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The story of rRNA expansion segments: Finding functionality amidst diversity
Abstract
Expansion segments (ESs) are multinucleotide insertions present across phyla at specific conserved positions in eukaryotic rRNAs. ESs are generally absent in bacterial rRNAs with some exceptions, while the archaeal rRNAs have microexpansions at regions that coincide with those of eukaryotic ESs. Although there is an increasing prominence of ribosomes, especially the ribosomal proteins, in fine‐tuning gene expression through translation regulation, the role of rRNA ESs is relatively underexplored. While rRNAs have been established as the major catalytic hub in ribosome function, the presence of ESs widens their scope as a species‐specific regulatory hub of protein synthesis. In this comprehensive review, we have elaborately discussed the current understanding of the functional aspects of rRNA ESs of cytoplasmic eukaryotic ribosomes and discuss their past, present, and future.
This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems
Translation > Ribosome Structure/Function
Translation > Regulation
... Nevertheless, kingdom-or species-specific features have been acquired over time, with S. cerevisiae having evolved an extra 908 nucleotide (nt) and humans 2650 nt in addition to the 4567 nt that make up the conserved rRNA core [1,5,16,17]. Some of these sequences have formed what is known as 'expansion segments' (ESs), large rRNA insertions that are specific to eukaryotic ribosomes and vary greatly in length and sequence between species [18]. However, the evolution of rRNA has also included more subtle changes in only a small number of nucleotides in some places as well as new sites and types of rRNA modifications, which were proposed to have contributed to the precise fine-tuning of ribosome function in diverse environments, from single-cell organisms to complex tissues and organisms. ...
... An evolutionary increase in rRNA size and complexity has most commonly been observed through the insertion of socalled expansion segments (ESs), GC-rich insertions of varying lengths into the rDNA sequence [18]. Guided by information from both high-resolution 3D and secondary structure modelling, a model has emerged, termed 'the accretion model', which implies a general tendency towards molecular growth, i.e., expansion, of rRNA [39]. ...
... According to its principles, rRNA expanded iteratively and incrementally in size during evolution without a substantial remodelling of the ribosome's core or common trunk; as such, ES sites have increased as organisms became more complex. While most commonly observed in eukaryotes, ESs have been identified in the 5S rRNA of 36 bacterial strains and Archaea, which possess ESs with most insertions ranging between~5-20 nt in length at sites that coincide with those of eukaryotic ESs [18,39]. An exception to these short Archaeal ESs, are the Asgard ES39 and ES9, which have been discovered in the LSU rRNA Asgard archaeal phyla and, ranging in length from 50 to 200 nt, are closer to eukaryotic ESs, where ES39S varies between 80 nt in protists and 138 nt in S.cerevisiae to 178 nt in Drosophila and 231 nt in humans [40]. ...
For over 40 years, ribosomes were considered monolithic machines that translate the genetic code indiscriminately. However, over the past two decades, there have been a growing number of studies that suggest ribosomes to have a degree of compositional and functional adaptability in response to tissue type, cell environment and stimuli, cell cycle or development state. In such form, ribosomes themselves take an active part in translation regulation through an intrinsic adaptability provided by evolution, which furnished ribosomes with a dynamic plasticity that confers another layer of gene expression regulation. Yet despite the identification of various sources that give rise to ribosomal heterogeneity both at the protein and RNA level, its functional relevance is still debated, and many questions remain. Here, we will review aspects, including evolutionary ones, of ribosome heterogeneity emerging at the nucleic acid level, and aim to reframe ribosome ‘heterogeneity’ as an adaptive and dynamic process of plasticity.The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.
... Compared to bacterial ribosomes, eukaryotic ribosomes contain an ad tional 1-2 MDa of mass (depending on species) that represents the extension of some b terial RPs and the expansion of bacterial rRNA called eukaryotic "expansion segmen (ESs) [16][17][18]. Eukaryotic rRNA expansion segments vary between species and even different tissues of the same species [19,20]. Expansion segments play numerous roles ribosome biogenesis and translation, including translation initiation, co-translational p tein folding, endoplasmic reticulum (ER) attachment and protein translocation, and n cent peptide processing [19,21]. ...
... Eukaryotic rRNA expansion segments vary between species and even different tissues of the same species [19,20]. Expansion segments play numerous roles ribosome biogenesis and translation, including translation initiation, co-translational p tein folding, endoplasmic reticulum (ER) attachment and protein translocation, and n cent peptide processing [19,21]. Compared to yeast, the human ribosomal large subu . ...
