Evolutionary and functional insights into the Ski2-like helicase family in Archaea: a comparison of Thermococcales ASH-Ski2 and Hel308 activities.

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The Ski2-like proteins are primordial helicases that play an active role in eukaryotic RNA homeostasis pathways, with multiple homologs having specialized functions. The significance of the expansion and diversity of Ski2-like proteins in Archaea, the third domain of life, has not yet been established. Here, by studying the phylogenetic diversity of Ski2-like helicases among archaeal genomes and the enzymatic activities of those in Thermococcales, we provide further evidence of the function of this protein family in archaeal metabolism of nucleic acids. We show that, in the course of evolution, ASH-Ski2 and Hel308-Ski2, the two main groups of Ski2-like proteins, have diverged in their biological functions. Whereas Hel308 has been shown to mainly act on DNA, we show that ASH-Ski2, previously described to be associated with the 5'-3' aRNase J exonuclease, acts on RNA by supporting an efficient annealing activity, but also an RNA unwinding with a 3'-5' polarity. To gain insights into the function of Ski2, we also analyse the transcriptome of Thermococcus barophilus ΔASH-Ski2 mutant strain and provide evidence of the importance of ASH-Ski2 in cellular metabolism pathways related to translation.

Characterization of a small tRNA-binding protein that interacts with the archaeal proteasome complex.

The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with the translational machinery remains to be described. Here, we characterized a small orphan protein, Q9UZY3 (UniProt ID), conserved in Thermococcales. The protein was identified in native pull-down experiments using the proteasome regulatory complex (proteasome-activating nucleotidase [PAN]) as bait. X-ray crystallography and small-angle X-ray scattering experiments revealed that the protein is monomeric and adopts a ß-barrel core structure with an oligonucleotide/oligosaccharide-binding (OB)-fold, typically found in translation elongation factors. Mobility shift experiment showed that Q9UZY3 displays transfer ribonucleic acid (tRNA)-binding properties. Pull-downs, co-immunoprecipitation and isothermal titration calorimetry (ITC) studies revealed that Q9UZY3 interacts in vitro with PAN. Native pull-downs and proteomic analysis using different versions of Q9UZY3 showed that the protein interacts with the assembled PAN-20S proteasome machinery in Pyrococcus abyssi (Pa) cellular extracts. The protein was therefore named Pbp11, for Proteasome-Binding Protein of 11 kDa. Interestingly, the interaction network of Pbp11 also includes ribosomal proteins, tRNA-processing enzymes and exosome subunits dependent on Pbp11's N-terminal domain that was found to be essential for tRNA binding. Together these data suggest that Pbp11 participates in an interface between the proteasome and the translational machinery.

Phylogenetic Diversity of Lhr Proteins and Biochemical Activities of the Thermococcales aLhr2 DNA/RNA Helicase.

Helicase proteins are known to use the energy of ATP to unwind nucleic acids and to remodel protein-nucleic acid complexes. They are involved in almost every aspect of DNA and RNA metabolisms and participate in numerous repair mechanisms that maintain cellular integrity. The archaeal Lhr-type proteins are SF2 helicases that are mostly uncharacterized. They have been proposed to be DNA helicases that act in DNA recombination and repair processes in Sulfolobales and Methanothermobacter. In Thermococcales, a protein annotated as an Lhr2 protein was found in the network of proteins involved in RNA metabolism. To investigate this, we performed in-depth phylogenomic analyses to report the classification and taxonomic distribution of Lhr-type proteins in Archaea, and to better understand their relationship with bacterial Lhr. Furthermore, with the goal of envisioning the role(s) of aLhr2 in Thermococcales cells, we deciphered the enzymatic activities of aLhr2 from Thermococcus barophilus (Tbar). We showed that Tbar-aLhr2 is a DNA/RNA helicase with a significant annealing activity that is involved in processes dependent on DNA and RNA transactions.

