Ribosomal RNA expansion segments and their role in ribosome biology.

Ribosomes are universally conserved cellular machines that catalyze protein biosynthesis. The active sites underly immense evolutionary conservation resulting in virtually identical core structures of ribosomes in all domains of life including organellar ribosomes. However, more peripheral structures of cytosolic ribosomes changed during evolution accommodating new functions and regulatory options. The expansion occurred at the riboprotein level, including more and larger ribosomal proteins and at the RNA level increasing the length of ribosomal RNA. Expansions within the ribosomal RNA occur as clusters at conserved sites that face toward the periphery of the cytosolic ribosome. Recent biochemical and structural work has shed light on how rRNA-specific expansion segments (ESs) recruit factors during translation and how they modulate translation dynamics in the cytosol. Here we focus on recent work on yeast, human and trypanosomal cytosolic ribosomes that explores the role of two specific rRNA ESs within the small and large subunit respectively. While no single regulatory strategy exists, the absence of ESs has consequences for proteomic stability and cellular fitness, rendering them fascinating evolutionary tools for tailored protein biosynthesis.

Evolving precision: rRNA expansion segment 7S modulates translation velocity and accuracy in eukaryal ribosomes.

Ribosome-enhanced translational miscoding of the genetic code causes protein dysfunction and loss of cellular fitness. During evolution, open reading frame length increased, necessitating mechanisms for enhanced translation fidelity. Indeed, eukaryal ribosomes are more accurate than bacterial counterparts, despite their virtually identical, conserved active centers. During the evolution of eukaryotic organisms ribosome expansions at the rRNA and protein level occurred, which potentially increases the options for translation regulation and cotranslational events. Here we tested the hypothesis that ribosomal RNA expansions can modulate the core function of the ribosome, faithful protein synthesis. We demonstrate that a short expansion segment present in all eukaryotes' small subunit, ES7S, is crucial for accurate protein synthesis as its presence adjusts codon-specific velocities and guarantees high levels of cognate tRNA selection. Deletion of ES7S in yeast enhances mistranslation and causes protein destabilization and aggregation, dramatically reducing cellular fitness. Removal of ES7S did not alter ribosome architecture but altered the structural dynamics of inter-subunit bridges thus affecting A-tRNA selection. Exchanging the yeast ES7S sequence with the human ES7S increases accuracy whereas shortening causes the opposite effect. Our study demonstrates that ES7S provided eukaryal ribosomes with higher accuracy without perturbing the structurally conserved decoding center.

A ribosome-bound tRNA half stimulates mitochondrial translation during stress recovery in Trypanosoma brucei.

The protozoan parasite Trypanosoma brucei and its disease-causing relatives are among the few organisms that barely regulate the transcription of protein-coding genes. Yet, alterations in its gene expression are essential to survive in different host environments. Recently, tRNA-derived RNAs have been implicated as regulators of many cellular processes within and beyond translation. Previously, we identified the tRNAThr-3'-half (AGU) as a ribosome-associated non-coding RNA able to enhance global translation. Here we report that the tRNAThr-3'-half is generated upon starvation inside the mitochondria. The tRNAThr-3'-half associates with mitochondrial ribosomes and stimulates translation during stress recovery, positively affecting mitochondrial activity and, consequently, cellular energy production capacity. Our results describe an organelle ribosome-associated ncRNA involved in translation regulation to boost the central hub of energy metabolism as an immediate stress recovery response.

The stationary phase-specific sRNA FimR2 is a multifunctional regulator of bacterial motility, biofilm formation and virulence.

Bacterial pathogens employ a plethora of virulence factors for host invasion, and their use is tightly regulated to maximize infection efficiency and manage resources in a nutrient-limited environment. Here we show that during Escherichia coli stationary phase the 3' UTR-derived small non-coding RNA FimR2 regulates fimbrial and flagellar biosynthesis at the post-transcriptional level, leading to biofilm formation as the dominant mode of survival under conditions of nutrient depletion. FimR2 interacts with the translational regulator CsrA, antagonizing its functions and firmly tightening control over motility and biofilm formation. Generated through RNase E cleavage, FimR2 regulates stationary phase biology by fine-tuning target mRNA levels independently of the chaperones Hfq and ProQ. The Salmonella enterica orthologue of FimR2 induces effector protein secretion by the type III secretion system and stimulates infection, thus linking the sRNA to virulence. This work reveals the importance of bacterial sRNAs in modulating various aspects of bacterial physiology including stationary phase and virulence.

Small but Powerful: The Human Vault RNAs as Multifaceted Modulators of Pro-Survival Characteristics and Tumorigenesis.

