Alexandre Smirnov

All living beings use ribosomes to synthesize proteins from α-amino acids. Inherited from our common ancestor, a few universally conserved ribosome biogenesis steps, introducing essential ribosomal RNA modifications, have survived to our days. However, it was unknown whether similar primordial core mechanisms could also target ribosomal proteins. Here we show that the assembly and the function of ribosomes depend on the installation of an unusual β-amino acid, isoaspartate, within the ribosomal protein uS11. In bacteria, mitochondria, and plastids, this modification is catalyzed by the metzincin-like hydrolase YbeY, whereas in archaea and eukaryotes, it is introduced by the atypical kinase Fap7. The formation of this strategically positioned isoaspartate enables correct maturation of the small ribosomal subunit to support protein synthesis and normal cell physiology.

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.

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.

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 nondegradable 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.

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)

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.

• An information theory framework reveals salient features of RNA-protein networks. • Targets interacting with a common RNA-binding hub share information. • Competitive communication between targets drives noticeable biochemical effects. • Changes in target and hub abundances differentially affect crosstalk between them. • Information partially spreads to distal nodes in multi-hub networks.

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 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.