RNA processing machineries in Archaea: the 5'-3' exoribonuclease aRNase J of the ß-CASP family is engaged specifically with the helicase ASH-Ski2 and the 3'-5' exoribonucleolytic RNA exosome machinery.

A network of RNA helicases, endoribonucleases and exoribonucleases regulates the quantity and quality of cellular RNAs. To date, mechanistic studies focussed on bacterial and eukaryal systems due to the challenge of identifying the main drivers of RNA decay and processing in Archaea. Here, our data support that aRNase J, a 5'-3' exoribonuclease of the ß-CASP family conserved in Euryarchaeota, engages specifically with a Ski2-like helicase and the RNA exosome to potentially exert control over RNA surveillance, at the vicinity of the ribosome. Proteomic landscapes and direct protein-protein interaction analyses, strengthened by comprehensive phylogenomic studies demonstrated that aRNase J interplay with ASH-Ski2 and a cap exosome subunit. Finally, Thermococcus barophilus whole-cell extract fractionation experiments provide evidences that an aRNase J/ASH-Ski2 complex might exist in vivo and hint at an association of aRNase J with the ribosome that is emphasised in absence of ASH-Ski2. Whilst aRNase J homologues are found among bacteria, the RNA exosome and the Ski2-like RNA helicase have eukaryotic homologues, underlining the mosaic aspect of archaeal RNA machines. Altogether, these results suggest a fundamental role of ß-CASP RNase/helicase complex in archaeal RNA metabolism.

Physical and functional interplay between PCNA DNA clamp and Mre11-Rad50 complex from the archaeon Pyrococcus furiosus.

Several archaeal species prevalent in extreme environments are particularly exposed to factors likely to cause DNA damages. These include hyperthermophilic archaea (HA), living at temperatures >70°C, which arguably have efficient strategies and robust genome guardians to repair DNA damage threatening their genome integrity. In contrast to Eukarya and other archaea, homologous recombination appears to be a vital pathway in HA, and the Mre11-Rad50 complex exerts a broad influence on the initiation of this DNA damage response process. In a previous study, we identified a physical association between the Proliferating Cell Nuclear Antigen (PCNA) and the Mre11-Rad50 (MR) complex. Here, by performing co-immunoprecipitation and SPR analyses, we identified a short motif in the C- terminal portion of Pyrococcus furiosus Mre11 involved in the interaction with PCNA. Through this work, we revealed a PCNA-interaction motif corresponding to a variation on the PIP motif theme which is conserved among Mre11 sequences of Thermococcale species. Additionally, we demonstrated functional interplay in vitro between P. furiosus PCNA and MR enzymatic functions in the DNA end resection process. At physiological ionic strength, PCNA stimulates MR nuclease activities for DNA end resection and promotes an endonucleolytic incision proximal to the 5' strand of double strand DNA break.

Insights into RNA-processing pathways and associated RNA-degrading enzymes in Archaea.

RNA-processing pathways are at the centre of regulation of gene expression. All RNA transcripts undergo multiple maturation steps in addition to covalent chemical modifications to become functional in the cell. This includes destroying unnecessary or defective cellular RNAs. In Archaea, information on mechanisms by which RNA species reach their mature forms and associated RNA-modifying enzymes are still fragmentary. To date, most archaeal actors and pathways have been proposed in light of information gathered from Bacteria and Eukarya. In this context, this review provides a state of the art overview of archaeal endoribonucleases and exoribonucleases that cleave and trim RNA species and also of the key small archaeal proteins that bind RNAs. Furthermore, synthetic up-to-date views of processing and biogenesis pathways of archaeal transfer and ribosomal RNAs as well as of maturation of stable small non-coding RNAs such as CRISPR RNAs, small C/D and H/ACA box guide RNAs, and other emerging classes of small RNAs are described. Finally, prospective post-transcriptional mechanisms to control archaeal messenger RNA quality and quantity are discussed.

