scholarly journals RNA base pairing complexity in living cells visualized by correlated chemical probing

2019 ◽  
Author(s):  
Anthony M. Mustoe ◽  
Nicole Lama ◽  
Patrick S. Irving ◽  
Samuel W. Olson ◽  
Kevin M. Weeks
Keyword(s):  
Single Molecule ◽  
Rna Structure ◽  
Dimethyl Sulfate ◽  
Detection Methods ◽  
Base Pairing ◽  
Base Pairs ◽  
Chemical Probing ◽  
Pairing Interactions ◽  
In Cells

ABSTRACTRNA structure and dynamics are critical to biological function. However, strategies for determining RNA structure in vivo are limited, with established chemical probing and newer duplex detection methods each having notable deficiencies. Here we convert the common reagent dimethyl sulfate (DMS) into a useful probe of all four RNA nucleotides. Building on this advance, we introduce PAIR-MaP, which uses single-molecule correlated chemical probing to directly detect base pairing interactions in cells. PAIR-MaP has superior resolution and accuracy compared to alternative experiments, can resolve alternative pairing interactions of structurally dynamic RNAs, and enables highly accurate structure modeling, including of RNAs containing multiple pseudoknots and extensively bound by proteins. Application of PAIR-MaP to human RNase MRP and two bacterial mRNA 5'-UTRs reveals new functionally important and complex structures undetectable by conventional analyses. PAIR-MaP is a powerful, experimentally concise, and broadly applicable strategy for directly visualizing RNA base pairs and dynamics in cells.

scholarly journals RNA base-pairing complexity in living cells visualized by correlated chemical probing

2019 ◽  
Vol 116 (49) ◽  
pp. 24574-24582 ◽  
Author(s):  
Anthony M. Mustoe ◽  
Nicole N. Lama ◽  
Patrick S. Irving ◽  
Samuel W. Olson ◽  
Kevin M. Weeks
Keyword(s):  
Single Molecule ◽  
Rna Structure ◽  
Messenger Rna ◽  
Dimethyl Sulfate ◽  
Detection Methods ◽  
Base Pairing ◽  
Chemical Probing ◽  
Pairing Interactions ◽  
In Cells

RNA structure and dynamics are critical to biological function. However, strategies for determining RNA structure in vivo are limited, with established chemical probing and newer duplex detection methods each having deficiencies. Here we convert the common reagent dimethyl sulfate into a useful probe of all 4 RNA nucleotides. Building on this advance, we introduce PAIR-MaP, which uses single-molecule correlated chemical probing to directly detect base-pairing interactions in cells. PAIR-MaP has superior resolution compared to alternative experiments, can resolve multiple sets of pairing interactions for structurally dynamic RNAs, and enables highly accurate structure modeling, including of RNAs containing multiple pseudoknots and extensively bound by proteins. Application of PAIR-MaP to human RNase MRP and 2 bacterial messenger RNA 5′ untranslated regions reveals functionally important and complex structures undetected by prior analyses. PAIR-MaP is a powerful, experimentally concise, and broadly applicable strategy for directly visualizing RNA base pairs and dynamics in cells.

scholarly journals Best practices for genome-wide RNA structure analysis: combination of mutational profiles and drop-off information

2017 ◽  
Author(s):  
Eva Maria Novoa ◽  
Jean-Denis Beaudoin ◽  
Antonio J Giraldez ◽  
John S Mattick ◽  
Manolis Kellis
Keyword(s):  
Reverse Transcription ◽  
Rna Structure ◽  
Structural Information ◽  
Dimethyl Sulfate ◽  
Mutational Signatures ◽  
Chemically Modified ◽  
Chemical Probing ◽  
Genome Wide ◽  
Generation Sequencing

ABSTRACTGenome-wide RNA structure maps have recently become available through the coupling of in vivo chemical probing reagents with next-generation sequencing. Initial analyses relied on the identification of truncated reverse transcription reads to identify the chemically modified nucleotides, but recent studies have shown that mutational signatures can also be used. While these two methods have been employed interchangeably, here we show that they actually provide complementary information. Consequently, analyses using exclusively one of the two methodologies may disregard a significant portion of the structural information. We also show that the identity and sequence environment of the modified nucleotide greatly affect the odds of introducing a mismatch or causing reverse transcriptase drop-off. Finally, we identify specific mismatch signatures generated by dimethyl sulfate probing that can be exploited to remove false positives typically produced in RNA structurome analyses, and how these signatures vary depending on the reverse transcription enzyme used.

