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

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.

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Structure and functional implications of WYL-domain-containing transcription factor PafBC involved in the mycobacterial DNA damage response

10.1101/612655 ◽

2019 ◽

Author(s):

Andreas U. Müller ◽

Marc Leibundgut ◽

Nenad Ban ◽

Eilika Weber-Ban

Keyword(s):

Dna Damage ◽

Transcription Factor ◽

Transcription Factors ◽

Rna Binding ◽

Rna Binding Proteins ◽

Structural Information ◽

Rna Molecules ◽

Bacterial Transcription ◽

Arthrobacter Aurescens

AbstractIn mycobacteria, transcriptional activator PafBC is responsible for upregulating the majority of genes induced by DNA damage. Understanding the mechanism of PafBC activation is impeded by a lack of structural information on this transcription factor that contains a widespread, but poorly understood WYL domain frequently encountered in bacterial transcription factors. Here, we determined the crystal structure ofArthrobacter aurescensPafBC. The protein consists of two modules, each harboring an N-terminal helix-turn-helix DNA binding domain followed by a central WYL and a C-terminal extension (WCX) domain. The WYL domains exhibit Sm-folds, while the WCX domains adopt ferredoxin-like folds, both characteristic for RNA binding proteins. Our results suggest a mechanism of regulation in which WYL domain-containing transcription factors may be activated by binding RNA molecules. Using anin vivomutational screen inMycobacterium smegmatis, we identify potential co-activator binding sites on PafBC.

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Analysis of In Vivo Transcriptome of Intracellular Bacterial Pathogen Salmonella enterica serovar Typhmurium Isolated from Mouse Spleen

Pathogens ◽

10.3390/pathogens10070823 ◽

2021 ◽

Vol 10 (7) ◽

pp. 823

Author(s):

Na Sun ◽

Yanying Song ◽

Cong Liu ◽

Mengda Liu ◽

Lanping Yu ◽

...

Keyword(s):

Salmonella Enterica ◽

Mammalian Cells ◽

High Throughput Sequencing ◽

Bacterial Pathogen ◽

Virulence Gene ◽

Sequencing Analysis ◽

Virulence Gene Expression ◽

Serovar Typhimurium ◽

Survival And Reproduction

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important intracellular pathogen that poses a health threat to humans. This study tries to clarify the mechanism of Salmonella survival and reproduction in the host. In this study, high-throughput sequencing analysis was performed on RNA extracted from the strains isolated from infected mouse spleens and an S. Typhimurium reference strain (ATCC 14028) based on the BGISEQ-500 platform. A total of 1340 significant differentially expressed genes (DEGs) were screened. Functional annotation revealed DEGs associated with regulation, metabolism, transport and binding, pathogenesis, and motility. Through data mining and literature retrieval, 26 of the 58 upregulated DEGs (FPKM > 10) were not reported to be related to the adaptation to intracellular survival and were classified as candidate key genes (CKGs) for survival and proliferation in vivo. Our data contribute to our understanding of the mechanisms used by Salmonella to regulate virulence gene expression whilst replicating inside mammalian cells.

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RNA modifications detection by comparative Nanopore direct RNA sequencing

Nature Communications ◽

10.1038/s41467-021-27393-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Adrien Leger ◽

Paulo P. Amaral ◽

Luca Pandolfini ◽

Charlotte Capitanchik ◽

Federica Capraro ◽

...

Keyword(s):

Rna Sequencing ◽

High Throughput Sequencing ◽

Control Sample ◽

Analytical Framework ◽

Rna Modifications ◽

Rna Molecules ◽

Oxford Nanopore ◽

Experimental Approaches

AbstractRNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. In recent years, a growing number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework that identifies modifications from these data. Our strategy compares an RNA sample of interest against a non-modified control sample, not requiring a training set and allowing the use of replicates. We show that Nanocompore can detect different RNA modifications with position accuracy in vitro, and we apply it to profile m6A in vivo in yeast and human RNAs, as well as in targeted non-coding RNAs. We confirm our results with orthogonal methods and provide novel insights on the co-occurrence of multiple modified residues on individual RNA molecules.

