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CRISPR-Cas systems provide RNA-guided adaptive immunity in prokaryotes. We report that the multisubunit CRISPR effector Cascade transcriptionally regulates a toxin-antitoxin RNA pair, CreTA. CreT (Cascade-repressed toxin) is a bacteriostatic RNA that sequesters the rare arginine tRNA UCU (transfer RNA with anticodon UCU). CreA is a CRISPR RNA-resembling antitoxin RNA, which requires Cas6 for maturation. The partial complementarity between CreA and the creT promoter directs Cascade to repress toxin transcription. Thus, CreA becomes antitoxic only in the presence of Cascade. In CreTA-deleted cells, cascade genes become susceptible to disruption by transposable elements. We uncover several CreTA analogs associated with diverse archaeal and bacterial CRISPR- cas loci. Thus, toxin-antitoxin RNA pairs can safeguard CRISPR immunity by making cells addicted to CRISPR-Cas, which highlights the multifunctionality of Cas proteins and the intricate mechanisms of CRISPR-Cas regulation., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Prokaryotes encounter constant and often brutal modifications to their environment. In order to survive, they need to maintain fitness, which includes adapting their protein expression patterns. Many factors control gene expression but this review focuses on just one, namely antisense RNAs (asRNAs), a class of non-coding RNAs (ncRNAs) characterized by their location in cis and their perfect complementarity with their targets. asRNAs were considered for a long time to be trivial and only to be found on mobile genetic elements. However, recent advances in methodology have revealed that their abundance and potential activities have been underestimated. This review aims to illustrate the role of asRNA in various physiologically crucial functions in both archaea and bacteria, which can be regrouped in three categories: cell maintenance, horizontal gene transfer and virulence. A literature survey of asRNAs demonstrates the difficulties to characterize and assign a role to asRNAs. With the aim of facilitating this task, we describe recent technological advances that could be of interest to identify new asRNAs and to discover their function., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Many years of research in RNA biology have soundly established the importance of RNA-based regulation far beyond most early traditional presumptions. Importantly, the advances in "wet" laboratory techniques have produced unprecedented amounts of data that require efficient and precise computational analysis schemes and algorithms. Hence, many in silico methods that attempt topological and functional classification of novel putative RNA-based regulators are available. In this review, we technically outline thermodynamics-based standard RNA secondary structure and RNA-RNA interaction prediction approaches that have proven valuable to the RNA research community in the past and present. For these, we highlight their usability with a special focus on prokaryotic organisms and also briefly mention recent advances in whole-genome interactomics and how this may influence the field of predictive RNA research.

The rRNA is the largest and most abundant RNA in bacterial and archaeal cells. It is also one of the best-characterized RNAs in terms of its structural motifs and sequence variation. Production of ribosome components including >50 ribosomal proteins (r-proteins) consumes significant cellular resources. Thus, RNA cis -regulatory structures that interact with r-proteins to repress further r-protein synthesis play an important role in maintaining appropriate stoichiometry between r-proteins and rRNA. Classically, such mRNA structures were thought to directly mimic the rRNA. However, more than 30 years of research has demonstrated that a variety of different recognition and regulatory paradigms are present. This review will demonstrate how structural mimicry between the rRNA and mRNA cis -regulatory structures may take many different forms. The collection of mRNA structures that interact with r-proteins to regulate r-protein operons are best characterized in Escherichia coli , but are increasingly found within species from nearly all phyla of bacteria and several archaea. Furthermore, they represent a unique opportunity to assess the plasticity of RNA structure in the context of RNA-protein interactions. The binding determinants imposed by r-proteins to allow regulation can be fulfilled in many ways. Some r-protein-interacting mRNAs are immediately obvious as rRNA mimics from primary sequence similarity, others are identifiable only after secondary or tertiary structure determination, and some show no obvious similarity. In addition, across different bacterial species a host of different mechanisms of action have been characterized, showing that there is no simple one-size-fits-all solution.