The importance of non-coding RNAs for regulating gene expression has been uncovered in model systems spanning all three domains of life. More recently, their involvement in modulating signal transduction, cell proliferation, tumorigenesis and cancer progression has also made them promising tools and targets for oncotherapy. Recent studies revealed a class of highly conserved small ncRNAs, namely vault RNAs, as regulators of several cellular homeostasis mechanisms. The human genome encodes four vault RNA paralogs that share significant sequence and structural similarities, yet they seem to possess distinct roles in mammalian cells. The alteration of vault RNA expression levels has frequently been observed in cancer tissues, thus hinting at a putative role in orchestrating pro-survival characteristics. Over the last decade, significant advances have been achieved in clarifying the relationship between vault RNA and cellular mechanisms involved in cancer development. It became increasingly clear that vault RNAs are involved in controlling apoptosis, lysosome biogenesis and function, as well as autophagy in several malignant cell lines, most likely by modulating signaling pathways (e.g., the pro-survival MAPK cascade). In this review, we discuss the identified and known functions of the human vault RNAs in the context of cell proliferation, tumorigenesis and chemotherapy resistance.

Ribosome-Associated ncRNAs (rancRNAs) Adjust Translation and Shape Proteomes.

The regulation of protein synthesis is of extreme importance for cell survival in challenging environmental conditions. Modulating gene expression at the level of translation allows a swift and low-energy-cost response to external stimuli. In the last decade, an emerging class of regulatory ncRNAs, namely ribosome-associated non-coding RNAs (rancRNAs), has been discovered. These rancRNAs have proven to be efficient players in the regulation of translation as a first wave of stress adaptation by directly targeting the ribosome, the central enzyme of protein production. This underlying principle appears to be highly conserved, since rancRNAs are present in all three domains of life. Here, we review the major findings and mechanistic peculiarities of rancRNAs, a class of transcripts that is providing new and broader perspectives on the complexity of the ribosome and translation regulation.

Mammalian In Vitro Translation Systems.

Under cellular stress, tight and coordinated regulation of the gene expression allows to minimize cellular damage, maintains cellular homeostasis, and ensures cell survival. Among stress-induced cellular responses, alteration of translation rates represents one of the most effective and rapid regulatory mechanisms available for cells. Here we report on detailed protocols of mammalian in vitro translation systems. While most of the available in vitro translation methods are based on bacterial or yeast components, tailor-made and robust mammalian systems are sparse. Our protocols allow measuring global translation of the total mRNA pool as well as translation of one specific reporter mRNA. Furthermore, it provides access to measuring translational activity of isolated ribosomes combined with non-ribosomal cytosolic fractions using reduced amounts of biological starting material. The herein described method can be applied to (1) investigate the effects of stress-dependent soluble factors regulating translation (such as tRNA fragments or ribosome-associated ncRNAs), (2) compare translational activity and translational fidelity of different ribosomes supplemented with the same non-ribosomal fractions, and (3) to investigate protein biosynthesis in various mammalian cell lines as well as tissue samples.

The human vault RNA enhances tumorigenesis and chemoresistance through the lysosome in hepatocellular carcinoma.

The small non-coding VTRNA1-1 (vault RNA 1-1) is known to confer resistance to apoptosis in several malignant cell lines and to also modulate the macroautophagic/autophagic flux in hepatocytes, thus highlighting its pro-survival role. Here we describe a new function of VTRNA1-1 in regulating in vitro and in vivo tumor cell proliferation, tumorigenesis and chemoresistance. Knockout (KO) of VTRNA1-1 in human hepatocellular carcinoma cells reduced nuclear localization of TFEB (transcription factor EB), leading to a downregulation of the coordinated lysosomal expression and regulation (CLEAR) network genes and lysosomal compartment dysfunction. We demonstrate further that impaired lysosome function due to loss of VTRNA1-1 potentiates the anticancer effect of conventional chemotherapeutic drugs. Finally, loss of VTRNA1-1 reduced drug lysosomotropism allowing higher intracellular compound availability and thereby significantly reducing tumor cell proliferation in vitro and in vivo. These findings reveal a so far unknown role of VTRNA1-1 in the intracellular catabolic compartment and describe its contribution to lysosome-mediated chemotherapy resistance.Abbreviations: ATP6V0D2: ATPase H+ transporting V0 subunit d2; BafA: bafilomycin A1; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; DMSO: dimethyl sulfoxide; GST-BHMT: glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase; HCC: hepatocellular carcinoma; LAMP1: lysosomal associated membrane protein 1; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAPK: mitogen-activated protein kinase; MITF: melanocyte inducing transcription factor; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ncRNA: non-coding RNA; RNP: ribonucleoprotein; SF: sorafenib; SQSTM1/p62: sequestosome 1; STS: staurosporine; tdRs: tRNA-derived RNAs; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; vtRNA: vault RNA transcript.