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Insights into the structure-driven protein interactivity of RNA molecules

10.1101/436279 ◽

2018 ◽

Author(s):

Natalia Sanchez de Groot ◽

Alexandros Armaos ◽

Ricardo Graña Montes ◽

Marion Alriquet ◽

Giulia Calloni ◽

...

Keyword(s):

Protein Interactions ◽

Rna Structure ◽

High Throughput Sequencing ◽

Interaction Network ◽

Recent Experimental Data ◽

Rna Molecules ◽

Rna Granules ◽

Complex Interdependence

ABSTRACTThe combination of high-throughput sequencing and in vivo crosslinking approaches leads to the progressive uncovering of the complex interdependence between cellular transcriptome and proteome. Yet the molecular determinants that govern interactions in protein-RNA networks are poorly known at present. Here we used the most recent experimental data to investigate the relationship between RNA structure and protein interactions. Our results show that, independently of the particular technique, the amount of structure in RNA molecules correlates with the capacity of binding to proteins in vitro and in vivo. To validate this observation, we generated an in vitro network that mimics the composition of phase-separated RNA granules. We observed that RNA, when structured, competes with protein binding and can rearrange the interaction network. The simplicity of the principle bears great potential to boost the understanding and modelling of cellular processes involving RNA-protein interactions.

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Insights into the global effect on Staphylococcus aureus growth arrest by induction of the endoribonuclease MazF toxin

Nucleic Acids Research ◽

10.1093/nar/gkaa617 ◽

2020 ◽

Vol 48 (15) ◽

pp. 8545-8561

Author(s):

Roberto Sierra ◽

Julien Prados ◽

Olesya O Panasenko ◽

Diego O Andrey ◽

Betty Fleuchot ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Ribosome Biogenesis ◽

Molecular Mechanisms ◽

Growth Arrest ◽

Global Scale ◽

Cleavage Sites ◽

Rna Turnover ◽

Rna Molecules ◽

Transcript Cleavage

Abstract A crucial bacterial strategy to avoid killing by antibiotics is to enter a growth arrested state, yet the molecular mechanisms behind this process remain elusive. The conditional overexpression of mazF, the endoribonuclease toxin of the MazEF toxin–antitoxin system in Staphylococcus aureus, is one approach to induce bacterial growth arrest, but its targets remain largely unknown. We used overexpression of mazF and high-throughput sequence analysis following the exact mapping of non-phosphorylated transcriptome ends (nEMOTE) technique to reveal in vivo toxin cleavage sites on a global scale. We obtained a catalogue of MazF cleavage sites and unearthed an extended MazF cleavage specificity that goes beyond the previously reported one. We correlated transcript cleavage and abundance in a global transcriptomic profiling during mazF overexpression. We observed that MazF affects RNA molecules involved in ribosome biogenesis, cell wall synthesis, cell division and RNA turnover and thus deliver a plausible explanation for how mazF overexpression induces stasis. We hypothesize that autoregulation of MazF occurs by directly modulating the MazEF operon, such as the rsbUVW genes that regulate the sigma factor SigB, including an observed cleavage site on the MazF mRNA that would ultimately play a role in entry and exit from bacterial stasis.

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The in vivo RNA structurome of the malaria parasite Plasmodium falciparum, a protozoan with an A/T-rich transcriptome

10.1101/2021.04.29.441925 ◽

2021 ◽

Author(s):

F Dumetz ◽

AJ Enright ◽

J Zhao ◽

CK Kwok ◽

CJ Merrick

Keyword(s):

Plasmodium Falciparum ◽

Protozoan Parasite ◽

Structural Features ◽

Translation Efficiency ◽

Rna Molecules ◽

Transcript Stability ◽

Parasite Plasmodium ◽

Parasite Plasmodium Falciparum ◽

Transcriptomic Level

AbstractPlasmodium falciparum, a protozoan parasite and causative agent of human malaria, has one of the most A/T-biased genomes sequenced to date. This may give the genome and the transcriptome unusual structural features. Recent progress in sequencing techniques has made it possible to study the secondary structures of RNA molecules at the transcriptomic level. Accordingly, in this study we produced the first in vivo RNA structurome of a protozoan parasite, and the first of a highly A/U-biased transcriptome. We showed that it is possible to probe the secondary structure of P. falciparum RNA molecules in vivo using two different chemical probes, and obtained structures for more than half of all transcripts in the transcriptome. These showed greater stability (lower free energy) than the same structures modelled in silico, and structural features appeared to influence translation efficiency and RNA decay. Finally, we compared the P. falciparum RNA structurome with the predicted RNA structurome of an A/T-balanced species, P. knowlesi, finding a bias towards lower overall transcript stability and more hairpins and multi-stem loops in P. falciparum. This first protozoan RNA structurome will provide a basis for similar studies in other protozoans and also in other unusual genomes.

