RNase W, a conserved ribonuclease family with a novel active site.

Ribosome biogenesis is a complex process requiring multiple precursor ribosomal RNA (rRNA) cleavage steps. In archaea, the full set of ribonucleases (RNases) involved in rRNA processing remains to be discovered. A previous study suggested that FAU-1, a conserved protein containing an RNase G/E-like protein domain fused to a domain of unknown function (DUF402), acts as an RNase in archaea. However, the molecular basis of this activity remained so far elusive. Here, we report two X-ray crystallographic structures of RNase G/E-like-DUF402 hybrid proteins from Pyrococcus furiosus and Sulfolobus acidocaldarius, at 2.1 and 2.0 Å, respectively. The structures highlight a structural homology with the 5' RNA recognition domain of Escherichia coli RNase E but no homology with other known catalytic nuclease domains. Surprisingly, we demonstrate that the C-terminal domain of this hybrid protein, annotated as a putative diphosphatase domain, harbors the RNase activity. Our functional analysis also supports a model by which the RNase G/E-like domain acts as a regulatory subunit of the RNase activity. Finally, in vivo experiments in Haloferax volcanii suggest that this RNase participates in the maturation of pre-16S rRNA. Together, our study defines a new RNase family, which we termed the RNase W family, as the first archaea-specific contributor to archaeal ribosome biogenesis.

Transcriptional and translational dynamics underlying heat shock response in the thermophilic crenarchaeon Sulfolobus acidocaldarius.

IMPORTANCE: Heat shock response is the ability to respond adequately to sudden temperature increases that could be harmful for cellular survival and fitness. It is crucial for microorganisms living in volcanic hot springs that are characterized by high temperatures and large temperature fluctuations. In this study, we investigated how S. acidocaldarius, which grows optimally at 75°C, responds to heat shock by altering its gene expression and protein production processes. We shed light on which cellular processes are affected by heat shock and propose a hypothesis on underlying regulatory mechanisms. This work is not only relevant for the organism's lifestyle, but also with regard to its evolutionary status. Indeed, S. acidocaldarius belongs to the archaea, an ancient group of microbes that is more closely related to eukaryotes than to bacteria. Our study thus also contributes to a better understanding of the early evolution of heat shock response.

Nanopore-based RNA sequencing deciphers the formation, processing, and modification steps of rRNA intermediates in archaea.

Ribosomal RNA (rRNA) maturation in archaea is a complex multistep process that requires well-defined endo- and exoribonuclease activities to generate fully mature linear rRNAs. However, technical challenges prevented detailed mapping of rRNA processing steps and a systematic analysis of rRNA maturation pathways across the tree of life. In this study, we used long-read (PCR)-cDNA and direct RNA nanopore-based sequencing to study rRNA maturation in three archaeal model organisms, namely the Euryarchaea Haloferax volcanii and Pyrococcus furiosus and the Crenarchaeon Sulfolobus acidocaldarius Compared to standard short-read protocols, nanopore sequencing facilitates simultaneous readout of 5'- and 3'-positions, which is required for the classification of rRNA processing intermediates. More specifically, we (i) accurately detect and describe rRNA maturation stages by analysis of terminal read positions of cDNA reads and thereupon (ii) explore the stage-dependent installation of the KsgA-mediated dimethylations in H. volcanii using base-calling and signal characteristics of direct RNA reads. Due to the single-molecule sequencing capacity of nanopore sequencing, we could detect hitherto unknown intermediates with high confidence, revealing details about the maturation of archaea-specific circular rRNA intermediates. Taken together, our study delineates common principles and unique features of rRNA processing in euryarchaeal and crenarchaeal representatives, thereby significantly expanding our understanding of rRNA maturation pathways in archaea.

The dark side of the ribosome life cycle.

Thanks to genetics, biochemistry, and structural biology many features of the ribosome´s life cycles in models of bacteria, eukaryotes, and some organelles have been revealed to near-atomic details. Collectively, these studies have provided a very detailed understanding of what are now well-established prototypes for ribosome biogenesis and function as viewed from a 'classical' model organisms perspective. However, very important challenges remain ahead to explore the functional and structural diversity of both ribosome biogenesis and function across the biological diversity on earth. Particularly, the 'third domain of life', the archaea, and also many non-model bacterial and eukaryotic organisms have been comparatively neglected. Importantly, characterizing these additional biological systems will not only offer a yet untapped window to enlighten the evolution of ribosome biogenesis and function but will also help to unravel fundamental principles of molecular adaptation of these central cellular processes.