... Compared to bacterial ribosomes, eukaryotic ribosomes contain an additional 1-2 MDa of mass (depending on species) that represents the extension of some bacterial RPs and the expansion of bacterial rRNA called eukaryotic "expansion segments" (ESs) [16][17][18]. Eukaryotic rRNA expansion segments vary between species and even in different tissues of the same species [19,20]. Expansion segments play numerous roles in ribosome biogenesis and translation, including translation initiation, co-translational protein folding, endoplasmic reticulum (ER) attachment and protein translocation, and nascent peptide processing [19,21]. ...
Ribosomal heterogeneity exists within cells and between different cell types, at specific developmental stages, and occurs in response to environmental stimuli. Mounting evidence supports the existence of specialized ribosomes, or specific changes to the ribosome that regulate the translation of a specific group of transcripts. These alterations have been shown to affect the affinity of ribosomes for certain mRNAs or change the cotranslational folding of nascent polypeptides at the exit tunnel. The identification of specialized ribosomes requires evidence of the incorporation of different ribosomal proteins or of modifications to rRNA and/or protein that lead(s) to physiologically relevant changes in translation. In this review, we summarize ribosomal heterogeneity and specialization in mammals and discuss their relevance to several human diseases.
... Notably, ES are variable in sequence and size across eukaryotes and their functions remain poorly understood. Recent studies showed that ES might be required for proper cell growth and participate in ribosome biogenesis, translation fidelity, protein folding, ER membrane binding and interactions with the mRNA and auxiliary proteins (reviewed in 23 ). However, most of the studies were performed using S. cerevisiae as a model, which has shorter ES compared to higher eukaryotes 1 . ...
... The rRNA ES are considered to play a regulatory role in translation, and their proportion progressively increases from lower to higher eukaryotes 23 . In addition, the natural habitat of the organism is likely to influence the amount of ribosomal regulatory elements, especially expansion segments 28 . ...
Ribosomes translate genetic information into primary structure. During translation, various cofactors transiently bind to the ribosome that undergoes prominent conformational and structural changes. Different translational states of ribosomes have been well characterized in vitro. However, to which extent the known translational states are representative of the native situation inside cells has thus far only been addressed in prokaryotes. Here, we apply cryo-electron tomography to cryo-FIB milled Dictyostelium discoideum cells combined with subtomogram averaging and classification. We obtain an in situ structure that is locally resolved up to 3 Angstrom, the distribution of eukaryotic ribosome translational states, and unique arrangement of rRNA expansion segments. Our work demonstrates the use of in situ structural biology techniques for identifying distinct ribosome states within the cellular environment. The translational states of eukaryotic ribosomes have so far been only investigated in vitro. Here, authors obtained the 3.8 Å in situ 80S ribosome structure, the distribution of translational states and unique arrangement of rRNA expansion segments.
... It is made machineries. Expansion segments are extra sequences found in eukaryotic rRNA, which are believed to reflect the complex regulation of protein synthesis associated with organismal complexity (144). Previous studies suggested that expansion segments can serve as interaction platforms for ribosome-associated factors, binding with specific mRNAs, regulating translation fidelity, and controlling compartment-specific translation (145)(146)(147)(148)(149). ES7L, the largest eukaryotic expansion segment, contains a highly conserved core signature fold and long tentacle-like extensions that undergo rapid evolutionary changes (150). ...
Mapping of the epitranscriptome has revealed the chemical diversity of RNA modifications and their functional importance in regulating gene expression. Transfer RNAs (tRNAs) are one of the most modified cellular RNAs, containing on average 10-13 modifications per molecule. These modifications have been shown to be critical for several aspects of tRNA functions, such as decoding, folding, and stability. Here we report that the human RNA methyltransferase TRMT1L associates with components of the Rix1 ribosome biogenesis complex and co-sediments with pre-60S ribosomes. Using eCLIP-Seq, we show that TRMT1L binds to a subset of tRNAs and to the 28S rRNA. Additionally, we demonstrate that TRMT1L is responsible for catalyzing N2, N2-dimethylguanosine (m ² 2 G) solely at position 27 of tRNA-Tyr-GUA by Nano-tRNAseq and RNA LC-MS. Surprisingly, TRMT1L depletion also impaired the deposition of acp ³ U and dihydrouridine on tRNA-Tyr-GUA, Cys-GCA, and Ala-CGC. TRMT1L knockout cells have a marked decrease in tRNA-Tyr-GUA levels, coinciding with a reduction in global translation rates and hypersensitivity of oxidative stress. Our results establish TRMT1L as the elusive methyltransferase catalyzing the m ² 2 G27 modification on tRNA Tyr, resolving a long-standing gap of knowledge and highlighting its potential role in a tRNA modification circuit crucial for translation regulation and stress response.
