GTPase Era at the heart of ribosome assembly.

Ribosome biogenesis is a key process in all organisms. It relies on coordinated work of multiple proteins and RNAs, including an array of assembly factors. Among them, the GTPase Era stands out as an especially deeply conserved protein, critically required for the assembly of bacterial-type ribosomes from Escherichia coli to humans. In this review, we bring together and critically analyze a wealth of phylogenetic, biochemical, structural, genetic and physiological data about this extensively studied but still insufficiently understood factor. We do so using a comparative and, wherever possible, synthetic approach, by confronting observations from diverse groups of bacteria and eukaryotic organelles (mitochondria and chloroplasts). The emerging consensus posits that Era intervenes relatively early in the small subunit biogenesis and is essential for the proper shaping of the platform which, in its turn, is a prerequisite for efficient translation. The timing of Era action on the ribosome is defined by its interactions with guanosine nucleotides [GTP, GDP, (p)ppGpp], ribosomal RNA, and likely other factors that trigger or delay its GTPase activity. As a critical nexus of the small subunit biogenesis, Era is subject to sophisticated regulatory mechanisms at the transcriptional, post-transcriptional, and post-translational levels. Failure of these mechanisms or a deficiency in Era function entail dramatic generalized consequences for the protein synthesis and far-reaching, pleiotropic effects on the organism physiology, such as the Perrault syndrome in humans.

Controlled Level of Contamination Coupled to Deep Sequencing (CoLoC-seq) Probes the Global Localisation Topology of Organelle Transcriptomes.

Information on RNA localisation is essential for understanding physiological and pathological processes, such as gene expression, cell reprogramming, host-pathogen interactions, and signalling pathways involving RNA transactions at the level of membrane-less or membrane-bounded organelles and extracellular vesicles. In many cases, it is important to assess the topology of RNA localisation, i.e., to distinguish the transcripts encapsulated within an organelle of interest from those merely attached to its surface. This allows establishing which RNAs can, in principle, engage in local molecular interactions and which are prevented from interacting by membranes or other physical barriers. The most widely used techniques interrogating RNA localisation topology are based on the treatment of isolated organelles with RNases with subsequent identification of the surviving transcripts by northern blotting, qRT-PCR, or RNA-seq. However, this approach produces incoherent results and many false positives. Here, we describe Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq), a more refined subcellular transcriptomics approach that overcomes these pitfalls. CoLoC-seq starts by the purification of organelles of interest. They are then either left intact or lysed and subjected to a gradient of RNase concentrations to produce unique RNA degradation dynamics profiles, which can be monitored by northern blotting or RNA-seq. Through straightforward mathematical modelling, CoLoC-seq distinguishes true membrane-enveloped transcripts from degradable and non-degradable contaminants of any abundance. The method has been implemented in the mitochondria of HEK293 cells, where it outperformed alternative subcellular transcriptomics approaches. It is applicable to other membrane-bounded organelles, e.g., plastids, single-membrane organelles of the vesicular system, extracellular vesicles, or viral particles. Key features • Tested on human mitochondria; potentially applicable to cell cultures, non-model organisms, extracellular vesicles, enveloped viruses, tissues; does not require genetic manipulations or highly pure organelles. • In the case of human cells, the required amount of starting material is ~2,500 cm2 of 80% confluent cells (or ~3 × 108 HEK293 cells). • CoLoC-seq implements a special RNA-seq strategy to selectively capture intact transcripts, which requires RNases generating 5'-hydroxyl and 2'/3'-phosphate termini (e.g., RNase A, RNase I). • Relies on nonlinear regression software with customisable exponential functions.

FinO/ProQ-family proteins: an evolutionary perspective.

