The archaeal Lsm protein from Pyrococcus furiosus binds co-transcriptionally to poly(U)-rich target RNAs.

Posttranscriptional processes in Bacteria include the association of small regulatory RNAs (sRNA) with a target mRNA. The sRNA/mRNA annealing process is often mediated by an RNA chaperone called Hfq. The functional role of bacterial and eukaryotic Lsm proteins is partially understood, whereas knowledge about archaeal Lsm proteins is scarce. Here, we used the genetically tractable archaeal hyperthermophile Pyrococcus furiosus to identify the protein interaction partners of the archaeal Sm-like proteins (PfuSmAP1) using mass spectrometry and performed a transcriptome-wide binding site analysis of PfuSmAP1. Most of the protein interaction partners we found are part of the RNA homoeostasis network in Archaea including ribosomal proteins, the exosome, RNA-modifying enzymes, but also RNA polymerase subunits, and transcription factors. We show that PfuSmAP1 preferentially binds messenger RNAs and antisense RNAs recognizing a gapped poly(U) sequence with high affinity. Furthermore, we found that SmAP1 co-transcriptionally associates with target RNAs. Our study reveals that in contrast to bacterial Hfq, PfuSmAP1 does not affect the transcriptional activity or the pausing behaviour of archaeal RNA polymerases. We propose that PfuSmAP1 recruits antisense RNAs to target mRNAs and thereby executes its putative regulatory function on the posttranscriptional level.

Small RNA-Sequencing Library Preparation for the Halophilic Archaeon Haloferax volcanii.

Posttranscriptional regulation actuated by small RNAs (sRNAs) plays essential roles in a wide variety of cellular processes, especially in stress responses and environmental signaling. Hundreds of sRNAs have recently been discovered in archaea using genome-wide approaches but the molecular mechanisms of only a few have been characterized experimentally. Here, we describe how to build sRNA sequencing libraries using size-selected total RNA in the model archaeon, Haloferax volcanii , to provide a tool to further characterize sRNAs in archaea.

Methylation guide RNAs without box C/D motifs.

Box C/D RNAs guide site-specific 2'-O-methylation of RNAs in archaea and eukaryotes. The defining feature of methylation guide RNAs is two sets of box C and D motifs that form kink-turn structures specifically recognized by L7Ae family proteins. Here, we engineered a new type of methylation guide that lacks C/D motifs and requires no L7Ae for assembly and function. We determined a crystal structure of a bipartite C/D-free guide RNA in complex with Nop5, fibrillarin and substrate in the active form at 2.2 Å resolution. The stems of new guide RNAs functionally replace C/D motifs in Nop5 binding, precisely placing the substrate for site-specific modification. We also found that the bipartite architecture and association of L7Ae with C/D motifs enhance modification when association of guide RNAs or substrates is weak. Our study provides insights into the variations, robustness and possible evolutionary path of methylation guide RNAs.

Comparative genomic analysis reveals metabolic flexibility of Woesearchaeota.

The archaeal phylum Woesearchaeota, within the DPANN superphylum, includes phylogenetically diverse microorganisms that inhabit various environments. Their biology is poorly understood due to the lack of cultured isolates. Here, we analyze datasets of Woesearchaeota 16S rRNA gene sequences and metagenome-assembled genomes to infer global distribution patterns, ecological preferences and metabolic capabilities. Phylogenomic analyses indicate that the phylum can be classified into ten subgroups, termed A-J. While a symbiotic lifestyle is predicted for most, some members of subgroup J might be host-independent. The genomes of several Woesearchaeota, including subgroup J, encode putative [FeFe] hydrogenases (known to be important for fermentation in other organisms), suggesting that these archaea might be anaerobic fermentative heterotrophs.

Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P.

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg2+-dependent cleavage of the 5' leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250-500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to ∼10-20 mM and improves the rate and fidelity of cleavage. To understand the Mg2+- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPRC domain and Cy5-RPRS domain), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg2+-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg2+ cooperativity. Our findings highlight how Mg2+ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.