... In animals, rRNA gene size and complexity have been observed to increase in evolution by inserting GC-rich sequences called expansion segments (48)(49)(50). A recent computational study revealed the insertion of the expansion segments in the 5S rRNA genes of some disparate bacterial and archaeal genomes (51). ...
Background
The correlation between GC content and halophilicity has received limited attention, despite the numerous environmental factors associated with GC content evolution. While higher GC content has been linked to halophiles in a few archaeal cases, it is widely believed that selective pressure from high-intensity ultraviolet radiation in halophilic archaea drives GC content increase, as it prevents DNA photoproduct formation. However, this assumption has not been statistically analyzed in a phylogenetically independent manner prior to our study.
Results
Using phylogenetic generalized least squares (PGLS), we investigated the relationship between GC content and halophilicity in 1226 bacteria and 181 archaea. Our analysis found significant positive correlations in bacteria but not in archaea. Our resampling analysis indicates that the absence of significant correlation in archaea may be due to the relatively small sample size. We also observed that the strength of the correlation is negatively influenced by the functional constraint of genomic components. Additionally, we examined the relationship between GC content and photoreactivity, a measure of DNA vulnerability to ultraviolet radiation, but found no significant correlation in either bacteria or archaea.
Conclusions
We propose that the correlation between bacterial GC content and salinity can be attributed to a GC-biased DNA repair process, a response to DNA double-stranded breaks caused by high salt concentrations.
... Eukaryotic rRNAs display several large rRNA expansion segments with largely unexplored function. The accretion of these expansion segments bears the major contribution to the increase in rRNA length and ribosome size from yeast to vertebrates (Table 1) (Hariharan et al, 2022). Furthermore, eukaryotic ribosomes contain additional RPs, and most RPs of the conserved core harbor extensions and insertions (Spahn et al, 2001;Armache et al, 2010;Ben-Shem et al, 2011;Klinge et al, 2011;Rabl et al, 2011;Melnikov et al, 2012;Khatter et al, 2015). ...
The assembly of ribosomal subunits is a highly orchestrated process that involves a huge cohort of accessory factors. Most eukaryotic ribosome biogenesis factors were first identified by genetic screens and proteomic approaches of pre-ribosomal particles in Saccharomyces cerevisiae. Later, research on human ribosome synthesis not only demonstrated that the requirement for many of these factors is conserved in evolution, but also revealed the involvement of additional players, reflecting a more complex assembly pathway in mammalian cells. Yet, it remained a challenge for the field to assign a function to many of the identified factors and to reveal their molecular mode of action. Over the past decade, structural, biochemical, and cellular studies have largely filled this gap in knowledge and led to a detailed understanding of the molecular role that many of the players have during the stepwise process of ribosome maturation. Such detailed knowledge of the function of ribosome biogenesis factors will be key to further understand and better treat diseases linked to disturbed ribosome assembly, including ribosomopathies, as well as different types of cancer.
... Experimental manipulation of rRNA of eukaryotic ribosomes in vivo is problematic, complicating the determination of functions (76). Nevertheless, expansion segment ES7 is associated with multiple functions, many of which have been recently highlighted for fungal and protist ribosomes (Group 1). ...
The ribosomal core is universally conserved across the tree of life. However, eukaryotic ribosomes contain diverse rRNA expansion segments (ESs) on their surfaces. Sites of ES insertions are predicted from sites of insertion of micro-ESs in archaea. Expansion segment 7 (ES7) is one of the most diverse regions of the ribosome, emanating from a short stem loop and ranging to over 750 nucleotides in mammals. We present secondary and full-atom 3D structures of ES7 from species spanning eukaryotic diversity. Our results are based on experimental 3D structures, the accretion model of ribosomal evolution, phylogenetic relationships, multiple sequence alignments, RNA folding algorithms and 3D modeling by RNAComposer. ES7 contains a distinct motif, the ‘ES7 Signature Fold’, which is generally invariant in 2D topology and 3D structure in all eukaryotic ribosomes. We establish a model in which ES7 developed over evolution through a series of elementary and recursive growth events. The data are sufficient to support an atomic-level accretion path for rRNA growth. The non-monophyletic distribution of some ES7 features across the phylogeny suggests acquisition via convergent processes. And finally, illustrating the power of our approach, we constructed the 2D and 3D structure of the entire LSU rRNA of Mus musculus.