RNA-binding proteins are key actors of post-transcriptional networks. Almost exclusively studied in the light of their interactions with RNA ligands and the associated functional events, they are still poorly understood as evolutionary units. In this review, we discuss the FinO/ProQ family of bacterial RNA chaperones, how they evolve and spread across bacterial populations and what properties and opportunities they provide to their host cells. We reflect on major conserved and divergent themes within the family, trying to understand how the same ancestral RNA-binding fold, augmented with additional structural elements, could yield either highly specialised proteins or, on the contrary, globally acting regulatory hubs with a pervasive impact on gene expression. We also consider dominant convergent evolutionary trends that shaped their RNA chaperone activity and recurrently implicated the FinO/ProQ-like proteins in bacterial DNA metabolism, translation and virulence. Finally, we offer a new perspective in which FinO/ProQ-family regulators emerge as active evolutionary players with both negative and positive roles, significantly impacting the evolutionary modes and trajectories of their bacterial hosts.

CoLoC-seq probes the global topology of organelle transcriptomes.

Proper RNA localisation is essential for physiological gene expression. Various kinds of genome-wide approaches permit to comprehensively profile subcellular transcriptomes. Among them, cell fractionation methods, that couple RNase treatment of isolated organelles to the sequencing of protected transcripts, remain most widely used, mainly because they do not require genetic modification of the studied system and can be easily implemented in any cells or tissues, including in non-model species. However, they suffer from numerous false-positives since incompletely digested contaminant RNAs can still be captured and erroneously identified as resident transcripts. Here we introduce Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq) as a new subcellular transcriptomics approach that efficiently bypasses this caveat. CoLoC-seq leverages classical enzymatic kinetics and tracks the depletion dynamics of transcripts in a gradient of an exogenously added RNase, with or without organellar membranes. By means of straightforward mathematical modelling, CoLoC-seq infers the localisation topology of RNAs and robustly distinguishes between genuinely resident, luminal transcripts and merely abundant surface-attached contaminants. Our generic approach performed well on human mitochondria and is in principle applicable to other membrane-bounded organelles, including plastids, compartments of the vacuolar system, extracellular vesicles, and viral particles.

How global RNA-binding proteins coordinate the behaviour of RNA regulons: An information approach.

RNA-binding proteins are central players in post-transcriptional regulation. Some of them, such as the well-studied bacterial RNA chaperones Hfq and ProQ or the eukaryotic RNAi factor Argonaute, interact with hundreds-to-thousands of different RNAs and thereby globally affect gene expression. As a shared yet limited resource, these and other RNA-binding hubs drive strong competition between their multiple ligands. This creates a ground for significant cross-communication between RNA targets, which enables them to share information, "synchronise" their behaviour, and produce interesting biochemical effects, sometimes propagating across the highly connected RNA-protein network. This property is likely universally present in hub-centred networks and plays a key role in global gene expression programmes. It is also an important factor in biotechnology and synthetic biology applications of RNA/protein-based circuits. However, few studies so-far focused on describing and explaining this phenomenon from first principles. Here we introduce an information theory-based framework to comprehensively and exactly describe information flow in hub-centred networks. We show that information sharing can achieve significant levels in relatively small networks, provided the hub is present in limiting concentrations. The transmitted information is sufficient to noticeably affect the binding probabilities of competing targets but drops exponentially along the network. Target overexpression can disrupt communication between other targets, while hub sequestration boosts the crosstalk. We also find that overlaps between the interactomes of two different hubs create both entropic challenges and new forms of long-range communication between RNAs and proteins.

Research Progress in RNA-Binding Proteins.

RNA-binding proteins are everywhere and accompany RNA molecules at every stage of their molecular life, from "birth" (transcription) through "growing up" (maturation), "active life" (molecular function) until "death" (turnover) [...].

Suborganellar Localization of Mitochondrial Proteins and Transcripts in Human Cells.

Mitochondria have complex ultrastructure which includes continuous subcompartments, such as matrix, intermembrane space, and two membranes, as well as focal structures, such as nucleoids, RNA granules, and mitoribosomes. Comprehensive studies of the spatial distribution of proteins and RNAs inside the mitochondria are necessary to understand organellar gene expression processes and macromolecule targeting pathways. Here we give examples of distribution analysis of mitochondrial proteins and transcripts by conventional microscopy and the super-resolution technique 3D STORM. We provide detailed protocols and discuss limitations of immunolabeling of mitochondrial proteins and newly synthesized mitochondrial RNAs by bromouridine incorporation and single-molecule RNA FISH in hepatocarcinoma cells.

