◽  
Structural Heterogeneities

ABSTRACTThe structural interconversions that mediate the gene regulatory functions of RNA molecules may be different from classic models of allostery, but the relevant structural correlations have remained elusive in even intensively studied systems. Here, we present a four-dimensional expansion of chemical mapping called lock-mutate-map-rescue (LM2R), which integrates multiple layers of mutation with nucleotide-resolution chemical mapping. This technique resolves the core mechanism of the adenine-responsive V. vulnificus add riboswitch, a paradigmatic system for which both Monod-Wyman-Changeux (MWC) conformational selection models and non-MWC alternatives have been proposed. To discriminate amongst these models, we locked each functionally important helix through designed mutations and assessed formation or depletion of other helices via compensatory rescue evaluated by chemical mapping. These LM2R measurements give strong support to the pre-existing correlations predicted by MWC models, disfavor alternative models, and suggest additional structural heterogeneities that may be general across ligand-free riboswitches.

scholarly journals Allosteric mechanism of the V. vulnificus adenine riboswitch resolved by four-dimensional chemical mapping

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Siqi Tian ◽  
Wipapat Kladwang ◽  
Rhiju Das
Keyword(s):  
Strong Support ◽  
Chemical Mapping ◽  
Rna Molecules ◽  
Conformational Selection ◽  
Allosteric Mechanism ◽  
Ligand Free ◽  
Gene Regulatory ◽  
Nucleotide Resolution ◽  
Regulatory Functions ◽  
Structural Heterogeneities

The structural interconversions that mediate the gene regulatory functions of RNA molecules may be different from classic models of allostery, but the relevant structural correlations have remained elusive in even intensively studied systems. Here, we present a four-dimensional expansion of chemical mapping called lock-mutate-map-rescue (LM2R), which integrates multiple layers of mutation with nucleotide-resolution chemical mapping. This technique resolves the core mechanism of the adenine-responsive V. vulnificus add riboswitch, a paradigmatic system for which both Monod-Wyman-Changeux (MWC) conformational selection models and non-MWC alternatives have been proposed. To discriminate amongst these models, we locked each functionally important helix through designed mutations and assessed formation or depletion of other helices via compensatory rescue evaluated by chemical mapping. These LM2R measurements give strong support to the pre-existing correlations predicted by MWC models, disfavor alternative models, and suggest additional structural heterogeneities that may be general across ligand-free riboswitches.

scholarly journals Computational Design of Asymmetric Three-dimensional RNA Structures and Machines

2017 ◽  
Author(s):  
Joseph D. Yesselman ◽  
Daniel Eiler ◽  
Erik D. Carlson ◽  
Alexandra N. Ooms ◽  
Wipapat Kladwang ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Computational Design ◽  
Chemical Mapping ◽  
Rna Structures ◽  
Single Nucleotide ◽  
Rna Molecules ◽  
Rna Nanotechnology ◽  
X Ray Scattering ◽  
Nucleotide Resolution ◽  
Large Sections

AbstractThe emerging field of RNA nanotechnology seeks to create nanoscale 3D machines by repurposing natural RNA modules, but successes have been limited to symmetric assemblies of single repeating motifs. We present RNAMake, a suite that automates design of RNA molecules with complex 3D folds. We first challenged RNAMake with the paradigmatic problem of aligning a tetraloop and sequence-distal receptor, previously only solved via symmetry. Single-nucleotide-resolution chemical mapping, native gel electrophoresis, and solution x-ray scattering confirmed that 11 of the 16 ‘miniTTR’ designs successfully achieved clothespin-like folds. A 2.55 Å diffraction-resolution crystal structure of one design verified formation of the target asymmetric nanostructure, with large sections achieving near-atomic accuracy (< 2.0 Å). Finally, RNAMake designed asymmetric segments to tether the 16S and 23S rRNAs together into a synthetic singlestranded ribosome that remains uncleaved by ribonucleases and supports life in Escherichia coli, a challenge previously requiring several rounds of trial-and-error.

scholarly journals Role of Methylation in the Induced and Spontaneous Expression of the Avian Endogenous Virusev-1: DNA Structure and Gene Products

