Circularization restores signal recognition particle RNA functionality in Thermoproteus.

Signal recognition particles (SRPs) are universal ribonucleoprotein complexes found in all three domains of life that direct the cellular traffic and secretion of proteins. These complexes consist of SRP proteins and a single, highly structured SRP RNA. Canonical SRP RNA genes have not been identified for some Thermoproteus species even though they contain SRP19 and SRP54 proteins. Here, we show that genome rearrangement events in Thermoproteus tenax created a permuted SRP RNA gene. The 5'- and 3'-termini of this SRP RNA are located close to a functionally important loop present in all known SRP RNAs. RNA-Seq analyses revealed that these termini are ligated together to generate circular SRP RNA molecules that can bind to SRP19 and SRP54. The circularization site is processed by the tRNA splicing endonuclease. This moonlighting activity of the tRNA splicing machinery permits the permutation of the SRP RNA and creates highly stable and functional circular RNA molecules.

Interference activity of a minimal Type I CRISPR-Cas system from Shewanella putrefaciens.

Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs.

DNA and RNA interference mechanisms by CRISPR-Cas surveillance complexes.

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) adaptive immune systems use small guide RNAs, the CRISPR RNAs (crRNAs), to mark foreign genetic material, e.g. viral nucleic acids, for degradation. Archaea and bacteria encode a large variety of Cas proteins that bind crRNA molecules and build active ribonucleoprotein surveillance complexes. The evolution of CRISPR-Cas systems has resulted in a diversification of cas genes and a classification of the systems into three types and additional subtypes characterized by distinct surveillance and interfering complexes. Recent crystallographic and biochemical advances have revealed detailed insights into the assembly and DNA/RNA targeting mechanisms of the various complexes. Here, we review our knowledge on the molecular mechanism involved in the DNA and RNA interference stages of type I (Cascade: CRISPR-associated complex for antiviral defense), type II (Cas9) and type III (Csm, Cmr) CRISPR-Cas systems. We further highlight recently reported structural and mechanistic themes shared among these systems.

In Vitro Co-reconstitution of Cas Protein Complexes.

CRISPR-Cas systems employ diverse and often multimeric CRISPR-associated (Cas) protein effector complexes to mediate antiviral defense. The elucidation of the mechanistic details and the protein interaction partners requires production of recombinant Cas proteins. However, these proteins are often produced as inactive inclusion bodies. Here, we present a detailed protocol for the isolation and purification of insoluble Cas proteins. Guidelines for their solubilization via co-reconstitution strategies and procedures to upscale the production of soluble multimeric Cas protein complexes are provided.

DNA binding properties of the small cascade subunit Csa5.

CRISPR-Cas systems provide immunity against viral attacks in archaeal and bacterial cells. Type I systems employ a Cas protein complex termed Cascade, which utilizes small CRISPR RNAs to detect and degrade the exogenic DNA. A small sequence motif, the PAM, marks the foreign substrates. Previously, a recombinant type I-A Cascade complex from the archaeon Thermoproteus tenax was shown to target and degrade DNA in vitro, dependent on a native PAM sequence. Here, we present the biochemical analysis of the small subunit, Csa5, of this Cascade complex. T. tenax Csa5 preferentially bound ssDNA and mutants that showed decreased ssDNA-binding and reduced Cascade-mediated DNA cleavage were identified. Csa5 oligomerization prevented DNA binding. Specific recognition of the PAM sequence was not observed. Phylogenetic analyses identified Csa5 as a universal member of type I-A systems and revealed three distinct groups. A potential role of Csa5 in R-loop stabilization is discussed.

In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems of type I use a Cas ribonucleoprotein complex for antiviral defense (Cascade) to mediate the targeting and degradation of foreign DNA. To address molecular features of the archaeal type I-A Cascade interference mechanism, we established the in vitro assembly of the Thermoproteus tenax Cascade from six recombinant Cas proteins, synthetic CRISPR RNAs (crRNAs) and target DNA fragments. RNA-Seq analyses revealed the processing pattern of crRNAs from seven T. tenax CRISPR arrays. Synthetic crRNA transcripts were matured by hammerhead ribozyme cleavage. The assembly of type I-A Cascade indicates that Cas3' and Cas3'' are an integral part of the complex, and the interference activity was shown to be dependent on the crRNA and the matching target DNA. The reconstituted Cascade was used to identify sequence motifs that are required for efficient DNA degradation and to investigate the role of the subunits Cas7 and Cas3'' in the interplay with other Cascade subunits.

Exploiting CRISPR/Cas: interference mechanisms and applications.

The discovery of biological concepts can often provide a framework for the development of novel molecular tools, which can help us to further understand and manipulate life. One recent example is the elucidation of the prokaryotic adaptive immune system, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) that protects bacteria and archaea against viruses or conjugative plasmids. The immunity is based on small RNA molecules that are incorporated into versatile multi-domain proteins or protein complexes and specifically target viral nucleic acids via base complementarity. CRISPR/Cas interference machines are utilized to develop novel genome editing tools for different organisms. Here, we will review the latest progress in the elucidation and application of prokaryotic CRISPR/Cas systems and discuss possible future approaches to exploit the potential of these interference machineries.

Substrate generation for endonucleases of CRISPR/cas systems.