YbeY, éminence grise of ribosome biogenesis.

YbeY is an ultraconserved small protein belonging to the unique heritage shared by most existing bacteria and eukaryotic organelles of bacterial origin, mitochondria and chloroplasts. Studied in more than a dozen of evolutionarily distant species, YbeY is invariably critical for cellular physiology. However, the exact mechanisms by which it exerts such penetrating influence are not completely understood. In this review, we attempt a transversal analysis of the current knowledge about YbeY, based on genetic, structural, and biochemical data from a wide variety of models. We propose that YbeY, in association with the ribosomal protein uS11 and the assembly GTPase Era, plays a critical role in the biogenesis of the small ribosomal subunit, and more specifically its platform region, in diverse genetic systems of bacterial type.

The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches.

Macromolecular complexes of proteins and RNAs are essential building blocks of cells. These stable supramolecular particles can be viewed as minimal biochemical units whose structural organization, i.e., the way the RNA and the protein interact with each other, is directly linked to their biological function. Whether those are dynamic regulatory ribonucleoproteins (RNPs) or integrated molecular machines involved in gene expression, the comprehensive knowledge of these units is critical to our understanding of key molecular mechanisms and cell physiology phenomena. Such is the goal of diverse complexomic approaches and in particular of the recently developed gradient profiling by sequencing (Grad-seq). By separating cellular protein and RNA complexes on a density gradient and quantifying their distributions genome-wide by mass spectrometry and deep sequencing, Grad-seq charts global landscapes of native macromolecular assemblies. In this review, we propose a function-based ontology of stable RNPs and discuss how Grad-seq and related approaches transformed our perspective of bacterial and eukaryotic ribonucleoproteins by guiding the discovery of new RNA-binding proteins and unusual classes of noncoding RNAs. We highlight some methodological aspects and developments that permit to further boost the power of this technique and to look for exciting new biology in understudied and challenging biological models.

YBEY is an essential biogenesis factor for mitochondrial ribosomes.

Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3'-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter helps to properly incorporate uS11m into the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.

Import of Non-Coding RNAs into Human Mitochondria: A Critical Review and Emerging Approaches.

Mitochondria harbor their own genetic system, yet critically depend on the import of a number of nuclear-encoded macromolecules to ensure their expression. In all eukaryotes, selected non-coding RNAs produced from the nuclear genome are partially redirected into the mitochondria, where they participate in gene expression. Therefore, the mitochondrial RNome represents an intricate mixture of the intrinsic transcriptome and the extrinsic RNA importome. In this review, we summarize and critically analyze data on the nuclear-encoded transcripts detected in human mitochondria and outline the proposed molecular mechanisms of their mitochondrial import. Special attention is given to the various experimental approaches used to study the mitochondrial RNome, including some recently developed genome-wide and in situ techniques.

Discovery of new RNA classes and global RNA-binding proteins.

The identification of new RNA functions and the functional annotation of transcripts in genomes represent exciting yet challenging endeavours of modern biology. Crucial insights into the biological roles of RNA molecules can be gained from the identification of the proteins with which they form specific complexes. Modern interactome techniques permit to profile RNA-protein interactions in a genome-wide manner and identify new RNA classes associated with globally acting RNA-binding proteins. Applied to a variety of organisms, these methods are already revolutionising our understanding of RNA-mediated biological processes. Here, we focus on one such approach-Gradient sequencing or Grad-seq-which has recently guided the discovery of protein ProQ and its associated small RNAs as a new domain of post-transcriptional control in bacteria.

Molecular mechanism of mRNA repression in trans by a ProQ-dependent small RNA.