... ESs still remain under-explored, even though they were discovered nearly four decades back. They may potentially have roles in diverse cellular processes such as ribosome biogenesis, post-transcriptional gene regulation and RNA metabolism (107). Therefore we think it is imperative to expand the repertoire of techniques required to probe the function of all ESs, especially because of the potential they hold in expanding the regulatory landscape of ribosomes. ...
Ribosomes, the molecular machines that are central to protein synthesis, have gradually been gaining prominence for their potential regulatory role in translation. Eukaryotic cytosolic ribosomes are typically larger than the bacterial ones, partly due to multi-nucleotide insertions at specific conserved positions in the ribosomal RNAs (rRNAs). Such insertions called expansion segments (ESs) are present primarily on the ribosomal surface, with their role in translation and its regulation remaining under-explored. One such ES in the ribosomal large subunit (LSU) is ES30L, which is observed mostly in mammals and birds among eukaryotes. In this study, we show that ES30L possesses complementarity to many protein-coding transcripts in humans and that the complementarity is enriched around the start codon, indicating a possible role in translation regulation. Further, our in silico analysis analyses and in vitro pull-down assays show that ES30L may bind to secondary structures in the 5′ UTR of several transcripts and RNA binding proteins (RBPs) that are essential for translation. Thus, we have identified a potential regulatory role for ES30L in translation.
Ribosomal DNA (rDNA) repeat units are organized into tandem clusters in eukaryotic cells. In mice, these clusters are located on at least eight chromosomes and show extensive variation in the number of repeats between mouse genomes. To analyze intra- and inter-genomic variation of mouse rDNA repeats, we selectively isolated 25 individual rDNA units using Transformation-Associated Recombination (TAR) cloning. Long-read sequencing and subsequent comparative sequence analysis revealed that each full-length unit comprises an intergenic spacer (IGS) and a ∼13.4 kb long transcribed region encoding the three rRNAs, but with substantial variability in rDNA unit size, ranging from ∼35 to ∼46 kb. Within the transcribed regions of rDNA units, we found 209 variants, 70 of which are in external transcribed spacers (ETSs); but the rDNA size differences are driven primarily by IGS size heterogeneity, due to indels containing repetitive elements and some functional signals such as enhancers. Further evolutionary analysis categorized rDNA units into distinct clusters with characteristic IGS lengths; numbers of enhancers; and presence/absence of two common SNPs in promoter regions, one of which is located within promoter (p)RNA and may influence pRNA folding stability. These characteristic features of IGSs also correlated significantly with 5′ETS variant patterns described previously and associated with differential expression of rDNA units. Our results suggest that variant rDNA units are differentially regulated and open a route to investigate the role of rDNA variation on nucleolar formation and possible associations with pathology.
Here, we present the high‐resolution structure of the Gallus gallus 80S ribosome obtained from cold‐treated chicken embryos. The translationally inactive ribosome complex contains elongation factor eEF2 with GDP, SERPINE1 mRNA binding protein 1 (SERBP1) and deacylated tRNA in the P/E position, showing common features with complexes already described in mammals. Modeling of most expansion segments of G. gallus 28S ribosomal RNA allowed us to identify specific features in their structural organization and to describe areas where a marked difference between mammalian and avian ribosomes could shed light on the evolution of the expansion segments. This study provides the first structure of an avian ribosome, establishing a model for future structural and functional studies on the translational machinery in Aves.
Cardiovascular disease (CVD) is a major contributor to increasing morbidity and mortality worldwide and seriously threatens human health and life. Cardiomyocyte death is considered the pathological basis of various CVDs, including myocardial infarction, heart failure, and aortic dissection. Multiple mechanisms, such as ferroptosis, necrosis, and apoptosis, contribute to cardiomyocyte death. Among them, ferroptosis is an iron-dependent form of programmed cell death that plays a vital role in various physiological and pathological processes, from development and aging to immunity and CVD. The dysregulation of ferroptosis has been shown to be closely associated with CVD progression, yet its underlying mechanisms are still not fully understood. In recent years, a growing amount of evidence suggests that non-coding RNAs (ncRNAs), particularly microRNAs, long non-coding RNAs, and circular RNAs, are involved in the regulation of ferroptosis, thus affecting CVD progression. Some ncRNAs also exhibit potential value as biomarker and/or therapeutic target for patients with CVD. In this review, we systematically summarize recent findings on the underlying mechanisms of ncRNAs involved in ferroptosis regulation and their role in CVD progression. We also focus on their clinical applications as diagnostic and prognostic biomarkers as well as therapeutic targets in CVD treatment. DATA AVAILABILITY: No new data were created or analyzed in this study. Data sharing is not applicable to this article.