Archaeal fibrillarin-Nop5 heterodimer 2'-O-methylates RNA independently of the C/D guide RNP particle.

Archaeal fibrillarin (aFib) is a well-characterized S-adenosyl methionine (SAM)-dependent RNA 2'-O-methyltransferase that is known to act in a large C/D ribonucleoprotein (RNP) complex together with Nop5 and L7Ae proteins and a box C/D guide RNA. In the reaction, the guide RNA serves to direct the methylation reaction to a specific site in tRNA or rRNA by sequence complementarity. Here we show that a Pyrococcus abyssi aFib-Nop5 heterodimer can alone perform SAM-dependent 2'-O-methylation of 16S and 23S ribosomal RNAs in vitro independently of L7Ae and C/D guide RNAs. Using tritium-labeling, mass spectrometry, and reverse transcription analysis, we identified three in vitro 2'-O-methylated positions in the 16S rRNA of P. abyssi, positions lying outside of previously reported pyrococcal C/D RNP methylation sites. This newly discovered stand-alone activity of aFib-Nop5 may provide an example of an ancestral activity retained in enzymes that were recruited to larger complexes during evolution.

Universal RNA-degrading enzymes in Archaea: Prevalence, activities and functions of ß-CASP ribonucleases.

ß-CASP ribonucleases are widespread in all three domains of life. They catalyse both 5'-3' exoribonucleolytic RNA trimming and/or endoribonucleolytic RNA cleavage using a unique active site coordinated by two zinc ions. These fascinating enzymes have a key role in 3' end processing in Eukarya and in RNA decay and ribosomal RNA maturation in Bacteria. The recent recognition of ß-CASP ribonucleases as major players in Archaea is an important contribution towards identifying RNA-degrading enzymes in the third domain of life. Three ß-CASP orthologous groups, aCPSF1, aCPSF2, aCPSF1b, are closely related to the eukaryal CPSF73 termination factor and one, aRNase J, is ortholog of the bacterial RNase J. The endo- and 5'-3' exoribonucleolytic activities carried by archaeal ß-CASP enzymes are strictly conserved throughout archaeal phylogeny suggesting essential roles in maturation and/or degradation of RNA. The recent progress in understanding the prevalence, activities and functions of archaeal ß-CASP ribonucleases is the focus of this review. The current status of our understanding of RNA processing pathways in Archaea is covered in light of this new knowledge on ß-CASP ribonucleases.

Tips and tricks to probe the RNA-degrading activities of hyperthermophilic archaeal ß-CASP ribonucleases.

The importance of ribonucleases in posttranscriptional control of gene expression has been established in Eukarya and Bacteria for over a decade. However, this process has been overlooked in Archaea, which are of universal importance to elucidate fundamental biological mechanisms and to study the evolution of life on Earth. Very few ribonucleolytic activities have been reported in Archaea, and RNA metabolism pathways wait to be described. Recently we have identified two major groups of archaeal ribonucleases, aCPSF1 and aRNase J, which are members of the ß-CASP metallo-ß-lactamase family. Here, we describe in vitro methods to characterize the endo- and exoribonucleolytic activities of hyperthermophilic archaeal ß-CASP ribonucleases. The use of various labeled RNA substrates allows defining the specificity of RNA cleavage and the directionality of the exoribonucleolytic trimming activity of the archaeal enzymes which work at high temperature. Elucidating in vitro ribonucleolytic activities is one step toward the understanding of the role of ß-CASP ribonucleases in RNA metabolism pathways in archaeal cells.

Emergence of the ß-CASP ribonucleases: highly conserved and ubiquitous metallo-enzymes involved in messenger RNA maturation and degradation.