Research into post-transcriptional control of mRNAs by small noncoding RNAs (sRNAs) in the model bacteria Escherichia coli and Salmonella enterica has mainly focused on sRNAs that associate with the RNA chaperone Hfq. However, the recent discovery of the protein ProQ as a common binding partner that stabilizes a distinct large class of structured sRNAs suggests that additional RNA regulons exist in these organisms. The cellular functions and molecular mechanisms of these new ProQ-dependent sRNAs are largely unknown. Here, we report in Salmonella Typhimurium the mode-of-action of RaiZ, a ProQ-dependent sRNA that is made from the 3' end of the mRNA encoding ribosome-inactivating protein RaiA. We show that RaiZ is a base-pairing sRNA that represses in trans the mRNA of histone-like protein HU-α. RaiZ forms an RNA duplex with the ribosome-binding site of hupA mRNA, facilitated by ProQ, to prevent 30S ribosome loading and protein synthesis of HU-α. Similarities and differences between ProQ- and Hfq-mediated regulation will be discussed.

Grad-seq guides the discovery of ProQ as a major small RNA-binding protein.

The functional annotation of transcriptomes and identification of noncoding RNA (ncRNA) classes has been greatly facilitated by the advent of next-generation RNA sequencing which, by reading the nucleotide order of transcripts, theoretically allows the rapid profiling of all transcripts in a cell. However, primary sequence per se is a poor predictor of function, as ncRNAs dramatically vary in length and structure and often lack identifiable motifs. Therefore, to visualize an informative RNA landscape of organisms with potentially new RNA biology that are emerging from microbiome and environmental studies requires the use of more functionally relevant criteria. One such criterion is the association of RNAs with functionally important cognate RNA-binding proteins. Here we analyze the full ensemble of cellular RNAs using gradient profiling by sequencing (Grad-seq) in the bacterial pathogen Salmonella enterica, partitioning its coding and noncoding transcripts based on their network of RNA-protein interactions. In addition to capturing established RNA classes based on their biochemical profiles, the Grad-seq approach enabled the discovery of an overlooked large collective of structured small RNAs that form stable complexes with the conserved protein ProQ. We show that ProQ is an abundant RNA-binding protein with a wide range of ligands and a global influence on Salmonella gene expression. Given its generic ability to chart a functional RNA landscape irrespective of transcript length and sequence diversity, Grad-seq promises to define functional RNA classes and major RNA-binding proteins in both model species and genetically intractable organisms.

Induced tRNA import into human mitochondria: implication of a host aminoacyl-tRNA-synthetase.

In human cell, a subset of small non-coding RNAs is imported into mitochondria from the cytosol. Analysis of the tRNA import pathway allowing targeting of the yeast tRNA(Lys)(CUU) into human mitochondria demonstrates a similarity between the RNA import mechanisms in yeast and human cells. We show that the cytosolic precursor of human mitochondrial lysyl-tRNA synthetase (preKARS2) interacts with the yeast tRNA(Lys)(CUU) and small artificial RNAs which contain the structural elements determining the tRNA mitochondrial import, and facilitates their internalization by isolated human mitochondria. The tRNA import efficiency increased upon addition of the glycolytic enzyme enolase, previously found to be an actor of the yeast RNA import machinery. Finally, the role of preKARS2 in the RNA mitochondrial import has been directly demonstrated in vivo, in cultured human cells transfected with the yeast tRNA and artificial importable RNA molecules, in combination with preKARS2 overexpression or downregulation by RNA interference. These findings suggest that the requirement of protein factors for the RNA mitochondrial targeting might be a conserved feature of the RNA import pathway in different organisms.

Mitochondrial targeting of recombinant RNAs modulates the level of a heteroplasmic mutation in human mitochondrial DNA associated with Kearns Sayre Syndrome.

Mitochondrial mutations, an important cause of incurable human neuromuscular diseases, are mostly heteroplasmic: mutated mitochondrial DNA is present in cells simultaneously with wild-type genomes, the pathogenic threshold being generally >70% of mutant mtDNA. We studied whether heteroplasmy level could be decreased by specifically designed oligoribonucleotides, targeted into mitochondria by the pathway delivering RNA molecules in vivo. Using mitochondrially imported RNAs as vectors, we demonstrated that oligoribonucleotides complementary to mutant mtDNA region can specifically reduce the proportion of mtDNA bearing a large deletion associated with the Kearns Sayre Syndrome in cultured transmitochondrial cybrid cells. These findings may be relevant to developing of a new tool for therapy of mtDNA associated diseases.

Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18.

5S rRNA is an essential component of ribosomes of all living organisms, the only known exceptions being mitochondrial ribosomes of fungi, animals, and some protists. An intriguing situation distinguishes mammalian cells: Although the mitochondrial genome contains no 5S rRNA genes, abundant import of the nuclear DNA-encoded 5S rRNA into mitochondria was reported. Neither the detailed mechanism of this pathway nor its rationale was clarified to date. In this study, we describe an elegant molecular conveyor composed of a previously identified human 5S rRNA import factor, rhodanese, and mitochondrial ribosomal protein L18, thanks to which 5S rRNA molecules can be specifically withdrawn from the cytosolic pool and redirected to mitochondria, bypassing the classic nucleolar reimport pathway. Inside mitochondria, the cytosolic 5S rRNA is shown to be associated with mitochondrial ribosomes.

Mitochondrial enzyme rhodanese is essential for 5 S ribosomal RNA import into human mitochondria.

5 S rRNA is an essential component of ribosomes. In eukaryotic cells, it is distinguished by particularly complex intracellular traffic, including nuclear export and re-import. The finding that in mammalian cells 5 S rRNA can eventually escape its usual circuit toward nascent ribosomes to get imported into mitochondria has made the scheme more complex, and it has raised questions about both the mechanism of 5 S rRNA mitochondrial targeting and its function inside the organelle. Previously, we showed that import of 5 S rRNA into mitochondria requires unknown cytosolic proteins. Here, one of them was identified as mitochondrial thiosulfate sulfurtransferase, rhodanese. Rhodanese in its misfolded form was found to possess a strong and specific 5 S rRNA binding activity, exploiting sites found earlier to function as signals of 5 S rRNA mitochondrial localization. The interaction with 5 S rRNA occurs cotranslationally and results in formation of a stable complex in which rhodanese is preserved in a compact enzymatically inactive conformation. Human 5 S rRNA in a branched Mg(2+)-free form, upon its interaction with misfolded rhodanese, demonstrates characteristic functional traits of Hsp40 cochaperones implicated in mitochondrial precursor protein targeting, suggesting that it may use this mechanism to ensure its own mitochondrial localization. Finally, silencing of the rhodanese gene caused not only a proportional decrease of 5 S rRNA import but also a general inhibition of mitochondrial translation, indicating the functional importance of the imported 5 S rRNA inside the organelle.

Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria.

RNA import into mitochondria is a widespread phenomenon. Studied in details for yeast, protists, and plants, it still awaits thorough investigation for human cells, in which the nuclear DNA-encoded 5S rRNA is imported. Only the general requirements for this pathway have been described, whereas specific protein factors needed for 5S rRNA delivery into mitochondria and its structural determinants of import remain unknown. In this study, a systematic analysis of the possible role of human 5S rRNA structural elements in import was performed. Our experiments in vitro and in vivo show that two distinct regions of the human 5S rRNA molecule are needed for its mitochondrial targeting. One of them is located in the proximal part of the helix I and contains a conserved uncompensated G:U pair. The second and most important one is associated with the loop E-helix IV region with several noncanonical structural features. Destruction or even destabilization of these sites leads to a significant decrease of the 5S rRNA import efficiency. On the contrary, the beta-domain of the 5S rRNA was proven to be dispensable for import, and thus it can be deleted or substituted without affecting the 5S rRNA importability. This finding was used to demonstrate that the 5S rRNA can function as a vector for delivering heterologous RNA sequences into human mitochondria. 5S rRNA-based vectors containing a substitution of a part of the beta-domain by a foreign RNA sequence were shown to be much more efficiently imported in vivo than the wild-type 5S rRNA.