Post-transcriptional regulation of redox homeostasis by the small RNA SHOxi in haloarchaea.

While haloarchaea are highly resistant to oxidative stress, a comprehensive understanding of the processes regulating this remarkable response is lacking. Oxidative stress-responsive small non-coding RNAs (sRNAs) have been reported in the model archaeon, Haloferax volc anii, but targets and mechanisms have not been elucidated. Using a combination of high throughput and reverse molecular genetic approaches, we elucidated the functional role of the most up-regulated intergenic sRNA during oxidative stress in H. volcanii, named Small RNA in Haloferax Oxidative Stress (SHOxi). SHOxi was predicted to form a stable secondary structure with a conserved stem-loop region as the potential binding site for trans-targets. NAD-dependent malic enzyme mRNA, identified as a putative target of SHOxi, interacted directly with a putative 'seed' region within the predicted stem loop of SHOxi. Malic enzyme catalyzes the oxidative decarboxylation of malate into pyruvate using NAD+ as a cofactor. The destabilization of malic enzyme mRNA, and the decrease in the NAD+/NADH ratio, resulting from the direct RNA-RNA interaction between SHOxi and its trans-target was essential for the survival of H. volcanii to oxidative stress. These findings indicate that SHOxi likely regulates redox homoeostasis during oxidative stress by the post-transcriptional destabilization of malic enzyme mRNA. SHOxi-mediated regulation provides evidence that the fine-tuning of metabolic cofactors could be a core strategy to mitigate damage from oxidative stress and confer resistance. This study is the first to establish the regulatory effects of sRNAs on mRNAs during the oxidative stress response in Archaea.

Expansion segments in bacterial and archaeal 5S ribosomal RNAs.

The large ribosomal RNAs of eukaryotes frequently contain expansion sequences that add to the size of the rRNAs but do not affect their overall structural layout and are compatible with major ribosomal function as an mRNA translation machine. The expansion of prokaryotic ribosomal RNAs is much less explored. In order to obtain more insight into the structural variability of these conserved molecules, we herein report the results of a comprehensive search for the expansion sequences in prokaryotic 5S rRNAs. Overall, 89 expanded 5S rRNAs of 15 structural types were identified in 15 archaeal and 36 bacterial genomes. Expansion segments ranging in length from 13 to 109 residues were found to be distributed among 17 insertion sites. The strains harboring the expanded 5S rRNAs belong to the bacterial orders Clostridiales, Halanaerobiales, Thermoanaerobacterales, and Alteromonadales as well as the archael order Halobacterales When several copies of a 5S rRNA gene are present in a genome, the expanded versions may coexist with normal 5S rRNA genes. The insertion sequences are typically capable of forming extended helices, which do not seemingly interfere with folding of the conserved core. The expanded 5S rRNAs have largely been overlooked in 5S rRNA databases.

Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS.

Post-transcriptional RNA modifications play an important role in cellular metabolism with homoeostatic disturbances manifesting as a wide repertoire of phenotypes, reduced stress tolerance and translational perturbation, developmental defects, and diseases, such as type II diabetes, leukaemia, and carcinomas. Hence, there has been an intense effort to develop various methods for investigating RNA modifications and their roles in various organisms, including sequencing-based approaches and, more frequently, liquid chromatography-mass spectrometry (LC-MS)-based methods. Although LC-MS offers numerous advantages, such as being highly sensitive and quantitative over a broad detection range, some stationary phase chemistries struggle to resolve positional isomers. Furthermore, the demand for detailed analyses of complex biological samples often necessitates long separation times, hampering sample-to-sample turnover and making multisample analyses time consuming. To overcome this limitation, we have developed an ultra-performance LC-MS (UPLC-MS) method that uses an octadecyl carbon chain (C18)-bonded silica matrix for the efficient separation of 50 modified ribonucleosides, including positional isomers, in a single 9-min sample-to-sample run. To validate the performance and versatility of our method, we analysed tRNA modification patterns of representative microorganisms from each domain of life, namely Archaea (Methanosarcina acetivorans), Bacteria (Pseudomonas syringae), and Eukarya (Saccharomyces cerevisiae). Additionally, our method is flexible and readily applicable for detection and relative quantification using stable isotope labelling and targeted approaches like multiple reaction monitoring (MRM). In conclusion, this method represents a fast and robust tool for broad-range exploration and quantification of ribonucleosides, facilitating future homoeostasis studies of RNA modification in complex biological samples.

