In vivo probing of nascent RNA structures reveals principles of cotranscriptional folding

Abstract RNA structures that impede ribosome binding or subsequent scanning of the 5′-untranslated region (5′-UTR) for the AUG initiation codon reduce translation efficiency. Yeast DEAD-box RNA helicase Ded1 appears to promote translation by resolving 5′-UTR structures, but whether its paralog, Dbp1, performs similar functions is unknown. Furthermore, direct in vivo evidence was lacking that Ded1 or Dbp1 resolves 5′-UTR structures that impede attachment of the 43S preinitiation complex (PIC) or scanning. Here, profiling of translating 80S ribosomes reveals that the translational efficiencies of many more mRNAs are reduced in a ded1-ts dbp1Δ double mutant versus either single mutant, becoming highly dependent on Dbp1 or Ded1 only when the other helicase is impaired. Such ‘conditionally hyperdependent’ mRNAs contain unusually long 5′-UTRs with heightened propensity for secondary structure and longer transcript lengths. Consistently, overexpressing Dbp1 in ded1 cells improves the translation of many such Ded1-hyperdependent mRNAs. Importantly, Dbp1 mimics Ded1 in conferring greater acceleration of 48S PIC assembly in a purified system on mRNAs harboring structured 5′-UTRs. Profiling 40S initiation complexes in ded1 and dbp1 mutants provides direct evidence that Ded1 and Dbp1 cooperate to stimulate both PIC attachment and scanning on many Ded1/Dbp1-hyperdependent mRNAs in vivo.

Download Full-text

Transcriptional Pausing in Vivo: A Nascent RNA Hairpin Restricts Lateral Movements of RNA Polymerase in Both Forward and Reverse Directions

Journal of Molecular Biology ◽

10.1016/j.jmb.2005.05.052 ◽

2005 ◽

Vol 351 (1) ◽

pp. 39-51 ◽

Cited By ~ 18

Author(s):

Francine Toulmé ◽

Christine Mosrin-Huaman ◽

Irina Artsimovitch ◽

A. Rachid Rahmouni

Keyword(s):

Rna Polymerase ◽

Transcriptional Pausing ◽

Nascent Rna ◽

Rna Hairpin

Download Full-text

Self-assembly of large RNA structures: learning from DNA nanotechnology

DNA and RNA Nanotechnology ◽

10.1515/rnan-2015-0002 ◽

2016 ◽

Vol 2 (1) ◽

Cited By ~ 3

Author(s):

Jaimie Marie Stewart ◽

Elisa Franco

Keyword(s):

Self Assembly ◽

De Novo ◽

Dna Nanotechnology ◽

De Novo Design ◽

Dna Nanostructures ◽

Rna Structures ◽

Functional Nanostructures ◽

Rna And Dna ◽

Unique Chemical

AbstractNucleic acid nanotechnology offers many methods to build self-assembled structures using RNA and DNA. These scaffolds are valuable in multiple applications, such as sensing, drug delivery and nanofabrication. Although RNA and DNA are similar molecules, they also have unique chemical and structural properties. RNA is generally less stable than DNA, but it folds into a variety of tertiary motifs that can be used to produce complex and functional nanostructures. Another advantage of using RNA over DNA is its ability to be encoded into genes and to be expressed in vivo. Here we review existing approaches for the self-assembly of RNA and DNA nanostructures and specifically methods to assemble large RNA structures. We describe de novo design approaches used in DNA nanotechnology that can be ported to RNA. Lastly, we discuss some of the challenges yet to be solved to build micron-scale, multi stranded RNA scaffolds.

Download Full-text

Comprehensive in-vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms

10.1101/2020.07.10.197079 ◽

2020 ◽

Cited By ~ 14

Author(s):

Nicholas C. Huston ◽

Han Wan ◽

Rafael de Cesaris Araujo Tavares ◽

Craig Wilen ◽

Anna Marie Pyle

Keyword(s):