In all organisms, several distinct stand-alone pseudouridine synthase (PUS) family enzymes are expressed to isomerize uridine into pseudouridine (Ψ) by specific recognition of RNAs. In addition, Ψs are generated in Archaea and Eukaryotes by PUS enzymes which are organized as ribonucleoprotein particles (RNP)--the box H/ACA s/snoRNPs. For this modification system, a unique TruB-like catalytic PUS subunit is associated with various RNA guides which specifically target and secure substrate RNAs by base-pairing. The archaeal Cbf5 PUS displays the special feature of exhibiting both RNA guide-dependent and -independent activities. Structures of substrate-bound TruB and H/ACA sRNP revealed the importance of histidines in positioning the target uridine in the active site. To analyze the respective role of H60 and H77, we have generated variants carrying alanine substitutions at these positions. The impact of the mutations was analyzed for unguided modifications U(55) in tRNA and U2603 in 23S rRNA, and for activity of the box H/ACA Pab91 sRNP enzyme. H77 (H43 in TruB), but not H60, appeared to be crucial for the RNA guide-independent activity. In contrast to earlier suggestions, H60 was found to be noncritical for the activity of the H/ACA sRNP, but contributes together with H77 to the full activity of H/ACA sRNPs. The data suggest that a similar catalytic process was conserved in the two divergent pseudouridylation systems., (© 2015 Tillault et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for targeted elimination of foreign nucleic acids. These immune systems have been divided into three main types, and the first atomic-resolution structure of a type III RNA-guided immune complex provides new insights into the mechanisms of nucleic acid degradation. Here we compare the crystal structure of a type III complex to recently determined structures of DNA-targeting type I CRISPR complexes. Structural comparisons support previous assertions that type I and type III systems share a common ancestor and reveal how a conserved structural chassis is used to support RNA-, DNA-, or both RNA- and DNA-targeting mechanisms., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs) are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq) and a primary-transcript enriched (dRNA-seq) strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (∼7% of the annotated genome). Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.

All cellular systems evolve ways to combat predators and genomic parasites. In bacteria and archaea, numerous resistance mechanisms have developed against phage. Our understanding of this defensive repertoire has recently been expanded to include the CRISPR system of clustered, regularly interspaced short palindromic repeats. In this remarkable pathway, short sequence tags from invading genetic elements are actively incorporated into the host's CRISPR locus to be transcribed and processed into a set of small RNAs that guide the destruction of foreign genetic material. Here we review the inner workings of this adaptable and heritable immune system and draw comparisons to small RNA-guided defense mechanisms in eukaryotic cells., (Copyright 2010 Elsevier Inc. All rights reserved.)

Clusters of regularly spaced direct repeats, separated by unconserved spacer sequences, are ubiquitous in archaeal chromosomes and occur in some plasmids. Some clusters constitute around 1% of chromosomal DNA. Similarly structured clusters, generally smaller, also occur in some bacterial chromosomes. Although early studies implicated these clusters in segregation/partition functions, recent evidence suggests that the spacer sequences derive from extrachromosomal elements, and, primarily, viruses. This has led to the proposal that the clusters provide a defence against viral propagation in cells, and that both the mode of inhibition of viral propagation and the mechanism of adding spacer-repeat units to clusters, are dependent on RNAs transcribed from the clusters. Moreover, the putative inhibitory apparatus (piRNA-based) may be evolutionarily related to the interference RNA systems (siRNA and miRNA), which are common in eukarya. Here, we analyze all the current data on archaeal repeat clusters and provide some new insights into their diverse structures, transcriptional properties and mode of structural development. The results are consistent with larger cluster transcripts being processed at the centers of the repeat sequences and being further trimmed by exonucleases to yield a dominant, intracellular RNA species, which corresponds approximately to the size of a spacer. Furthermore, analysis of the extensive clusters of Sulfolobus solfataricus strains P1 and P2B provides support for the presence of a flanking sequence adjoining a cluster being a prerequisite for the incorporation of new spacer-repeat units, which occurs between the flanking sequence and the cluster. An archaeal database summarizing the data will be maintained at http://dac.molbio.ku.dk/dbs/SRSR/.