The ß-CASP ribonucleases, which are found in the three domains of life, have in common a core of 460 residues containing seven conserved sequence motifs involved in the tight binding of two catalytic zinc ions. A hallmark of these enzymes is their ability to catalyze both endo- and exo-ribonucleolytic degradation. Exo-ribonucleolytic degradation proceeds in the 5' to 3' direction and is sensitive to the phosphorylation state of the 5' end of a transcript. Recent phylogenomic analyses have shown that the ß-CASP ribonucleases can be partitioned into two major subdivisions that correspond to orthologs of eukaryal CPSF73 and bacterial RNase J. We discuss the known functions of the CPSF73 and RNase J orthologs, their association into complexes, and their structure as it relates to mechanism of action. Eukaryal CPSF73 is part of a large multiprotein complex that is involved in the maturation of the 3' end of RNA Polymerase II transcripts and the polyadenylation of messenger RNA. RNase J1 and J2 are paralogs in Bacillus subtilis that are involved in the degradation of messenger RNA and the maturation of non-coding RNA. RNase J1 and J2 co-purify as a heteromeric complex and there is recent evidence that they interact with other enzymes to form a bacterial RNA degradosome. Finally, we speculate on the evolutionary origin of ß-CASP ribonucleases and on their functions in Archaea. Orthologs of CPSF73 with endo- and exo-ribonuclease activity are strictly conserved throughout the archaea suggesting a role for these enzymes in the maturation and/or degradation of messenger RNA. This article is part of a Special Issue entitled: RNA Decay mechanisms.

Diversity of CRISPR systems in the euryarchaeal Pyrococcales.

Pyrococcales are members of the order Thermococcales, a group of hyperthermophilic euryarchaea that are frequently found in deep sea hydrothermal vents. Infectious genetic elements, such as plasmids and viruses, remain a threat even in this remote environment and these microorganisms have developed several ways to fight their genetic invaders. Among these are the recently discovered CRISPR systems. In this review, we have combined and condensed available information on genetic elements infecting the Thermococcales and on the multiple CRISPR systems found in the Pyrococcales to fight them. Their organization and mode of action will be presented with emphasis on the Type III-B system that is the only CRISPR system known to target RNA molecules in a process reminiscent of RNA interference. The intriguing case of Pyrococcus abyssi, which is among the rare strains to present a CRISPR system devoid of the universal cas1 and cas2 genes, is also discussed.

Archaeal ß-CASP ribonucleases of the aCPSF1 family are orthologs of the eukaryal CPSF-73 factor.

Bacterial RNase J and eukaryal cleavage and polyadenylation specificity factor (CPSF-73) are members of the ß-CASP family of ribonucleases involved in mRNA processing and degradation. Here we report an in-depth phylogenomic analysis that delineates aRNase J and archaeal CPSF (aCPSF) as distinct orthologous groups and establishes their repartition in 110 archaeal genomes. The aCPSF1 subgroup, which has been inherited vertically and is strictly conserved, is characterized by an N-terminal extension with two K homology (KH) domains and a C-terminal motif involved in dimerization of the holoenzyme. Pab-aCPSF1 (Pyrococcus abyssi homolog) has an endoribonucleolytic activity that preferentially cleaves at single-stranded CA dinucleotides and a 5'-3' exoribonucleolytic activity that acts on 5' monophosphate substrates. These activities are the same as described for the eukaryotic cleavage and polyadenylation factor, CPSF-73, when engaged in the CPSF complex. The N-terminal KH domains are important for endoribonucleolytic cleavage at certain specific sites and the formation of stable high molecular weight ribonucleoprotein complexes. Dimerization of Pab-aCPSF is important for exoribonucleolytic activity and RNA binding. Altogether, our results suggest that aCPSF1 performs an essential function and that an enzyme with similar activities was present in the last common ancestor of Archaea and Eukarya.

Programmable sequence-specific click-labeling of RNA using archaeal box C/D RNP methyltransferases.