CRISPR-Cas adaptive immune systems in Sulfolobales: genetic studies and molecular mechanisms.

CRISPR-Cas systems provide the small RNA-based adaptive immunity to defend against invasive genetic elements in archaea and bacteria. Organisms of Sulfolobales, an order of thermophilic acidophiles belonging to the Crenarchaeotal Phylum, usually contain both type I and type III CRISPR-Cas systems. Two species, Saccharolobus solfataricus and Sulfolobus islandicus, have been important models for CRISPR study in archaea, and knowledge obtained from these studies has greatly expanded our understanding of molecular mechanisms of antiviral defense in all three steps: adaptation, expression and crRNA processing, and interference. Four subtypes of CRISPR-Cas systems are common in these organisms, including I-A, I-D, III-B, and III-D. These cas genes form functional modules, e.g., all genes required for adaptation and for interference in the I-A immune system are clustered together to form aCas and iCas modules. Genetic assays have been developed to study mechanisms of adaptation and interference by different CRISPR-Cas systems in these model archaea, and these methodologies are useful in demonstration of the protospacer-adjacent motif (PAM)-dependent DNA interference by I-A interference modules and multiple interference activities by III-B Cmr systems. Ribonucleoprotein effector complexes have been isolated for Sulfolobales III-B and III-D systems, and their biochemical characterization has greatly enriched the knowledge of molecular mechanisms of these novel antiviral immune responses.

Structural Studies of HNA Substrate Specificity in Mutants of an Archaeal DNA Polymerase Obtained by Directed Evolution.

Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants.

iCLIP analysis of RNA substrates of the archaeal exosome.

BACKGROUND: The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3' to 5' direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3'-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging. RESULTS: To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon Sulfolobus solfataricus. A relatively high proportion (17-19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5' parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3'-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5'-ends of RNAs was detected. CONCLUSIONS: In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5'-ends of genes suggests simultaneous binding of both RNA ends by the S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3'-5' direction.

Comparative patterns of modified nucleotides in individual tRNA species from a mesophilic and two thermophilic archaea.

To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale), and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations, and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius Notable observations are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs, especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii.

A newly identified duplex RNA unwinding activity of archaeal RNase J depends on processive exoribonucleolysis coupled steric occlusion by its structural archaeal loops.

RNase J is a prokaryotic 5'-3' exo/endoribonuclease that functions in mRNA decay and rRNA maturation. Here, we report a novel duplex unwinding activity of mpy-RNase J, an archaeal RNase J from Methanolobus psychrophilus, which enables it to degrade duplex RNAs with hairpins up to 40 bp when linking a 5' single-stranded overhangs of ≥ 7 nt, corresponding to the RNA channel length. A 6-nt RNA-mpy-RNase J-S247A structure reveals the RNA-interacting residues and a steric barrier at the RNA channel entrance comprising two archaeal loops and two helices. Mutagenesis of the residues key to either exoribonucleolysis or RNA translocation diminished the duplex unwinding activity. Substitution of the residues in the steric barrier yielded stalled degradation intermediates at the duplex RNA regions. Thus, an exoribonucleolysis-driven and steric occlusion-based duplex unwinding mechanism was identified. The duplex unwinding activity confers mpy-RNase J the capability of degrading highly structured RNAs, including the bacterial REP RNA, and archaeal mRNAs, rRNAs, tRNAs, SRPs, RNase P and CD-box RNAs, providing an indicative of the potential key roles of mpy-RNase J in pleiotropic RNA metabolisms. Hydrolysis-coupled duplex unwinding activity was also detected in a bacterial RNase J, which may use a shared but slightly different unwinding mechanism from archaeal RNase Js, indicating that duplex unwinding is a common property of the prokaryotic RNase Js.

Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping.

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.

Discovery of 20 novel ribosomal leader candidates in bacteria and archaea.

BACKGROUND: RNAs perform many functions in addition to supplying coding templates, such as binding proteins. RNA-protein interactions are important in multiple processes in all domains of life, and the discovery of additional protein-binding RNAs expands the scope for studying such interactions. To find such RNAs, we exploited a form of ribosomal regulation. Ribosome biosynthesis must be tightly regulated to ensure that concentrations of rRNAs and ribosomal proteins (r-proteins) match. One regulatory mechanism is a ribosomal leader (r-leader), which is a domain in the 5' UTR of an mRNA whose genes encode r-proteins. When the concentration of one of these r-proteins is high, the protein binds the r-leader in its own mRNA, reducing gene expression and thus protein concentrations. To date, 35 types of r-leaders have been validated or predicted. RESULTS: By analyzing additional conserved RNA structures on a multi-genome scale, we identified 20 novel r-leader structures. Surprisingly, these included new r-leaders in the highly studied organisms Escherichia coli and Bacillus subtilis. Our results reveal several cases where multiple unrelated RNA structures likely bind the same r-protein ligand, and uncover previously unknown r-protein ligands. Each r-leader consistently occurs upstream of r-protein genes, suggesting a regulatory function. That the predicted r-leaders function as RNAs is supported by evolutionary correlations in the nucleotide sequences that are characteristic of a conserved RNA secondary structure. The r-leader predictions are also consistent with the locations of experimentally determined transcription start sites. CONCLUSIONS: This work increases the number of known or predicted r-leader structures by more than 50%, providing additional opportunities to study structural and evolutionary aspects of RNA-protein interactions. These results provide a starting point for detailed experimental studies.

New proposal of nitrogen metabolism regulation by small RNAs in the extreme halophilic archaeon Haloferax mediterranei.

The regulatory networks involved in the uptake and metabolism of different nitrogen sources in response to their availability are crucial in all organisms. Nitrogen metabolism pathways have been studied in detail in archaea such as the extreme halophilic archaeon Haloferax mediterranei. However, knowledge about nitrogen metabolism regulation in haloarchaea is very scarce, and no transcriptional regulators involved in nitrogen metabolism have been identified to date. Advances in the molecular biology field have revealed that many small RNAs (sRNAs) are involved in the regulation of a diverse metabolic pathways. Surprisingly, no studies on regulation mediated by sRNAs have focused on the response to environmental fluctuations in nitrogen in haloarchaea. To identify sRNAs involved in the transcriptional regulation of nitrogen assimilation genes in Haloferax mediterranei and, thus, propose a novel regulatory mechanism, RNA-Seq was performed using cells grown in the presence of two different nitrogen sources. The differential transcriptional expression analysis of the RNA-Seq data revealed differences in the transcription patterns of 102 sRNAs according to the nitrogen source, and the molecular functions, cellular locations and biological processes with which the target genes were associated were predicted. These results enabled the identification of four sRNAs that could be directly related to the regulation of genes involved in nitrogen metabolism. This work provides the first proposed regulatory mechanism of nitrogen assimilation-related gene expression by sRNAs in haloarchaea as an alternative to transcriptional regulation mediated by proteins.

Identification of RNA 3´ ends and termination sites in Haloferax volcanii.