Estrogen-inducible binding of a nuclear factor to the vitellogenin upstream region

1988 ◽  
Vol 8 (10) ◽  
pp. 4557-4560
Author(s):  
O Bakker ◽  
J N Philipsen ◽  
B C Hennis ◽  
G Ab
Keyword(s):  
Dimethyl Sulfate ◽  
Upstream Region ◽  
Nuclear Factor ◽  
Base Pairs ◽  
Exonuclease Iii ◽  
Factor I ◽  
Nuclear Factor I ◽  
Vitellogenin Gene

The estrogen-dependent binding of a protein to the upstream region of the chicken vitellogenin gene was detected by using in vivo dimethyl sulfate, genomic DNase I, and in vitro exonuclease III footprinting. The site is located between base pairs -848 and -824, and its sequence resembles that of the nuclear factor I binding site. The results suggest that a nuclear factor binding to this site is involved in the regulation of the vitellogenin gene.

Cis- andtrans-acting factors regulating transcription of the BGT1 gene in response to hypertonicity

1998 ◽  
Vol 274 (4) ◽  
pp. F753-F761 ◽  
Author(s):  
Hiroshi Miyakawa ◽  
Seung Kyoon Woo ◽  
Ching-Pu Chen ◽  
Stephen C. Dahl ◽  
Joseph S. Handler ◽  
...  
Keyword(s):  
Dna Binding ◽  
Time Course ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Dimethyl Sulfate ◽  
Base Pairs ◽  
Biochemical Basis ◽  
Activation Of Transcription ◽  
Stimulation Of

We have previously identified a tonicity-responsive enhancer (TonE) in the promoter region of the canine BGT1 gene. TonE mediates hypertonicity-induced stimulation of transcription. Here, we characterize TonE and TonE binding proteins (TonEBPs) to provide a biochemical basis for cloning of the TonEBPs. Mutational analysis applied to both hypertonicity-induced stimulation of transcription and TonEBP binding reveals that TonE is 11 base pairs in length, with the consensus sequence of (C/T)GGAAnnn(C/T)n(C/T). Activity of the TonEBPs increases in response to hypertonicity with a time course similar to that of transcription of the BGT1 gene. Studies with inhibitors indicate that translation, but not transcription, is required for activation of the TonEBPs. Phosphorylation is required for the stimulation of transcription but not for activation of DNA binding by the TonEBPs. In vivo methylation by dimethyl sulfate reveals that the TonE site of the BGT1 gene is protected with a time course like that of activity of the TonEBPs and activation of transcription. Ultraviolet cross-linking indicates that the TonEBPs share a DNA binding subunit of 200 kDa.

scholarly journals Processing of Intron-Encoded Box C/D Small Nucleolar RNAs Lacking a 5′,3′-Terminal Stem Structure

2000 ◽  
Vol 20 (13) ◽  
pp. 4522-4531 ◽  
Author(s):  
Xavier Darzacq ◽  
Tamás Kiss
Keyword(s):  
Metabolic Stability ◽  
Small Nucleolar Rnas ◽  
Base Pairing ◽  
Flanking Sequences ◽  
Pairing Interactions ◽  
Precursor Rna ◽  
Nucleolar Rnas ◽  
Stem Structure

ABSTRACT The C and D box-containing (box C/D) small nucleolar RNAs (snoRNAs) function in the nucleolytic processing and 2′-O-methylation of precursor rRNA. In vertebrates, most box C/D snoRNAs are processed from debranched pre-mRNA introns by exonucleolytic activities. Elements directing accurate snoRNA excision are located within the snoRNA itself; they comprise the conserved C and D boxes and an adjoining 5′,3′-terminal stem. Although the terminal stem has been demonstrated to be essential for snoRNA accumulation, many snoRNAs lack a terminal helix. To identify thecis-acting elements supporting the accumulation of intron-encoded box C/D snoRNAs devoid of a terminal stem, we have investigated the in vivo processing of the human U46 snoRNA and an artificial snoRNA from the human β-globin pre-mRNA. We demonstrate that internal and/or external stem structures located within the snoRNA or in the intronic flanking sequences support the accumulation of mammalian box C/D snoRNAs lacking a canonical terminal stem. In the intronic precursor RNA, transiently formed external and/or stable internal base-pairing interactions fold the C and D boxes together and therefore facilitate the binding of snoRNP proteins. Since the external intronic stems are degraded during snoRNA processing, we propose that the C and D boxes alone can provide metabolic stability for the mature snoRNA.