Peptide-chain elongation during protein synthesis entails sequential aminoacyl-tRNA selection and translocation reactions that proceed rapidly (2–20 per second) and with a low error rate (around 10⁻³ to 10⁻⁵ at each step) over thousands of cycles¹. The cadence and fidelity of ribosome transit through mRNA templates in discrete codon increments is a paradigm for movement in biological systems that must hold for diverse mRNA and tRNA substrates across domains of life. Here we use single-molecule fluorescence methods to guide the capture of structures of early translocation events on the bacterial ribosome. Our findings reveal that the bacterial GTPase elongation factor G specifically engages spontaneously achieved ribosome conformations while in an active, GTP-bound conformation to unlock and initiate peptidyl-tRNA translocation. These findings suggest that processes intrinsic to the pre-translocation ribosome complex can regulate the rate of protein synthesis, and that energy expenditure is used later in the translocation mechanism than previously proposed.
Ribosomes of different species share an evolutionarily conserved core, exhibiting flexible shells formed partially by the addition of species-specific ribosomal RNAs (rRNAs) with largely unexplored functions. In this study, we showed that by swapping the Saccharomyces cerevisiae 25S rRNA genes with non-S. cerevisiae homologs, species-specific rRNA variations caused moderate to severe pre-rRNA processing defects. Specifically, rRNA substitution by the Candida albicans caused severe growth defects and deficient pre-rRNA processing. We observed that such defects could be attributed primarily to variations in expansion segment 7L (ES7L) and could be restored by an assembly factor Noc2p mutant (Noc2p-K384R). We showed that swapping ES7L attenuated the incorporation of Noc2p and other proteins (Erb1p, Rrp1p, Rpl6p and Rpl7p) into pre-ribosomes, and this effect could be compensated for by Noc2p-K384R. Furthermore, replacement of Noc2p with ortholog from C. albicans could also enhance the incorporation of Noc2p and the above proteins into pre-ribosomes and consequently restore normal growth. Taken together, our findings help to elucidate the roles played by the species-specific rRNA variations in ribosomal biogenesis and further provide evidence that coevolution of rRNA expansion segments and cognate assembly factors specialized the ribosome biogenesis pathway, providing further insights into the function and evolution of ribosome.
The most abundant cellular RNA species, ribosomal RNA (rRNA), appears to be a source of massive amounts of non-randomly generated fragments. We found rRNA fragments (rRFs) in immunoprecipitated Argonaute (Ago-IP) complexes in human and mouse cells and in small RNA sequencing datasets. In human Ago1-IP, guanine-rich rRFs were preferentially cut in single-stranded regions of mature rRNAs between pyrimidines and adenosine, and non-randomly paired with cellular transcripts in crosslinked chimeras. Numerous identical rRFs were found in the cytoplasm and nucleus in mouse Ago2-IP. We report specific interaction motifs enriched in rRF-target pairs. Locations of such motifs on rRFs were compatible with the Ago structural features and patterns of the Ago-RNA crosslinking in both species. Strikingly, many of these motifs may bind to double-stranded regions on target RNAs, suggesting a potential pathway for regulating translation by unwinding mRNAs. Occurring on either end of rRFs and matching intronic, untranslated or coding regions in targets, such interaction sites extend the concept of microRNA seed regions. Targeting both borders of certain short introns, rRFs may be involved in their biogenesis or function, facilitated by Ago. Frequently dismissed as noise, rRFs are poised to greatly enrich the known functional spectrum of small RNA regulation.
Evolution has led to a great diversity that ranges from elegant simplicity to ornate complexity. Many complex features are often assumed to be more functional or adaptive than their simpler alternatives. However, in 1999, Arlin Stolzfus published a paper in the Journal of Molecular Evolution that outlined a framework in which complexity can arise through a series of non-adaptive steps. He called this framework Constructive Neutral Evolution (CNE). Despite its two-decade-old roots, many evolutionary biologists still appear to be unaware of this explanastory framework for the origins of complexity. In this perspective piece, we explain the theory of CNE and how it changes the order of events in narratives that describe the evolution of complexity. We also provide an extensive list of cellular features that may have become more complex through CNE. We end by discussing strategies to determine whether complexity arose through neutral or adaptive processes.