A Comparative Perspective on Ribosome Biogenesis: Unity and Diversity Across the Tree of Life.

Ribosomes are universally conserved ribonucleoprotein complexes involved in the decoding of the genetic information contained in messenger RNAs into proteins. Accordingly, ribosome biogenesis is a fundamental cellular process required for functional ribosome homeostasis and to preserve satisfactory gene expression capability.Although the ribosome is universally conserved, its biogenesis shows an intriguing degree of variability across the tree of life . These differences also raise yet unresolved questions. Among them are (a) what are, if existing, the remaining ancestral common principles of ribosome biogenesis ; (b) what are the molecular impacts of the evolution history and how did they contribute to (re)shape the ribosome biogenesis pathway across the tree of life ; (c) what is the extent of functional divergence and/or convergence (functional mimicry), and in the latter case (if existing) what is the molecular basis; (d) considering the universal ribosome conservation, what is the capability of functional plasticity and cellular adaptation of the ribosome biogenesis pathway?In this review, we provide a brief overview of ribosome biogenesis across the tree of life and try to illustrate some potential and/or emerging answers to these unresolved questions.

Non-radioactive In Vivo Labeling of RNA with 4-Thiouracil.

RNA molecules and their expression dynamics play essential roles in the establishment of complex cellular phenotypes and/or in the rapid cellular adaption to environmental changes. Accordingly, analyzing RNA expression remains an important step to understand the molecular basis controlling the formation of cellular phenotypes, cellular homeostasis or disease progression. Steady-state RNA levels in the cells are controlled by the sum of highly dynamic molecular processes contributing to RNA expression and can be classified in transcription, maturation and degradation. The main goal of analyzing RNA dynamics is to disentangle the individual contribution of these molecular processes to the life cycle of a given RNA under different physiological conditions. In the recent years, the use of nonradioactive nucleotide/nucleoside analogs and improved chemistry, in combination with time-dependent and high-throughput analysis, have greatly expanded our understanding of RNA metabolism across various cell types, organisms, and growth conditions.In this chapter, we describe a step-by-step protocol allowing pulse labeling of RNA with the nonradioactive nucleotide analog, 4-thiouracil , in the eukaryotic model organism Saccharomyces cerevisiae and the model archaeon Haloferax volcanii .

Differential Translation Activity Analysis Using Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) in Archaea.

The study of protein production and degradation in a quantitative and time-dependent manner is a major challenge to better understand cellular physiological response. Among available technologies bioorthogonal noncanonical amino acid tagging (BONCAT) is an efficient approach allowing for time-dependent labeling of proteins through the incorporation of chemically reactive noncanonical amino acids like L-azidohomoalanine (L-AHA). The azide-containing amino-acid derivative enables a highly efficient and specific reaction termed click chemistry, whereby the azide group of the L-AHA reacts with a reactive alkyne derivate, like dibenzocyclooctyne (DBCO) derivatives, using strain-promoted alkyne-azide cycloaddition (SPAAC). Moreover, available DBCO containing reagents are versatile and can be coupled to fluorophore (e.g., Cy7) or affinity tag (e.g., biotin) derivatives, for easy visualization and affinity purification, respectively.Here, we describe a step-by-step BONCAT protocol optimized for the model archaeon Haloferax volcanii , but which is also suitable to harness other biological systems. Finally, we also describe examples of downstream visualization, affinity purification of L-AHA-labeled proteins and differential expression analysis.In conclusion, the following BONCAT protocol expands the available toolkit to explore proteostasis using time-resolved semiquantitative proteomic analysis in archaea .

Looking through the Lens of the Ribosome Biogenesis Evolutionary History: Possible Implications for Archaeal Phylogeny and Eukaryogenesis.