1982 ◽  
Vol 2 (6) ◽  
pp. 638-652 ◽  
Author(s):  
Kathleen F. Conklin ◽  
John M. Coffin ◽  
Harriet L. Robinson ◽  
Mark Groudine ◽  
Robert Eisenman
Keyword(s):  
Dna Methylation ◽  
Strong Support ◽  
Reverse Transcriptase Activity ◽  
Structural Defects ◽  
Long Terminal Repeats ◽  
Control Of Gene Expression ◽  
Rna Molecules ◽  
Hypersensitive Sites ◽  
High Level

The endogenous avian provirusev-1 is widespread in white leghorn chickens. Although it has no major structural defects,ev-1 has not been associated with any phenotype and is ordinarily expressed at a very low level. In this report, we describe a chicken embryo (Number 1836) cell culture containing bothev-1 andev-6 which spontaneously expressed theev-1 provirus. This culture released a high level of noninfectious virions containing a full complement of virion structural (gag) proteins but devoid of reverse transcriptase activity or antigen. These virions contained 70S RNA closely related to the genome of Rous-associated virus type 0, but identifiable as theev-1 genome by oligonucleotide mapping. A fraction of the RNA molecules in the 70S complex were unusual in that they were polyadenylated 100 to 200 nucleotides downstream of the usual polyadenylation site. Eight sibling embryo cultures did not share this unusual phenotype with 1836, indicating that it was not inherited. However, an identical phenotype was inducible in the sibling cultures by treatment with 5-azacytidine, an inhibitor of DNA methylation, and the induced expression was stable for more than 10 generations. Analysis of chromatin structure and DNA methylation of theev-1 provirus in 1836 cells revealed the presence (in a fraction of the proviruses) of both DNase I hypersensitive sites in the long terminal repeats and ingagand a pattern of cleavage sites for methyl-sensitive restriction endonuclease not found in a nonexpressing sibling. These results lend strong support to the role of DNA methylation in the control of gene expression. Additionally, they explain the lack of phenotype associated withev-1 as due to a combination of its low expression and defectiveness inpolandenv.

scholarly journals Exosomic microRNAs as emerging key regulators of intercellular communication in the tumor microenvironment and beyond

2016 ◽  
Vol 2 (1) ◽  
Author(s):  
Mariam Murtadha ◽  
Muller Fabbri
Keyword(s):  
Tumor Microenvironment ◽  
Cancer Patients ◽  
Intercellular Communication ◽  
Potential Role ◽  
Biological Fluids ◽  
Healthy Controls ◽  
Gene Regulatory ◽  
Non Coding Rnas ◽  
Regulatory Functions

AbstractMicroRNAs (miRs) are small non-coding RNAs with key gene regulatory functions. Recent evidence has shown that miRs have a central role in shaping the biology of the Tumor Microenvironment (TME). The discovery that some exosomes contain high levels of miR cargo that shuttle between cells and mediate intercellular cross-talk has shifted the focus of miR research towards understanding the biological role of exosomic miRs. In this review, we highlight the emerging role of exosomic miRs in molding the tumor microenvironment towards pro-tumor conditions by altering intercellular communication. We briefly discuss some mechanisms of selective loading of miRs into exosomes, as well as emerging evidence that exosomic miRs are present in all biological fluids. Furthermore, we describe the differences in the exosomic miR signatures between cancer patients and healthy controls, and the potential role of exosomic miRs as diagnostic, prognostic, and therapeutic biomarkers.

scholarly journals A method for RNA structure prediction shows evidence for structure in lncRNAs

2018 ◽  
Author(s):  
Riccardo Delli ponti ◽  
Alexandros Armaos ◽  
Stefanie Marti ◽  
Gian Gaetano Tartaglia
Keyword(s):  
Secondary Structure ◽  
Rna Structure ◽  
Structure Prediction ◽  
Sequence Information ◽  
Time Warping ◽  
Single Nucleotide ◽  
Rna Molecules ◽  
Rna Structure Prediction ◽  
Dynamic Time ◽  
Nucleotide Resolution