Secondary Structure ◽

Rna Virus ◽

Genomic Structure ◽

Structural Features ◽

Evolutionary Analysis ◽

Rna Structures ◽

Genomic Context ◽

Positive Sense ◽

Folded Rna

SummarySARS-CoV-2 is the positive-sense RNA virus that causes COVID-19, a disease that has triggered a major human health and economic crisis. The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form stable RNA structures and yet, as much as 97% of its 30 kilobases have not been structurally explored in the context of a viral infection. Our limited knowledge of SARS-CoV-2 genomic architecture is a fundamental limitation to both our mechanistic understanding of coronavirus life cycle and the development of COVID-19 RNA-based therapeutics. Here, we apply a novel long amplicon strategy to determine for the first time the secondary structure of the SARS-CoV-2 RNA genome probed in infected cells. In addition to the conserved structural motifs at the viral termini, we report new structural features like a conformationally flexible programmed ribosomal frameshifting pseudoknot, and a host of novel RNA structures, each of which highlights the importance of studying viral structures in their native genomic context. Our in-depth structural analysis reveals extensive networks of well-folded RNA structures throughout Orf1ab and reveals new aspects of SARS-CoV-2 genome architecture that distinguish it from other single-stranded, positive-sense RNA viruses. Evolutionary analysis of RNA structures in SARS-CoV-2 shows that several features of its genomic structure are conserved across beta coronaviruses and we pinpoint individual regions of well-folded RNA structure that merit downstream functional analysis. The native, complete secondary structure of SAR-CoV-2 presented here is a roadmap that will facilitate focused studies on mechanisms of replication, translation and packaging, and guide the identification of new RNA drug targets against COVID-19.

Download Full-text

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

Nucleic Acids Research ◽

10.1093/nar/gkaa1053 ◽

2020 ◽

Vol 48 (22) ◽

pp. 12436-12452 ◽

Cited By ~ 9

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.

Download Full-text

FRACTIONATION OF MAMMALIAN CHROMATIN

Acta Endocrinologica ◽

10.1530/acta.0.071s112 ◽

1972 ◽

Vol 71 (2_Suppla) ◽

pp. S112-S129 ◽

Cited By ~ 8

Author(s):

Michel Janowski ◽

DeLill S. Nasser ◽

Brian J. McCarthy

Keyword(s):

Chromatin Structure ◽

Active Fraction ◽

E Coli ◽

Chromatin Fraction ◽

Exclusion Chromatography ◽

Nascent Rna ◽

Active Peak ◽

Two Peaks

ABSTRACT Sheared chromatin may be separated into two peaks representing active and inactive fractions by gel exclusion chromatography on agarose columns. The active fraction is identified by the presence of nascent RNA chains synthesized in vivo or in vitro. Similarly, 3H labelled E. coli polymerase binds preferentially to the front active peak. Labelled hormones become associated with the chromatin of target tissues. Again this association occurs predominantly with the minor chromatin fraction identified as active by the above criteria. The basis for fractionation and the implications for our understanding of chromatin structure are discussed.

Download Full-text

Microtubule-dependent Organization of Vaccinia Virus Core–derivd Early mRNAs into Distinct Cytoplasmic Structures

Molecular Biology of the Cell ◽

10.1091/mbc.12.12.3875 ◽

2001 ◽

Vol 12 (12) ◽

pp. 3875-3891 ◽

Cited By ~ 46

Author(s):

Massimo Mallardo ◽

Sibylle Schleich ◽

Jacomine Krijnse Locker

Keyword(s):

Vaccinia Virus ◽

Rna Binding ◽

Rna Structures ◽

Viral Mrnas ◽

Virus Core ◽

Confocal Images ◽

Early Transcription ◽

Cytoplasmic Structures

Vaccinia virus (vv) early transcription can be reconstituted in vitro from purified virions; in this assay mRNAs are made inside the viral core and subsequently extruded. Although the in vitro process has been extensively characterized, relatively little is known about vv early transcription in vivo. In the present study the fate of vv early mRNAs in infected HeLa cells was followed by BrUTP transfection and confocal and electron microscopy. The extruded vv early mRNAs were found to be organized into unique granular cytoplasmic structures that reached a size up to 1 μm. By EM these structures appeared as amorphous electron-dense cytoplasmic aggregates that were surrounded by ribosomes. Confocal images showed that the RNA structures were located some distance away from intracellular cores and that both structures appeared to be aligned on microtubules (MTs), implying that MT tracks connected mRNAs and cores. Accordingly, intact MTs were found to be required for the typical punctate organization of viral mRNAs. Biochemical evidence supported the notion that vv mRNAs were MT associated and that MT depletion severely affected viral (but not cellular) mRNA synthesis and stability. By confocal microscopy the viral mRNA structures appeared to be surrounded by molecules of the translation machinery, showing that they were active in protein synthesis. Finally, our data suggest a role for a MT and RNA-binding viral protein of 25 kDa (gene L4R), in mRNA targeting away from intracellular cores to their sites of cytoplasmic accumulation.

Download Full-text

Deciphering Transcriptional Dynamics In Vivo by Counting Nascent RNA Molecules

PLoS Computational Biology ◽

10.1371/journal.pcbi.1004345 ◽

2015 ◽

Vol 11 (11) ◽

pp. e1004345 ◽

Cited By ~ 37

Author(s):

Sandeep Choubey ◽

Jane Kondev ◽

Alvaro Sanchez

Keyword(s):

Rna Molecules ◽

Transcriptional Dynamics ◽

Nascent Rna

Download Full-text