Our understanding of microbial diversity and its evolutionary relationships has increased substantially over the last decade. Such an understanding has been greatly fueled by culture-independent metagenomics analyses. However, the outcome of some of these studies and their biological and evolutionary implications, such as the origin of the eukaryotic lineage from the recently discovered archaeal Asgard superphylum, is debated. The sequences of the ribosomal constituents are amongst the most used phylogenetic markers. However, the functional consequences underlying the analysed sequence diversity and their putative evolutionary implications are essentially not taken into consideration. Here, we propose to exploit additional functional hallmarks of ribosome biogenesis to help disentangle competing evolutionary hypotheses. Using selected examples, such as the multiple origins of halophily in archaea or the evolutionary relationship between the Asgard archaea and Eukaryotes, we illustrate and discuss how function-aware phylogenetic framework can contribute to refining our understanding of archaeal phylogeny and the origin of eukaryotic cells.

Nanopore sequencing of RNA and cDNA molecules in Escherichia coli.

High-throughput sequencing dramatically changed our view of transcriptome architectures and allowed for ground-breaking discoveries in RNA biology. Recently, sequencing of full-length transcripts based on the single-molecule sequencing platform from Oxford Nanopore Technologies (ONT) was introduced and is widely used to sequence eukaryotic and viral RNAs. However, experimental approaches implementing this technique for prokaryotic transcriptomes remain scarce. Here, we present an experimental and bioinformatic workflow for ONT RNA-seq in the bacterial model organism Escherichia coli, which can be applied to any microorganism. Our study highlights critical steps of library preparation and computational analysis and compares the results to gold standards in the field. Furthermore, we comprehensively evaluate the applicability and advantages of different ONT-based RNA sequencing protocols, including direct RNA, direct cDNA, and PCR-cDNA. We find that (PCR)-cDNA-seq offers improved yield and accuracy compared to direct RNA sequencing. Notably, (PCR)-cDNA-seq is suitable for quantitative measurements and can be readily used for simultaneous and accurate detection of transcript 5' and 3' boundaries, analysis of transcriptional units, and transcriptional heterogeneity. In summary, based on our comprehensive study, we show nanopore RNA-seq to be a ready-to-use tool allowing rapid, cost-effective, and accurate annotation of multiple transcriptomic features. Thereby nanopore RNA-seq holds the potential to become a valuable alternative method for RNA analysis in prokaryotes.

Ribosome Biogenesis in Archaea.

Making ribosomes is a major cellular process essential for the maintenance of functional ribosome homeostasis and to ensure appropriate gene expression. Strikingly, although ribosomes are universally conserved ribonucleoprotein complexes decoding the genetic information contained in messenger RNAs into proteins, their biogenesis shows an intriguing degree of variability across the tree of life. In this review, we summarize our knowledge on the least understood ribosome biogenesis pathway: the archaeal one. Furthermore, we highlight some evolutionary conserved and divergent molecular features of making ribosomes across the tree of life.

Insights into synthesis and function of KsgA/Dim1-dependent rRNA modifications in archaea.

Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribosome biogenesis is a complex process which has been intensively analyzed in bacteria and eukaryotes. In contrast, our understanding of the in vivo archaeal ribosome biogenesis pathway remains less characterized. Here, we have analyzed the in vivo role of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 homolog in archaea. Our study reveals that KsgA/Dim1-dependent 16S rRNA dimethylation is dispensable for the cellular growth of phylogenetically distant archaea. However, proteomics and functional analyses suggest that archaeal KsgA/Dim1 and its rRNA modification activity (i) influence the expression of a subset of proteins and (ii) contribute to archaeal cellular fitness and adaptation. In addition, our study reveals an unexpected KsgA/Dim1-dependent variability of rRNA modifications within the archaeal phylum. Combining structure-based functional studies across evolutionary divergent organisms, we provide evidence on how rRNA structure sequence variability (re-)shapes the KsgA/Dim1-dependent rRNA modification status. Finally, our results suggest an uncoupling between the KsgA/Dim1-dependent rRNA modification completion and its release from the nascent small ribosomal subunit. Collectively, our study provides additional understandings into principles of molecular functional adaptation, and further evolutionary and mechanistic insights into an almost universally conserved step of ribosome synthesis.