AbstractTo compare the secondary structures of RNA molecules we developed the CROSSalign method. CROSSalign is based on the combination of the Computational Recognition Of Secondary Structure (CROSS) algorithm to predict the RNA secondary structure at single-nucleotide resolution using sequence information, and the Dynamic Time Warping (DTW) method to align profiles of different lengths. We applied CROSSalign to investigate the structural conservation of long non-coding RNAs such as XIST and HOTAIR as well as ssRNA viruses including HIV. In a pool of sequences with the same secondary structure CROSSalign accurately recognizes repeat A of XIST and domain D2 of HOTAIR and outperforms other methods based on covariance modelling. CROSSalign can be applied to perform pair-wise comparisons and is able to find homologues between thousands of matches identifying the exact regions of similarity between profiles of different lengths. The algorithm is freely available at the webpage http://service.tartaglialab.com//new_submission/CROSSalign.

scholarly journals miRTissue ce: extending miRTissue web service with the analysis of ceRNA-ceRNA interactions

2020 ◽  
Vol 21 (S8) ◽  
Author(s):  
Antonino Fiannaca ◽  
Laura La Paglia ◽  
Massimo La Rosa ◽  
Riccardo Rizzo ◽  
Alfonso Urso
Keyword(s):  
Web Service ◽  
Protein Translation ◽  
Cancer Tissue ◽  
Rna Molecules ◽  
Target Interaction ◽  
Regulation Mechanisms ◽  
Mirna Sponges ◽  
Regulatory Functions ◽  
Competitive Endogenous Rna

Abstract Background Non-coding RNAs include different classes of molecules with regulatory functions. The most studied are microRNAs (miRNAs) that act directly inhibiting mRNA expression or protein translation through the interaction with a miRNAs-response element. Other RNA molecules participate in the complex network of gene regulation. They behave as competitive endogenous RNA (ceRNA), acting as natural miRNA sponges to inhibit miRNA functions and modulate the expression of RNA messenger (mRNA). It became evident that understanding the ceRNA–miRNA–mRNA crosstalk would increase the functional information across the transcriptome, contributing to identify new potential biomarkers for translational medicine. Results We present miRTissue ce, an improvement of our original miRTissue web service. By introducing a novel computational pipeline, miRTissue ce provides an easy way to search for ceRNA interactions in several cancer tissue types. Moreover it extends the functionalities of previous miRTissue release about miRNA-target interaction in order to provide a complete insight about miRNA mediated regulation processes. miRTissue ce is freely available at http://tblab.pa.icar.cnr.it/mirtissue.html. Conclusions The study of ceRNA networks and its dynamics in cancer tissue could be applied in many fields of translational biology, as the investigation of new cancer biomarker, both diagnostic and prognostic, and also in the investigation of new therapeutic strategies of intervention. In this scenario, miRTissue ce can offer a powerful instrument for the analysis and characterization of ceRNA-ceRNA interactions in different tissue types, representing a fundamental step in order to understand more complex regulation mechanisms.

scholarly journals Combinatorial Gene Regulatory Functions Underlie Ultraconserved Elements in Drosophila

2016 ◽  
Vol 33 (9) ◽  
pp. 2294-2306 ◽  
Author(s):  
Maria Warnefors ◽  
Britta Hartmann ◽  
Stefan Thomsen ◽  
Claudio R. Alonso
Keyword(s):  
Ultraconserved Elements ◽  
Gene Regulatory ◽  
Regulatory Functions

scholarly journals Plasmid Negative Regulation of CPAF Expression Is Pgp4 Independent and Restricted to Invasive Chlamydia trachomatis Biovars

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Michael John Patton ◽  
Chih-Yu Chen ◽  
Chunfu Yang ◽  
Stuart McCorrister ◽  
Chris Grant ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Virulence Factor ◽  
Chlamydial Infection ◽  
Virulence Genes ◽  
Negative Regulation ◽  
Host Protein ◽  
Regulatory Function ◽  
Important Virulence Factor ◽  
Gene Regulatory ◽  
Regulatory Functions