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Secondary Structure of Chloroplast mRNAs In Vivo and In Vitro

Plants ◽

10.3390/plants9030323 ◽

2020 ◽

Vol 9 (3) ◽

pp. 323

Author(s):

Piotr Gawroński ◽

Aleksandra Pałac ◽

Lars B. Scharff

Keyword(s):

Gene Expression ◽

Secondary Structure ◽

Translation Initiation ◽

Structural Information ◽

Regulation Of Gene Expression ◽

Dimethyl Sulfate ◽

Translation Efficiency ◽

Mrna Secondary Structure

mRNA secondary structure can influence gene expression, e.g., by influencing translation initiation. The probing of in vivo mRNA secondary structures is therefore necessary to understand what determines the efficiency and regulation of gene expression. Here, in vivo mRNA secondary structure was analyzed using dimethyl sulfate (DMS)-MaPseq and compared to in vitro-folded RNA. We used an approach to analyze specific, full-length transcripts. To test this approach, we chose low, medium, and high abundant mRNAs. We included both monocistronic and multicistronic transcripts. Because of the slightly alkaline pH of the chloroplast stroma, we could probe all four nucleotides with DMS. The structural information gained was evaluated using the known structure of the plastid 16S rRNA. This demonstrated that the results obtained for adenosines and cytidines were more reliable than for guanosines and uridines. The majority of mRNAs analyzed were less structured in vivo than in vitro. The in vivo secondary structure of the translation initiation region of most tested genes appears to be optimized for high translation efficiency.

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

10.1101/2020.05.18.101402 ◽

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.

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Varying dependency of periplasmic peptidylprolyl cis–trans isomerases in promoting Yersinia pseudotuberculosis stress tolerance and pathogenicity

Biochemical Journal ◽

10.1042/bj20110767 ◽

2011 ◽

Vol 439 (2) ◽

pp. 321-332 ◽

Cited By ~ 27

Author(s):

Ikenna R. Obi ◽

Roland Nordfelth ◽

Matthew S. Francis

Keyword(s):

Yersinia Pseudotuberculosis ◽

Protein Profile ◽

Bacterial Pathogen ◽

Mammalian Host ◽

Ppiase Activity ◽

Rate Limiting Step ◽

Rate Limiting ◽

Extracellular Environment

Periplasmic PPIases (peptidylprolyl cis–trans isomerases) catalyse the cis–trans isomerization of peptidyl-prolyl bonds, which is a rate-limiting step during protein folding. We demonstrate that the surA, ppiA, ppiD, fkpA and fklB alleles each encode a periplasmic PPIase in the bacterial pathogen Yersinia pseudotuberculosis. Of these, four were purified to homogeneity. Purified SurA, FkpA and FklB, but not PpiD, displayed detectable PPIase activity in vitro. Significantly, only Y. pseudotuberculosis lacking surA caused drastic alterations to the outer membrane protein profile and FA (fatty acid) composition. They also exhibited aberrant cellular morphology, leaking LPS (lipopolysaccharide) into the extracellular environment. The SurA PPIase is therefore most critical for maintaining Y. pseudotuberculosis envelope integrity during routine culturing. On the other hand, bacteria lacking either surA or all of the genes ppiA, ppiD, fkpA and fklB were sensitive to hydrogen peroxide and were attenuated in mice infections. Thus Y. pseudotuberculosis exhibits both SurA-dependent and -independent requirements for periplasmic PPIase activity to ensure in vivo survival and a full virulence effect in a mammalian host.

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

10.1101/176883 ◽

2017 ◽

Cited By ~ 8

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.

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