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) (1-3). Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems (4). The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster (5). The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs (6-8). These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence (9). A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity(10). Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA (11,12) . These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases (4). Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

Small RNAs for defence and regulation in archaea.

Non-coding RNAs are key players in many cellular processes within organisms from all three domains of life. The range and diversity of small RNA functions beyond their involvement in translation and RNA processing was first recognized for eukaryotes and bacteria. Since then, small RNAs were also found to be abundant in archaea. Their functions include the regulation of gene expression and the establishment of immunity against invading mobile genetic elements. This review summarizes our current knowledge about small RNAs used for regulation and defence in archaea.

Characterization of the CRISPR/Cas subtype I-A system of the hyperthermophilic crenarchaeon Thermoproteus tenax.

CRISPR (clustered regularly interspaced short palindromic repeats) elements and cas (CRISPR-associated) genes are widespread in Bacteria and Archaea. The CRISPR/Cas system operates as a defense mechanism against mobile genetic elements (i.e., viruses or plasmids). Here, we investigate seven CRISPR loci in the genome of the crenarchaeon Thermoproteus tenax that include spacers with significant similarity not only to archaeal viruses but also to T. tenax genes. The analysis of CRISPR RNA (crRNA) transcription reveals transcripts of a length between 50 and 130 nucleotides, demonstrating the processing of larger crRNA precursors. The organization of identified cas genes resembles CRISPR/Cas subtype I-A, and the core cas genes are shown to be arranged on two polycistronic transcripts: cascis (cas4, cas1/2, and csa1) and cascade (csa5, cas7, cas5a, cas3, cas3', and cas8a2). Changes in the environmental parameters such as UV-light exposure or high ionic strength modulate cas gene transcription. Two reconstitution protocols were established for the production of two discrete multipartite Cas protein complexes that correspond to their operonic gene arrangement. These data provide insights into the specialized mechanisms of an archaeal CRISPR/Cas system and allow selective functional analyses of Cas protein complexes in the future.

The complete genome sequence of Thermoproteus tenax: a physiologically versatile member of the Crenarchaeota.

Here, we report on the complete genome sequence of the hyperthermophilic Crenarchaeum Thermoproteus tenax (strain Kra1, DSM 2078(T)) a type strain of the crenarchaeotal order Thermoproteales. Its circular 1.84-megabase genome harbors no extrachromosomal elements and 2,051 open reading frames are identified, covering 90.6% of the complete sequence, which represents a high coding density. Derived from the gene content, T. tenax is a representative member of the Crenarchaeota. The organism is strictly anaerobic and sulfur-dependent with optimal growth at 86°C and pH 5.6. One particular feature is the great metabolic versatility, which is not accompanied by a distinct increase of genome size or information density as compared to other Crenarchaeota. T. tenax is able to grow chemolithoautotrophically (CO2/H2) as well as chemoorganoheterotrophically in presence of various organic substrates. All pathways for synthesizing the 20 proteinogenic amino acids are present. In addition, two presumably complete gene sets for NADH:quinone oxidoreductase (complex I) were identified in the genome and there is evidence that either NADH or reduced ferredoxin might serve as electron donor. Beside the typical archaeal A0A1-ATP synthase, a membrane-bound pyrophosphatase is found, which might contribute to energy conservation. Surprisingly, all genes required for dissimilatory sulfate reduction are present, which is confirmed by growth experiments. Mentionable is furthermore, the presence of two proteins (ParA family ATPase, actin-like protein) that might be involved in cell division in Thermoproteales, where the ESCRT system is absent, and of genes involved in genetic competence (DprA, ComF) that is so far unique within Archaea.

Phosphoenolpyruvate synthetase and pyruvate, phosphate dikinase of Thermoproteus tenax: key pieces in the puzzle of archaeal carbohydrate metabolism.

The interconversion of phosphoenolpyruvate and pyruvate represents an important control point of the Embden-Meyerhof-Parnas (EMP) pathway in Bacteria and Eucarya, but little is known about this site of regulation in Archaea. Here we report on the coexistence of phosphoenolpyruvate synthetase (PEPS) and the first described archaeal pyruvate, phosphate dikinase (PPDK), which, besides pyruvate kinase (PK), are involved in the catalysis of this reaction in the hyperthermophilic crenarchaeote Thermoproteus tenax. The genes encoding T. tenax PEPS and PPDK were cloned and expressed in Escherichia coli, and the enzymic and regulatory properties of the recombinant gene products were analysed. Whereas PEPS catalyses the unidirectional conversion of pyruvate to phosphoenolpyruvate, PPDK shows a bidirectional activity with a preference for the catabolic reaction. In contrast to PK of T. tenax, which is regulated on transcript level but exhibits only limited regulatory potential on protein level, PEPS and PPDK activities are modulated by adenosine phosphates and intermediates of the carbohydrate metabolism. Additionally, expression of PEPS is regulated on transcript level in response to the offered carbon source as revealed by Northern blot analyses. The combined action of the differently regulated enzymes PEPS, PPDK and PK represents a novel way of controlling the interconversion of phosphoenolpyruvate and pyruvate in the reversible EMP pathway, allowing short-term and long-term adaptation to different trophic conditions. Comparative genomic analyses indicate the coexistence of PEPS, PPDK and PK in other Archaea as well, suggesting a similar regulation of the carbohydrate metabolism in these organisms.