Dynamic 23S rRNA modification ho5C2501 benefits Escherichia coli under oxidative stress.

Post-transcriptional modifications are added to ribosomal RNAs (rRNAs) to govern ribosome biogenesis and to fine-tune protein biosynthesis. In Escherichia coli and related bacteria, RlhA uniquely catalyzes formation of a 5-hydroxycytidine (ho5C) at position 2501 of 23S rRNA. However, the molecular and biological functions as well as the regulation of ho5C2501 modification remain unclear. We measured growth curves with the modification-deficient ΔrlhA strain and quantified the extent of the modification during different conditions by mass spectrometry and reverse transcription. The levels of ho5C2501 in E. coli ribosomes turned out to be highly dynamic and growth phase-dependent, with the most effective hydroxylation yields observed in the stationary phase. We demonstrated a direct effect of ho5C2501 on translation efficiencies in vitro and in vivo. High ho5C2501 levels reduced protein biosynthesis which however turned out to be beneficial for E. coli for adapting to oxidative stress. This functional advantage was small under optimal conditions or during heat or cold shock, but becomes pronounced in the presence of hydrogen peroxide. Taken together, these data provided first functional insights into the role of this unique 23S rRNA modification for ribosome functions and bacterial growth under oxidative stress.

tRNA Synthetases Are Recruited to Yeast Ribosomes by rRNA Expansion Segment 7L but Do Not Require Association for Functionality.

Protein biosynthesis is essential for any organism, yet how this process is regulated is not fully understood at the molecular level. During evolution, ribosomal RNA expanded in specific regions, referred to as rRNA expansion segments (ES). First functional roles of these expansions have only recently been discovered. Here we address the role of ES7La located in the large ribosomal subunit for factor recruitment to the yeast ribosome and the potential consequences for translation. Truncation of ES7La has only minor effects on ribosome biogenesis, translation efficiency and cell doubling. Using yeast rRNA deletion strains coupled with ribosome-specific mass spectrometry we analyzed the interactome of ribosomes lacking ES7La. Three aminoacyl-tRNA synthetases showed reduced ribosome association. Synthetase activities however remained unaltered suggesting that the pool of aminoacylated tRNAs is unaffected by the ES deletion. These results demonstrated that aminoacylation activities of tRNA synthetases per se do not rely on ribosome association. These findings suggest a role of ribosome-associated aminoacyl-tRNA synthetase beyond their core enzymatic functions.

A small ribosome-associated ncRNA globally inhibits translation by restricting ribosome dynamics.

Ribosome-associated non-coding RNAs (rancRNAs) have been recognized as an emerging class of regulatory molecules capable of fine-tuning translation in all domains of life. RancRNAs are ideally suited for allowing a swift response to changing environments and are therefore considered pivotal during the first wave of stress adaptation. Previously, we identified an mRNA-derived 18 nucleotides long rancRNA (rancRNA_18) in Saccharomyces cerevisiae that rapidly downregulates protein synthesis during hyperosmotic stress. However, the molecular mechanism of action remained enigmatic. Here, we combine biochemical, genetic, transcriptome-wide and structural evidence, thus revealing rancRNA_18 as global translation inhibitor by targeting the E-site region of the large ribosomal subunit. Ribosomes carrying rancRNA_18 possess decreased affinity for A-site tRNA and impaired structural dynamics. Cumulatively, these discoveries reveal the mode of action of a rancRNA involved in modulating protein biosynthesis at a thus far unequalled precision.

Oxidative Stress in Bacteria and the Central Dogma of Molecular Biology.

Ever since the "great oxidation event," Earth's cellular life forms had to cope with the danger of reactive oxygen species (ROS) affecting the integrity of biomolecules and hampering cellular metabolism circuits. Consequently, increasing ROS levels in the biosphere represented growing stress levels and thus shaped the evolution of species. Whether the ROS were produced endogenously or exogenously, different systems evolved to remove the ROS and repair the damage they inflicted. If ROS outweigh the cell's capacity to remove the threat, we speak of oxidative stress. The injuries through oxidative stress in cells are diverse. This article reviews the damage oxidative stress imposes on the different steps of the central dogma of molecular biology in bacteria, focusing in particular on the RNA machines involved in transcription and translation.

Context-specific action of macrolide antibiotics on the eukaryotic ribosome.

Macrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.

Transcriptome-Wide Analysis of Stationary Phase Small ncRNAs in E. coli.