Splicing Endonuclease Is an Important Player in rRNA and tRNA Maturation in Archaea.

In all three domains of life, tRNA genes contain introns that must be removed to yield functional tRNA. In archaea and eukarya, the first step of this process is catalyzed by a splicing endonuclease. The consensus structure recognized by the splicing endonuclease is a bulge-helix-bulge (BHB) motif which is also found in rRNA precursors. So far, a systematic analysis to identify all biological substrates of the splicing endonuclease has not been carried out. In this study, we employed CRISPRi to repress expression of the splicing endonuclease in the archaeon Haloferax volcanii to identify all substrates of this enzyme. Expression of the splicing endonuclease was reduced to 1% of its normal level, resulting in a significant extension of lag phase in H. volcanii growth. In the repression strain, 41 genes were down-regulated and 102 were up-regulated. As an additional approach in identifying new substrates of the splicing endonuclease, we isolated and sequenced circular RNAs, which identified excised introns removed from tRNA and rRNA precursors as well as from the 5' UTR of the gene HVO_1309. In vitro processing assays showed that the BHB sites in the 5' UTR of HVO_1309 and in a 16S rRNA-like precursor are processed by the recombinant splicing endonuclease. The splicing endonuclease is therefore an important player in RNA maturation in archaea.

The Archaeal Proteome Project advances knowledge about archaeal cell biology through comprehensive proteomics.

While many aspects of archaeal cell biology remain relatively unexplored, systems biology approaches like mass spectrometry (MS) based proteomics offer an opportunity for rapid advances. Unfortunately, the enormous amount of MS data generated often remains incompletely analyzed due to a lack of sophisticated bioinformatic tools and field-specific biological expertise for data interpretation. Here we present the initiation of the Archaeal Proteome Project (ArcPP), a community-based effort to comprehensively analyze archaeal proteomes. Starting with the model archaeon Haloferax volcanii, we reanalyze MS datasets from various strains and culture conditions. Optimized peptide spectrum matching, with strict control of false discovery rates, facilitates identifying > 72% of the reference proteome, with a median protein sequence coverage of 51%. These analyses, together with expert knowledge in diverse aspects of cell biology, provide meaningful insights into processes such as N-terminal protein maturation, N-glycosylation, and metabolism. Altogether, ArcPP serves as an invaluable blueprint for comprehensive prokaryotic proteomics.

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.

A versatile cis-acting element reporter system to study the function, maturation and stability of ribosomal RNA mutants in archaea.

General molecular principles of ribosome biogenesis have been well explored in bacteria and eukaryotes. Collectively, these studies have revealed important functional differences and few similarities between these processes. Phylogenetic studies suggest that the information processing machineries from archaea and eukaryotes are evolutionary more closely related than their bacterial counterparts. These observations raise the question of how ribosome synthesis in archaea may proceed in vivo. In this study, we describe a versatile plasmid-based cis-acting reporter system allowing to analyze in vivo the consequences of ribosomal RNA mutations in the model archaeon Haloferax volcanii. Applying this system, we provide evidence that the bulge-helix-bulge motif enclosed within the ribosomal RNA processing stems is required for the formation of archaeal-specific circular-pre-rRNA intermediates and mature rRNAs. In addition, we have collected evidences suggesting functional coordination of the early steps of ribosome synthesis in H. volcanii. Together our investigation describes a versatile platform allowing to generate and functionally analyze the fate of diverse rRNA variants, thereby paving the way to better understand the cis-acting molecular determinants necessary for archaeal ribosome synthesis, maturation, stability and function.

Conformational proofreading of distant 40S ribosomal subunit maturation events by a long-range communication mechanism.

Eukaryotic ribosomes are synthesized in a hierarchical process driven by a plethora of assembly factors, but how maturation events at physically distant sites on pre-ribosomes are coordinated is poorly understood. Using functional analyses and cryo-EM, we show that ribosomal protein Rps20 orchestrates communication between two multi-step maturation events across the pre-40S subunit. Our study reveals that during pre-40S maturation, formation of essential contacts between Rps20 and Rps3 permits assembly factor Ltv1 to recruit the Hrr25 kinase, thereby promoting Ltv1 phosphorylation. In parallel, a deeply buried Rps20 loop reaches to the opposite pre-40S side, where it stimulates Rio2 ATPase activity. Both cascades converge to the final maturation steps releasing Rio2 and phosphorylated Ltv1. We propose that conformational proofreading exerted via Rps20 constitutes a checkpoint permitting assembly factor release and progression of pre-40S maturation only after completion of all earlier maturation steps.