Biophysical and mechanistic investigation of RNA function requires site-specific incorporation of spectroscopic and chemical probes, which is difficult to achieve using current technologies. We have in vitro reconstituted a functional box C/D small ribonucleoprotein RNA 2'-O-methyltransferase (C/D RNP) from the thermophilic archaeon Pyrococcus abyssi and demonstrated its ability to transfer a prop-2-ynyl group from a synthetic cofactor analog to a series of preselected target sites in model tRNA and pre-mRNA molecules. Target selection of the RNP was programmed by changing a dodecanucleotide guide sequence in a 64-nt C/D guide RNA leading to efficient derivatization of three out of four new targets in each RNA substrate. We also show that the transferred terminal alkyne can be further appended with a fluorophore using a bioorthogonal azide-alkyne 1,3-cycloaddition (click) reaction. The described approach for the first time permits synthetically tunable sequence-specific labeling of RNA with single-nucleotide precision.

Identification of CRISPR and riboswitch related RNAs among novel noncoding RNAs of the euryarchaeon Pyrococcus abyssi.

BACKGROUND: Noncoding RNA (ncRNA) has been recognized as an important regulator of gene expression networks in Bacteria and Eucaryota. Little is known about ncRNA in thermococcal archaea except for the eukaryotic-like C/D and H/ACA modification guide RNAs. RESULTS: Using a combination of in silico and experimental approaches, we identified and characterized novel P. abyssi ncRNAs transcribed from 12 intergenic regions, ten of which are conserved throughout the Thermococcales. Several of them accumulate in the late-exponential phase of growth. Analysis of the genomic context and sequence conservation amongst related thermococcal species revealed two novel P. abyssi ncRNA families. The CRISPR family is comprised of crRNAs expressed from two of the four P. abyssi CRISPR cassettes. The 5'UTR derived family includes four conserved ncRNAs, two of which have features similar to known bacterial riboswitches. Several of the novel ncRNAs have sequence similarities to orphan OrfB transposase elements. Based on RNA secondary structure predictions and experimental results, we show that three of the twelve ncRNAs include Kink-turn RNA motifs, arguing for a biological role of these ncRNAs in the cell. Furthermore, our results show that several of the ncRNAs are subjected to processing events by enzymes that remain to be identified and characterized. CONCLUSIONS: This work proposes a revised annotation of CRISPR loci in P. abyssi and expands our knowledge of ncRNAs in the Thermococcales, thus providing a starting point for studies needed to elucidate their biological function.

Euryarchaeal beta-CASP proteins with homology to bacterial RNase J Have 5'- to 3'-exoribonuclease activity.

In the Archaea only a handful of ribonucleases involved in RNA processing and degradation have been characterized. One potential group of archaeal ribonucleases are homologues of the bacterial RNase J family, which have a beta-CASP metallo-beta-lactamase fold. Here we show that beta-CASP proteins encoded in the genomes of the hyperthermophilic Euryarchaeota Pyrococcus abyssi and Thermococcus kodakaraensis are processive exoribonucleases with a 5' end dependence and a 5' to 3' directionality. We named these enzymes Pab-RNase J and Tk-RNase J, respectively. RNAs with 5'-monophosphate or 5'-hydroxyl ends are preferred substrates of Pab-RNase J, whereas circularized RNA is resistant to Pab-RNase J activity. Degradation of a 3' end-labeled synthetic RNA in which an internal nucleoside is substituted by three ethylene glycol units generates intermediates demonstrating 5' to 3' directionality. The substitution of conserved residues in Pab-RNase J predicted to be involved in the coordination of metal ions demonstrates their importance for ribonuclease activity, although the detailed geometry of the catalytic site is likely to differ from bacterial RNase J. This is the first identification of a 5'-exoribonuclease encoded in the genomes of the Archaea. Phylogenetic analysis shows that euryarchaeal RNase J has been inherited vertically, suggesting an ancient origin predating the separation of the Bacteria and the Archaea.

The K-loop, a general feature of the Pyrococcus C/D guide RNAs, is an RNA structural motif related to the K-turn.