Almost two-thirds of the microbiome's biomass has been predicted to be in a non-proliferating, and thus dormant, growth state. It is assumed that dormancy goes hand in hand with global downregulation of gene expression. However, it remains largely unknown how bacteria manage to establish this resting phenotype at the molecular level. Recently small non-protein-coding RNAs (sRNAs or ncRNAs) have been suggested to be involved in establishing the non-proliferating state in bacteria. Here, we have deep sequenced the small transcriptome of Escherichia coli in the exponential and stationary phases and analyzed the resulting reads by a novel biocomputational pipeline STARPA (Stable RNA Processing Product Analyzer). Our analysis reveals over 12,000 small transcripts enriched during both growth stages. Differential expression analysis reveals distinct sRNAs enriched in the stationary phase that originate from various genomic regions, including transfer RNA (tRNA) fragments. Furthermore, expression profiling by Northern blot and RT-qPCR analyses confirms the growth phase-dependent expression of several enriched sRNAs. Our study adds to the existing repertoire of bacterial sRNAs and suggests a role for some of these small molecules in establishing and maintaining stationary phase as well as the bacterial stress response. Functional characterization of these detected sRNAs has the potential of unraveling novel regulatory networks central for stationary phase biology.

RNA antitoxin SprF1 binds ribosomes to attenuate translation and promote persister cell formation in Staphylococcus aureus.

Persister cells are a subpopulation of transiently antibiotic-tolerant bacteria associated with chronic infection and antibiotic treatment failure. Toxin-antitoxin systems have been linked to persister cell formation but the molecular mechanisms leading to bacterial persistence are mostly unknown. Here, we show that SprF1, a type I antitoxin, associates with translating ribosomes from the major human pathogen Staphylococcus aureus to reduce the pathogen's overall protein synthesis during growth. Under hyperosmotic stress, SprF1 levels increase due to enhanced stability, accumulate on polysomes and attenuate protein synthesis. Using an internal 6-nucleotide sequence on its 5'-end, SprF1 binds ribosomes and interferes with initiator transfer RNA binding, thus reducing translation initiation. An excess of messenger RNA displaces the ribosome-bound antitoxin, freeing the ribosomes for new translation cycles; however, this RNA antitoxin can also displace ribosome-bound mRNA. This translation attenuation mechanism, mediated by an RNA antitoxin, promotes antibiotic persister cell formation. The untranslated SprF1 is a dual-function RNA antitoxin that represses toxin expression by its 3'-end and fine-tunes overall bacterial translation via its 5'-end. These findings demonstrate a general function for a bacterial RNA antitoxin beyond protection from toxicity. They also highlight an RNA-guided molecular process that influences antibiotic persister cell formation.

tRNA 3' shortening by LCCR4 as a response to stress in Trypanosoma brucei.

Sensing of environmental cues is crucial for cell survival. To adapt to changes in their surroundings cells need to tightly control the repertoire of genes expressed at any time. Regulation of translation is key, especially in organisms in which transcription is hardly controlled, like Trypanosoma brucei. In this study, we describe the shortening of the bulk of the cellular tRNAs during stress at the expense of the conserved 3' CCA-tail. This tRNA shortening is specific for nutritional stress and renders tRNAs unsuitable substrates for translation. We uncovered the nuclease LCCR4 (Tb927.4.2430), a homologue of the conserved deadenylase Ccr4, as being responsible for tRNA trimming. Once optimal growth conditions are restored tRNAs are rapidly repaired by the trypanosome tRNA nucleotidyltransferase thus rendering the recycled tRNAs amenable for translation. This mechanism represents a fast and efficient way to repress translation during stress, allowing quick reactivation with a low energy input.

Human vtRNA1-1 Levels Modulate Signaling Pathways and Regulate Apoptosis in Human Cancer Cells.

Regulatory non-protein coding RNAs perform a remarkable variety of complex biological functions. Previously, we demonstrated a role of the human non-coding vault RNA1-1 (vtRNA1-1) in inhibiting intrinsic and extrinsic apoptosis in several cancer cell lines. Yet on the molecular level, the function of the vtRNA1-1 is still not fully clear. Here, we created HeLa knock-out cell lines revealing that prolonged starvation triggers elevated levels of apoptosis in the absence of vtRNA1-1 but not in vtRNA1-3 knock-out cells. Next-generation deep sequencing of the mRNome identified the PI3K/Akt pathway and the ERK1/2 MAPK cascade, two prominent signaling axes, to be misregulated in the absence of vtRNA1-1 during starvation-mediated cell death conditions. Expression of vtRNA1-1 mutants identified a short stretch of 24 nucleotides of the vtRNA1-1 central domain as being essential for successful maintenance of apoptosis resistance. This study describes a cell signaling-dependent contribution of the human vtRNA1-1 to starvation-induced programmed cell death.