Impact of two neighbouring ribosomal protein clusters on biogenesis factor binding and assembly of yeast late small ribosomal subunit precursors.

Many of the small ribosomal subunit proteins are required for the stabilisation of late small ribosomal subunit (SSU) precursors and for final SSU rRNA processing in S. cerevisiae. Among them are ribosomal proteins (r-proteins) which form a protein cluster around rpS0 (uS2) at the "neck" of the SSU (S0-cluster) and others forming a nearby protein cluster around rpS3 (uS3) at the SSU "beak". Here we applied semi-quantitative proteomics together with complementary biochemical approaches to study how incomplete assembly of these two r-protein clusters affects binding and release of SSU maturation factors and assembly of other r-proteins in late SSU precursors in S. cerevisiae. For each of the two clusters specific impairment of the local r-protein assembly state was observed in Rio2 associated SSU precursors. Besides, cluster-specific effects on the association of biogenesis factors were detected. These suggested a role of S0-cluster formation for the efficient release of the two nuclear export factors Rrp12 and Slx9 from SSU precursors and for the correct incorporation of the late acting biogenesis factor Rio2. Based on our and on previous results we propose the existence of at least two different r-protein assembly checkpoints during late SSU maturation in S. cerevisiae. We discuss in the light of recent SSU precursor structure models how r-protein assembly states might be sensed by biogenesis factors at the S0-cluster checkpoint.

The Rio1 protein kinases/ATPases: conserved regulators of growth, division, and genomic stability.

The atypical Rio1 protein kinases/ATPases, which exist in most archaea and eukaryotes, have been studied intensely to understand how they promote small ribosomal subunit (SSU) maturation. However, mutant and knockdown phenotypes in various organisms suggested roles in activities beyond SSU biogenesis, including the regulation of cell cycle progression (DNA transcription, replication, condensation, and segregation), cell division, metabolism, physiology, and development. Recent work with budding yeast, indeed, revealed that Rio1 (RIOK1 in metazoans) manages a large signaling network at the protein and gene levels via which it stimulates or restricts growth and division in response to nutrient availability. We examine how these findings translate to human cells and suggest that RIOK1 over-expression or mutations, as observed in primary cancer cells, may cause a mis-regulation of its network, contributing to cancer initiation and progression. We also reflect on how targeting RIOK1 might eradicate hitherto incurable tumors in the clinic.

Integrating Rio1 activities discloses its nutrient-activated network in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae kinase/adenosine triphosphatase Rio1 regulates rDNA transcription and segregation, pre-rRNA processing and small ribosomal subunit maturation. Other roles are unknown. When overexpressed, human ortholog RIOK1 drives tumor growth and metastasis. Likewise, RIOK1 promotes 40S ribosomal subunit biogenesis and has not been characterized globally. We show that Rio1 manages directly and via a series of regulators, an essential signaling network at the protein, chromatin and RNA levels. Rio1 orchestrates growth and division depending on resource availability, in parallel to the nutrient-activated Tor1 kinase. To define the Rio1 network, we identified its physical interactors, profiled its target genes/transcripts, mapped its chromatin-binding sites and integrated our data with yeast's protein-protein and protein-DNA interaction catalogs using network computation. We experimentally confirmed network components and localized Rio1 also to mitochondria and vacuoles. Via its network, Rio1 commands protein synthesis (ribosomal gene expression, assembly and activity) and turnover (26S proteasome expression), and impinges on metabolic, energy-production and cell-cycle programs. We find that Rio1 activity is conserved to humans and propose that pathological RIOK1 may fuel promiscuous transcription, ribosome production, chromosomal instability, unrestrained metabolism and proliferation; established contributors to cancer. Our study will advance the understanding of numerous processes, here revealed to depend on Rio1 activity.