Roles for ribosomal RNA (rRNA) in gene regulation remain largely unexplored. With hundreds of rDNA units positioned across multiple loci, it is not possible to genetically modify rRNA in mammalian cells, hindering understanding of ribosome function. It remains elusive whether expansion segments (ESs), tentacle-like rRNA extensions that vary in sequence and size across eukaryotic evolution, may have functional roles in translation control. Here, we develop variable expansion segment-ligand chimeric ribosome immunoprecipitation RNA sequencing (VELCRO-IP RNA-seq), a versatile methodology to generate species-adapted ESs and to map specific mRNA regions across the transcriptome that preferentially associate with ESs. Application of VELCRO-IP RNA-seq to a mammalian ES, ES9S, identified a large array of transcripts that are selectively recruited to ribosomes via an ES. We further characterize a set of 5′ UTRs that facilitate cap-independent translation through ES9S-mediated ribosome binding. Thus, we present a technology for studying the enigmatic ESs of the ribosome, revealing their function in gene-specific translation.
SILVA (from Latin silva, forest, http://www.arb-silva.de) is a comprehensive web resource for up to date, quality-controlled databases of aligned ribosomal RNA (rRNA) gene sequences from the Bacteria, Archaea and Eukaryota domains and supplementary online services. The referred database release 111 (July 2012) contains 3 194 778 small subunit and 288 717 large subunit rRNA gene sequences. Since the initial description of the project, substantial new features have been introduced, including advanced quality control procedures, an improved rRNA gene aligner, online tools for probe and primer evaluation and optimized browsing, searching and downloading on the website. Furthermore, the extensively curated SILVA taxonomy and the new non-redundant SILVA datasets provide an ideal reference for high-throughput classification of data from next-generation sequencing approaches.
The large ribosomal RNAs of eukaryotes frequently contain expansion sequences that add to the size of the rRNAs but do not affect their overall structural layout and are compatible with major ribosomal function as an mRNA translation machine. The expansion of prokaryotic ribosomal RNAs is much less explored. In order to obtain more insight into the structural variability of these conserved molecules, we herein report the results of a comprehensive search for the expansion sequences in prokaryotic 5S rRNAs. Overall, 89 expanded 5S rRNAs of 15 structural types were identified in 15 archaeal and 36 bacterial genomes. Expansion segments ranging in length from 13 to 109 residues were found to be distributed among 17 insertion sites. The strains harboring the expanded 5S rRNAs belong to the bacterial orders Clostridiales, Halanaerobiales, Thermoanaerobacterales, and Alteromonadales as well as the archael order Halobacterales. When several copies of 5S rRNA gene are present in a genome, the expanded versions may co-exist with normal 5S rRNA genes. The insertion sequences are typically capable of forming extended helices, which do not seemingly interfere with folding of the conserved core. The expanded 5S rRNAs have largely been overlooked in 5S rRNA databases.
Mitoribosomes are specialized protein synthesis machineries in mitochondria. However, how mRNA binds to its dedicated channel, and tRNA moves as the mitoribosomal subunit rotate with respect to each other is not understood. We report models of the translating fungal mitoribosome with mRNA, tRNA and nascent polypeptide, as well as an assembly intermediate. Nicotinamide adenine dinucleotide (NAD) is found in the central protuberance of the large subunit, and the ATPase inhibitory factor 1 (IF1) in the small subunit. The models of the active mitoribosome explain how mRNA binds through a dedicated protein platform on the small subunit, tRNA is translocated with the help of the protein mL108, bridging it with L1 stalk on the large subunit, and nascent polypeptide paths through a newly shaped exit tunnel involving a series of structural rearrangements. An assembly intermediate is modeled with the maturation factor Atp25, providing insight into the biogenesis of the mitoribosomal large subunit and translation regulation.