scholarly journals Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences

1993 ◽  
Vol 13 (5) ◽  
pp. 2697-2705
Author(s):  
R H Schiestl ◽  
M Dominska ◽  
T D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Topoisomerase I ◽  
Target Sequence ◽  
Base Pairing ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Fragment

When the yeast Saccharomyces cerevisiae was transformed with DNA that shares no homology to the genome, three classes of transformants were obtained. In the most common class, the DNA was inserted as the result of a reaction that appears to require base pairing between the target sequence and the terminal few base pairs of the transforming DNA fragment. In the second class, no such homology was detected, and the transforming DNA was integrated next to a CTT or GTT in the target; it is likely that these integration events were mediated by topoisomerase I. The final class involved the in vivo ligation of transforming DNA with nucleus-localized linear fragments of mitochondrial DNA.

scholarly journals Hoogsteen base pairs increase the susceptibility of double-stranded DNA to cytotoxic damage

2020 ◽  
Vol 295 (47) ◽  
pp. 15933-15947
Author(s):  
Yu Xu ◽  
Akanksha Manghrani ◽  
Bei Liu ◽  
Honglue Shi ◽  
Uyen Pham ◽  
...  
Keyword(s):  
Dynamic Equilibrium ◽  
Alkylating Agents ◽  
Dimethyl Sulfate ◽  
Dot Blot ◽  
Base Pairs ◽  
Double Stranded Dna ◽  
Genome Wide ◽  
Damage Susceptibility

As the Watson–Crick faces of nucleobases are protected in dsDNA, it is commonly assumed that deleterious alkylation damage to the Watson–Crick faces of nucleobases predominantly occurs when DNA becomes single-stranded during replication and transcription. However, damage to the Watson–Crick faces of nucleobases has been reported in dsDNA in vitro through mechanisms that are not understood. In addition, the extent of protection from methylation damage conferred by dsDNA relative to ssDNA has not been quantified. Watson–Crick base pairs in dsDNA exist in dynamic equilibrium with Hoogsteen base pairs that expose the Watson–Crick faces of purine nucleobases to solvent. Whether this can influence the damage susceptibility of dsDNA remains unknown. Using dot-blot and primer extension assays, we measured the susceptibility of adenine-N1 to methylation by dimethyl sulfate (DMS) when in an A-T Watson–Crick versus Hoogsteen conformation. Relative to unpaired adenines in a bulge, Watson–Crick A-T base pairs in dsDNA only conferred ∼130-fold protection against adenine-N1 methylation, and this protection was reduced to ∼40-fold for A(syn)-T Hoogsteen base pairs embedded in a DNA-drug complex. Our results indicate that Watson–Crick faces of nucleobases are accessible to alkylating agents in canonical dsDNA and that Hoogsteen base pairs increase this accessibility. Given the higher abundance of dsDNA relative to ssDNA, these results suggest that dsDNA could be a substantial source of cytotoxic damage. The work establishes DMS probing as a method for characterizing A(syn)-T Hoogsteen base pairs in vitro and also lays the foundation for a sequencing approach to map A(syn)-T Hoogsteen and unpaired adenines genome-wide in vivo.

scholarly journals Genome-wide RNA structurome reprogramming by acute heat shock globally regulates mRNA abundance

2018 ◽  
Vol 115 (48) ◽  
pp. 12170-12175 ◽  
Author(s):  
Zhao Su ◽  
Yin Tang ◽  
Laura E. Ritchey ◽  
David C. Tack ◽  
Mengmeng Zhu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Rna Structure ◽  
Time Course ◽  
Structural Changes ◽  
Dimethyl Sulfate ◽  
Rna Degradation ◽  
Crop Species ◽  
Genome Wide