ABSTRACTChlamydia trachomatisis an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease.C. trachomatisisolates are classified into 2 biovars—lymphogranuloma venereum (LGV) and trachoma—which are distinguished biologically by their natural host cell infection tropism. LGV biovars infect macrophages and are invasive, whereas trachoma biovars infect oculo-urogenital epithelial cells and are noninvasive. TheC. trachomatisplasmid is an important virulence factor in the pathogenesis of these infections. Central to its pathogenic role is the transcriptional regulatory function of the plasmid protein Pgp4, which regulates the expression of plasmid and chromosomal virulence genes. As many gene regulatory functions are post-transcriptional, we employed a comparative proteomic study of cells infected with plasmid-curedC. trachomatisserovars A and D (trachoma biovar), a L2 serovar (LGV biovar), and the L2 serovar transformed with a plasmid containing a nonsense mutation inpgp4to more completely elucidate the effects of the plasmid on chlamydial infection biology. Our results show that the Pgp4-dependent elevations in the levels of Pgp3 and a conserved core set of chromosomally encoded proteins are remarkably similar for serovars within bothC. trachomatisbiovars. Conversely, we found a plasmid-dependent, Pgp4-independent, negative regulation in the expression of the chlamydial protease-like activity factor (CPAF) for the L2 serovar but not the A and D serovars. The molecular mechanism of plasmid-dependent negative regulation of CPAF expression in the LGV serovar is not understood but is likely important to understanding its macrophage infection tropism and invasive infection nature.IMPORTANCETheChlamydia trachomatisplasmid is an important virulence factor in the pathogenesis of chlamydial infection. It is known that plasmid protein 4 (Pgp4) functions in the transcriptional regulation of the plasmid virulence protein 3 (Pgp3) and multiple chromosomal loci of unknown function. Since many gene regulatory functions can be post-transcriptional, we undertook a comparative proteomic analysis to better understand the plasmid’s role in chlamydial and host protein expression. We report that Pgp4 is a potent and specific master positive regulator of a common core of plasmid and chromosomal virulence genes shared by multipleC. trachomatisserovars. Notably, we show that the plasmid is a negative regulator of the expression of the chlamydial virulence factor CPAF. The plasmid regulation of CPAF is independent of Pgp4 and restricted to aC. trachomatismacrophage-tropic strain. These findings are important because they define a previously unknown role for the plasmid in the pathophysiology of invasive chlamydial infection.

scholarly journals Noncoding RNAs: Emerging Players in Muscular Dystrophies

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Germana Falcone ◽  
Alessandra Perfetti ◽  
Beatrice Cardinali ◽  
Fabio Martelli
Keyword(s):  
Regulatory Networks ◽  
Noncoding Rnas ◽  
Skeletal Muscle Differentiation ◽  
Physiological Mechanisms ◽  
Rna Molecules ◽  
Disease Biomarkers ◽  
Cellular Processes ◽  
Sequencing Technologies ◽  
Regulatory Functions ◽  
Potential Use

The fascinating world of noncoding RNAs has recently come to light, thanks to the development of powerful sequencing technologies, revealing a variety of RNA molecules playing important regulatory functions in most, if not all, cellular processes. Many noncoding RNAs have been implicated in regulatory networks that are determinant for skeletal muscle differentiation and disease. In this review, we outline the noncoding RNAs involved in physiological mechanisms of myogenesis and those that appear dysregulated in muscle dystrophies, also discussing their potential use as disease biomarkers and therapeutic targets.

Towards In Vivo Nanomachines

2008 ◽  
Vol 58 ◽  
pp. 120-126
Author(s):  
Eike Friedrichs ◽  
Ralf Jungmann ◽  
Angeliki Tsokou ◽  
Stephan Renner ◽  
Friedrich C. Simmel
Keyword(s):  
Nucleic Acid ◽  
Molecular Devices ◽  
Rna Transcription ◽  
Rna Molecules ◽  
Regulatory Motifs ◽  
One Step ◽  
Gene Regulatory

DNA has been recently used to construct a variety of nanoscale machines and switches, among them devices which can translocate, compute, or bind and release molecules. For future applications it is interesting to investigate whether these "artificial" functions can also be implemented in vivo. As one step towards in vivo applications, we currently utilize RNA molecules for construction or control of molecular devices. RNA molecules can be transcribed from "artificial genes" and either fold into functional nanoassemblies themselves or drive other nucleic acid-based devices as a "fuel". Furthermore, coupling of nanomachines to RNA transcription enables control of their behavior using gene regulatory motifs.

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