The extreme halophile Halococcus morrhuae (ATCC® 17082) contains a 108‐nucleotide insertion in its 5S rRNA. Large rRNA expansions in Archaea are rare. This one almost doubles the length of the 5S rRNA. In order to understand how such an insertion is accommodated in the ribosome, we obtained a cryo‐electron microscopy reconstruction of the native large subunit at subnanometer resolution. The insertion site forms a four‐way junction that fully preserves the canonical 5S rRNA structure. Moving away from the junction site, the inserted region is conformationally flexible and does not pack tightly against the large subunit. The high salt requirement of the H. morrhuae ribosomes for their stability conflicted with the low salt threshold for cryo‐electron microscopy procedures. Despite this obstacle, this is the first cryo‐electron microscopy map of Halococcus ribosomes.
The ribosome's common core, comprised of ribosomal RNA (rRNA) and universal ribosomal proteins, connects all life back to a common ancestor and serves as a window to relationships among organisms. The rRNA of the common core is most similar to rRNA of extant bacteria. In eukaryotes, the rRNA of the common core is decorated by expansion segments (ESs) that vastly increase its size. Supersized ESs have not been observed previously in Archaea, and the origin of eukaryotic ESs remains enigmatic. We discovered that the large subunit (LSU) rRNA of two Asgard phyla, Lokiarchaeota and Heimdallarchaeota, considered to be the closest modern archaeal cell lineages to Eukarya, bridge the gap in size between prokaryotic and eukaryotic LSU rRNA. The elongated LSU rRNAs in Lokiarchaeota and Heimdallarchaeota stem from the presence of two supersized ESs, ES9 and ES39. We applied chemical footprinting experiments to study the structure of Lokiarchaeota ES39. Furthermore, we used covariation and sequence analysis to study the evolution of Asgard ES39s and ES9s. By defining the common eukaryotic ES39 signature fold, we found that Asgard ES39s have more and longer helices than eukaryotic ES39s. While Asgard ES39s have sequences and structures distinct from eukaryotic ES39s, we found overall conservation of a three-way junction across the Asgard species that matches eukaryotic ES39 topology, a result consistent with the accretion model of ribosomal evolution.
The in vitro formation of stable G-quadruplexes (G4s) in human ribosomal RNA (rRNA) was recently reported. However, their formation in cells and their cellular roles were not resolved. Here, by taking a chemical biology approach that integrates results from immunofluorescence, G4 ligands, heme affinity reagents, and a genetically encoded fluorescent heme sensor, we report that human ribosomes can form G4s in vivo that regulate heme bioavailability. Immunofluorescence experiments indicate that the vast majority of extra-nuclear G4s are associated with rRNA. Moreover, titrating human cells with a G4 ligand alters the ability of ribosomes to bind heme and disrupts cellular heme bioavailability as measured by a genetically encoded fluorescent heme sensor. Overall, these results suggest ribosomes play a role in regulating heme homeostasis.
SILVA (from Latin silva, forest, http://www.arb-silva.de) is a comprehensive web resource for up to date, quality-controlled databases of aligned ribosomal RNA (rRNA) gene sequences from the Bacteria, Archaea and Eukaryota domains and supplementary online services. The referred database release 111 (July 2012) contains 3 194 778 small subunit and 288 717 large subunit rRNA gene sequences. Since the initial description of the project, substantial new features have been introduced, including advanced quality control procedures, an improved rRNA gene aligner, online tools for probe and primer evaluation and optimized browsing, searching and downloading on the website. Furthermore, the extensively curated SILVA taxonomy and the new non-redundant SILVA datasets provide an ideal reference for high-throughput classification of data from next-generation sequencing approaches.
Cells continuously monitor protein synthesis to prevent accumulation of aberrant polypeptides. Insufficient capacity of cellular degradative systems, chaperone shortage or high levels of mistranslation by ribosomes can result in proteotoxic stress and endanger proteostasis. One of the least explored reasons for mistranslation is the incorrect functioning of the ribosome itself. To understand how cells deal with ribosome malfunction, we introduced mutations in the Expansion Segment 7 (ES7L) of 25S rRNA that allowed the formation of mature, translationally active ribosomes but induced proteotoxic stress and compromised cell viability. The ES7L-mutated ribosomes escaped nonfunctional rRNA Decay (NRD) and remained stable. Remarkably, ES7L-mutated ribosomes showed increased segregation into cytoplasmic foci containing soluble misfolded proteins. This ribosome entrapment pathway, termed TRAP (Translational Relocalization with Aberrant Polypeptides), was generalizable beyond the ES7L mutation, as wild-type ribosomes also showed increased relocalization into the same compartments in cells exposed to proteotoxic stressors. We propose that during TRAP, assembled ribosomes associated with misfolded nascent chains move into cytoplasmic compartments enriched in factors that facilitate protein quality control. In addition, TRAP may help to keep translation at its peak efficiency by preventing malfunctioning ribosomes from active duty in translation.