The exosome is a conserved eukaryotic enzymatic complex that plays an essential role in many pathways of RNA processing and degradation. Here, we describe the structural characterization of the predicted archaeal exosome in solution using small angle x-ray scattering. The structure model calculated from the small angle x-ray scattering pattern provides an indication of the existence of a disk-shaped structure, corresponding to the "RNases PH ring" complex formed by the proteins aRrp41 and aRrp42. The RNases PH ring complex corresponds to the core of the exosome, binds RNA, and has phosphorolytic and polymerization activities. Three additional molecules of the RNA-binding protein aRrp4 are attached to the core as extended and flexible arms that may direct the substrates to the active sites of the exosome. In the presence of aRrp4, the activity of the core complex is enhanced, suggesting a regulatory role for this protein. The results shown here also indicate the participation of the exosome in RNA metabolism in Archaea, as was established in Eukarya.

The signal recognition particle (SRP) RNA helix 6 of archaea and eukaryotes is essential for the binding of protein SRP19 and the assembly of a functional complex. The conserved adenosine at the third position of the tetraloop of helix 6 (A149) is crucial for the binding of protein SRP19 in the mammalian SRP. Here we investigated the significance of the equivalent adenosine residue at position 159 (A159) of Archaeoglobus fulgidus SRP RNA. The A159 of A. fulgidus and A149 of human SRP RNA were changed to C, G or U, and fragments containing helix 6 or helices 6 and 8 were synthesized by run-off transcription with T7 RNA polymerase. The ability of recombinant A. fulgidus and human SRP19 to form ribonucleoprotein complexes was measured in vitro. The simultaneous presence of A149 and helix 8 is required for the high-affinity binding of SRP19 to the human SRP RNA. In contrast, A. fulgidus SRP19 binds to the SRP RNA fragments with high affinity irrespective of the nature of the nucleotide, demonstrating that A159 does not directly participate in protein binding. Instead, as indicated by the resistance of the wild-type A. fulgidus RNA towards digestion by RNase A, this residue allows the formation of a tightly folded RNA molecule. The high affinity between A.fulgidus SRP 19 and RNA molecules that contain both helices 6 and 8 suggests that A159 is likely to initiate archaeal SRP assembly by forming a conserved tertiary RNA-RNA interaction.

The small nucleolar RNAs (snoRNAs) are an abundant class of trans-acting RNAs that function in ribosome biogenesis in the eukaryotic nucleolus. Elegant work has revealed that most known snoRNAs guide modification of pre-ribosomal RNA (pre-rRNA) by base pairing near target sites. Other snoRNAs are involved in cleavage of pre-rRNA by mechanisms that have not yet been detailed. Moreover, our appreciation of the cellular roles of the snoRNAs is expanding with new evidence that snoRNAs also target modification of small nuclear RNAs and messenger RNAs. Many snoRNAs are produced by unorthodox modes of biogenesis including salvage from introns of pre-mRNAs. The recent discovery that homologs of snoRNAs as well as associated proteins exist in the domain Archaea indicates that the RNA-guided RNA modification system is of ancient evolutionary origin. In addition, it has become clear that the RNA component of vertebrate telomerase (an enzyme implicated in cancer and cellular senescence) is related to snoRNAs. During its evolution, vertebrate telomerase RNA appears to have co-opted a snoRNA domain that is essential for the function of telomerase RNA in vivo. The unique properties of snoRNAs are now being harnessed for basic research and therapeutic applications.

In eukaryotes, the C/D box family of small nucleolar (sno)RNAs contain complementary guide regions that are used to direct 2'-O-ribose methylation to specific nucleotide positions within rRNA during the early stages of ribosome biogenesis. Direct cDNA cloning and computational genome searches have revealed homologues of C/D box snoRNAs (called sRNAs) in prokaryotic Archaea that grow at high temperature. The guide sequences within the sRNAs indicate that they are used to direct methylation to nucleotides in both rRNAs and tRNAs. The number of sRNA genes that are detectable within currently sequenced genomes correlates with the optimal growth temperature. We suggest that archaeal sRNAs may have two functions: to guide the deposition of methyl groups at the 2'-O position of ribose, which is an important determinant in RNA structural stability, and to serve as a molecular chaperones to help orchestrate the folding of rRNAs and tRNAs at high temperature.