The heat shock response is crucial for organism survival in natural environments. RNA structure is known to influence numerous processes related to gene expression, but there have been few studies on the global RNA structurome as it prevails in vivo. Moreover, how heat shock rapidly affects RNA structure genome-wide in living systems remains unknown. We report here in vivo heat-regulated RNA structuromes. We applied Structure-seq chemical [dimethyl sulfate (DMS)] structure probing to rice (Oryza sativa L.) seedlings with and without 10 min of 42 °C heat shock and obtained structural data on >14,000 mRNAs. We show that RNA secondary structure broadly regulates gene expression in response to heat shock in this essential crop species. Our results indicate significant heat-induced elevation of DMS reactivity in the global transcriptome, revealing RNA unfolding over this biological temperature range. Our parallel Ribo-seq analysis provides no evidence for a correlation between RNA unfolding and heat-induced changes in translation, in contrast to the paradigm established in prokaryotes, wherein melting of RNA thermometers promotes translation. Instead, we find that heat-induced DMS reactivity increases correlate with significant decreases in transcript abundance, as quantified from an RNA-seq time course, indicating that mRNA unfolding promotes transcript degradation. The mechanistic basis for this outcome appears to be mRNA unfolding at both 5′ and 3′-UTRs that facilitates access to the RNA degradation machinery. Our results thus reveal unexpected paradigms governing RNA structural changes and the eukaryotic RNA life cycle.

scholarly journals Implementation and application of FRET–FLIM technology

2019 ◽  
Vol 12 (05) ◽  
pp. 1930010 ◽  
Author(s):  
Shiqi Wang ◽  
Binglin Shen ◽  
Sheng Ren ◽  
Yihua Zhao ◽  
Silu Zhang ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Lifetime ◽  
Protein Interactions ◽  
Resonance Energy ◽  
Detection Methods ◽  
Fluorescence Lifetime Imaging ◽  
Donor And Acceptor ◽  
Fluorescent Molecules ◽  
In Cells

With the development of the new detection methods and the function of fluorescent molecule, researchers hope to further explore the internal mechanisms of organisms, monitor changes in the intracellular microenvironment, and dynamic processes of molecular interactions in cells. Fluorescence resonance energy transfer (FRET) describes the energy transfer process between donor fluorescent molecules and acceptor fluorescent molecules. It is an important means to detect protein–protein interactions and protein conformation changes in cells. Fluorescence lifetime imaging microscopy (FLIM) enables noninvasive measurement of the fluorescence lifetime of fluorescent particles in vivo. The FRET–FLIM technology, which is use FLIM to quantify and analyze FRET, enables real-time monitoring of dynamic changes of proteins in biological cells and analysis of protein interaction mechanisms. The distance between donor and acceptor and their respective fluorescent lifetime, which are of great importance for studying the mechanism of intracellular activity can be obtained by data analysis and algorithm fitting.

scholarly journals The RNA–RNA base pairing potential of human Dicer and Ago2 proteins

2019 ◽  
Vol 77 (16) ◽  
pp. 3231-3244 ◽  
Author(s):  
Maria Pokornowska ◽  
Marek C. Milewski ◽  
Kinga Ciechanowska ◽  
Agnieszka Szczepańska ◽  
Marta Wojnicka ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Rna Structure ◽  
Small Interfering Rnas ◽  
Base Pairing ◽  
Complementary Sequence ◽  
Control Of Gene Expression ◽  
Complementary Sequences

Abstract The ribonuclease Dicer produces microRNAs (miRNAs) and small interfering RNAs that are handed over to Ago proteins to control gene expression by targeting complementary sequences within transcripts. Interestingly, a growing number of reports have demonstrated that the activity of Dicer may extend beyond the biogenesis of small regulatory RNAs. Among them, a report from our latest studies revealed that human Dicer facilitates base pairing of complementary sequences present in two nucleic acids, thus acting as a nucleic acid annealer. Accordingly, in this manuscript, we address how RNA structure influences the annealing activity of human Dicer. We show that Dicer supports hybridization between a small RNA and a complementary sequence of a longer RNA in vitro, even when both complementary sequences are trapped within secondary structures. Moreover, we show that under applied conditions, human Ago2, a core component of RNA-induced silencing complex, displays very limited annealing activity. Based on the available data from new-generation sequencing experiments regarding the RNA pool bound to Dicer in vivo, we show that multiple Dicer-binding sites within mRNAs also contain miRNA targets. Subsequently, we demonstrate in vitro that Dicer but not Ago2 can anneal miRNA to its target present within mRNA. We hypothesize that not all miRNA duplexes are handed over to Ago proteins. Instead, miRNA-Dicer complexes could target specific sequences within transcripts and either compete or cooperate for binding sites with miRNA-Ago complexes. Thus, not only Ago but also Dicer might be directly involved in the posttranscriptional control of gene expression.

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