Archaeal genomes are densely packed; thus, correct transcription termination is an important factor for orchestrated gene expression. A systematic analysis of RNA 3´ termini, to identify transcription termination sites (TTS) using RNAseq data has hitherto only been performed in two archaea, Methanosarcina mazei and Sulfolobus acidocaldarius. In this study, only regions directly downstream of annotated genes were analysed, and thus, only part of the genome had been investigated. Here, we developed a novel algorithm (Internal Enrichment-Peak Calling) that allows an unbiased, genome-wide identification of RNA 3´ termini independent of annotation. In an RNA fraction enriched for primary transcripts by terminator exonuclease (TEX) treatment we identified 1,543 RNA 3´ termini. Approximately half of these were located in intergenic regions, and the remainder were found in coding regions. A strong sequence signature consistent with known termination events at intergenic loci indicates a clear enrichment for native TTS among them. Using these data we determined distinct putative termination motifs for intergenic (a T stretch) and coding regions (AGATC). In vivo reporter gene tests of selected TTS confirmed termination at these sites, which exemplify the different motifs. For several genes, more than one termination site was detected, resulting in transcripts with different lengths of the 3´ untranslated region (3´ UTR).

Archaeosine Modification of Archaeal tRNA: Role in Structural Stabilization.

Archaeosine (G+) is a structurally complex modified nucleoside found quasi-universally in the tRNA of Archaea and located at position 15 in the dihydrouridine loop, a site not modified in any tRNA outside the Archaea G+ is characterized by an unusual 7-deazaguanosine core structure with a formamidine group at the 7-position. The location of G+ at position 15, coupled with its novel molecular structure, led to a hypothesis that G+ stabilizes tRNA tertiary structure through several distinct mechanisms. To test whether G+ contributes to tRNA stability and define the biological role of G+, we investigated the consequences of introducing targeted mutations that disrupt the biosynthesis of G+ into the genome of the hyperthermophilic archaeon Thermococcus kodakarensis and the mesophilic archaeon Methanosarcina mazei, resulting in modification of the tRNA with the G+ precursor 7-cyano-7-deazaguansine (preQ0) (deletion of arcS) or no modification at position 15 (deletion of tgtA). Assays of tRNA stability from in vitro-prepared and enzymatically modified tRNA transcripts, as well as tRNA isolated from the T. kodakarensis mutant strains, demonstrate that G+ at position 15 imparts stability to tRNAs that varies depending on the overall modification state of the tRNA and the concentration of magnesium chloride and that when absent results in profound deficiencies in the thermophily of T. kodakarensisIMPORTANCE Archaeosine is ubiquitous in archaeal tRNA, where it is located at position 15. Based on its molecular structure, it was proposed to stabilize tRNA, and we show that loss of archaeosine in Thermococcus kodakarensis results in a strong temperature-sensitive phenotype, while there is no detectable phenotype when it is lost in Methanosarcina mazei Measurements of tRNA stability show that archaeosine stabilizes the tRNA structure but that this effect is much greater when it is present in otherwise unmodified tRNA transcripts than in the context of fully modified tRNA, suggesting that it may be especially important during the early stages of tRNA processing and maturation in thermophiles. Our results demonstrate how small changes in the stability of structural RNAs can be manifested in significant biological-fitness changes.

In Vivo RNA Chemical Footprinting Analysis in Archaea.

RNA structural conformation and dynamics govern the functional properties of all RNA/RNP. Accordingly, defining changes of RNA structure and dynamics in various conditions may provide detailed insight into how RNA structural properties regulate the function of RNA/RNP. Traditional chemical footprinting analysis using chemical modifiers allows to sample the dynamics and conformation landscape of diverse RNA/RNP. However, many chemical modifiers are limited in their capacity to provide unbiased information reflecting the in vivo RNA/RNP structural landscape. In the recent years, the development of selective-2'-hydroxyl acylation analyzed by primer extension (SHAPE) methodology that uses powerful new chemical modifiers has significantly improved in vitro and in vivo structural probing of secondary and tertiary interactions of diverse RNA species at the single nucleotide level.Although the original discovery of Archaea as an independent domain of life is intimately linked to the technological development of RNA analysis, our understanding of in vivo RNA structural conformation and dynamics in this domain of life remains scarce.This protocol describes the in vivo use of SHAPE chemistry in two evolutionary divergent model Archaea, Sulfolobus acidocaldarius and Haloferax volcanii.