The C/D guide RNAs predicted from the genomic sequences of three species of Pyrococcus delineate a family of small non-coding archaeal RNAs involved in the methylation of rRNA and tRNA. The C/D guides assemble into ribonucleoprotein (RNP) that contains the methyltransferase. The protein L7Ae, a key structural component of the RNP, binds to a Kink-turn (K-turn) formed by the C/D motif. The K-turn is a structure that consists of two RNA stems separated by a short asymmetric loop with a characteristic sharp bend (kink) between the two stems. The majority of the pyrococcal C/D guides contain a short 3 nt-spacer between the C'/D' motifs. We show here that conserved terminal stem-loops formed by the C'/D' motif of the Pyrococcus C/D RNAs are also L7Ae-binding sites. These stem-loops are related to the K-turn by sequence and structure, but they consist of a single stem closed by a terminal loop. We have named this structure the K-loop. We show that conserved non-canonical base pairs in the stem of the K-loop are necessary for L7Ae binding. For the C/D guides with a 3 nt-spacer we show that the sequence and length is also important. The K-loop could improve the stability of the C/D guide RNAs in Pyrococcal species, which are extreme hyperthermophiles.

Two different mechanisms for tRNA ribose methylation in Archaea: a short survey.

The biogenesis of tRNA involves multiple reactions including post-transcriptional modifications and pre-tRNA splicing. Among the three domains of life, only Archaea have two different mechanisms for tRNA ribose methylation: site-specific 2'-O-methyltransferases and C/D guided-RNA machinery. Recently, the first archaeal tRNA 2'-O-methyltransferase, aTrm56, has been characterized. This enzyme is found in all archaeal genomes sequenced so far except one and belongs to the SPOUT family (class IV) of RNA methyltransferases. Its substrate is the conserved C56 in the T-loop of archaeal tRNAs. In the crenarchaeon Pyrobaculum aerophylum, in which no homologue of this methyltransferase is found, a box C/D guide sRNP insures the ribose methylation of C56. Moreover, a new twist on tRNA processing is the finding, in most euryarchaeal tRNAtrp genes, of a box C/D guide RNA within their intron specifying methylation at two sites. Modification of tRNA is an integral part of the complex maturation process of primary tRNA transcripts. In addition to their role in modification, both modification enzymes and C/D guide RNPs may have a chaperone function insuring the precise folding of the mature, functional tRNA.

In vitro RNP assembly and methylation guide activity of an unusual box C/D RNA, cis-acting archaeal pre-tRNA(Trp).

Among the large family of C/D methylation guide RNAs, the intron of euryarchaeal pre-tRNA(Trp) represents an outstanding specimen able to guide in cis, instead of in trans, two 2'-O-methylations in the pre-tRNA exons. Remarkably, both sites of methylation involve nucleotides within the bulge-helix-bulge (BHB) splicing motif, while the RNA-guided methylation and pre-tRNA splicing events depend on mutually exclusive RNA folding patterns. Using the three recombinant core proteins of archaeal C/D RNPs, we have analyzed in vitro RNP assembly of the pre-tRNA and tested its site-specific methylation activity. Recognition by L7Ae of hallmark K-turns at the C/D and C'/D' motifs appears as a crucial assembly step required for subsequent binding of a Nop5p-aFib heterodimer at each site. Unexpectedly, however, even without L7Ae but at a higher concentration of Nop5p-aFib, a substantially active RNP complex can still form, possibly reflecting the higher propensity of the cis-acting system to form guide RNA duplex(es) relative to classical trans- acting C/D RNA guides. Moreover, footprinting data of RNPs, consistent with Nop5p interacting with the non-canonical stem of the K-turn, suggest that binding of Nop5p-aFib to the pre-tRNA-L7Ae complex might direct transition from a splicing-competent structure to an RNA conformer displaying the guide RNA duplexes required for site-specific methylation.