Human Ebp1 is a member of the proliferation-associated 2G4 (PA2G4) family and plays an important role in cancer regulation. Ebp1 shares the methionine aminopeptidase (MetAP) fold and binds to mature 80S ribosomes for translational control. Here, we present a cryo-EM single particle analysis reconstruction of Ebp1 bound to non-translating human 80S ribosomes at a resolution range from 3.3 to ~8 Å. Ebp1 blocks the tunnel exit with major interactions to the general uL23/uL29 docking site for nascent chain-associated factors complemented by eukaryote-specific eL19 and rRNA helix H59. H59 is defined as dynamic adaptor undergoing significant remodeling upon Ebp1 binding. Ebp1 recruits rRNA expansion segment ES27L to the tunnel exit via specific interactions with rRNA consensus sequences. The Ebp1-ribosome complex serves as a template for MetAP binding and provides insights into the structural principles for spatial coordination of co-translational events and molecular triage at the ribosomal tunnel exit.
Fine-tuned regulation of protein biosynthesis is crucial for cellular fitness and became even more vital when cellular and organismal complexity increased during the course of evolution. In order to cope with this augmented demand for translation control, eukaryal ribosomes have gained extensions both at the ribosomal protein and rRNA levels. Here we analyze the functional role of ES27L, an rRNA expansion segment in the large ribosomal subunit of Saccharomyces cerevisiae. Deletion of the b-arm of this expansion segment, called ES27Lb, did not hamper growth during optimal conditions, thus demonstrating that this 25S rRNA segment is not inherently crucial for ribosome functioning. However, reductive stress results in retarded growth and rendered unique protein sets prone to aggregation. Lack of ES27Lb negatively affects ribosome-association of known co-translational N-terminal processing enzymes which in turn contributes to the observed protein aggregation. Likely as a compensatory response to these challenges, the truncated ribosomes showed re-adjusted translation of specific sets of mRNAs and thus fine-tune the translatome in order to re-establish proteostasis. Our study gives comprehensive insight into how a highly conserved eukaryal rRNA expansion segment defines ribosomal integrity, co-translational protein maturation events and consequently cellular fitness.
Mammalian and bird ribosomes are nearly twice the mass of prokaryotic ribosomes in part because of their extraordinarily long rRNA tentacles. Human rRNA tentacles are not fully observable in current three-dimensional structures and their conformations remain to be fully resolved. In previous work we identified sequences that favor G-quadruplexes in silico and in vitro in rRNA tentacles of the human large ribosomal subunit. We demonstrated by experiment that these sequences form G-quadruplexes in vitro. Here, using a more recent motif definition, we report additional G-quadruplex sequences on surfaces of both subunits of the human ribosome. The revised sequence definition reveals expansive arrays of potential G-quadruplex sequences on LSU tentacles. In addition, we demonstrate by a variety of experimental methods that fragments of the small subunit rRNA form G-quadruplexes in vitro. Prior to this report rRNA sequences that form G-quadruplexes were confined to the large ribosomal subunit. Our combined results indicate that the surface of the assembled human ribosome contains numerous sequences capable of forming G-quadruplexes on both ribosomal subunits. The data suggest conversion between duplexes and G-quadru-plexes in response to association with proteins, ions, or other RNAs. In some systems it seems likely that the integrated population of RNA G-quadruplexes may be dominated by rRNA, which is the most abundant cellular RNA.
Hypotheses about the origin of eukaryotic cells are classically framed within the context of a universal ‘tree of life’ based on conserved core genes. Vigorous ongoing debate about eukaryote origins is based on assertions that the topology of the tree of life depends on the taxa included and the choice and quality of genomic data analysed. Here we have reanalysed the evidence underpinning those claims and apply more data to the question by using supertree and coalescent methods to interrogate >3,000 gene families in archaea and eukaryotes. We find that eukaryotes consistently originate from within the archaea in a two-domains tree when due consideration is given to the fit between model and data. Our analyses support a close relationship between eukaryotes and Asgard archaea and identify the Heimdallarchaeota as the current best candidate for the closest archaeal relatives of the eukaryotic nuclear lineage. The evolutionary origin of eukaryotes is under debate. Here, the authors conduct phylogenetic analyses using >3,000 gene families in archaea and eukaryotes and find support for an origin of eukaryotes from within the archaea.
Contrary to the widely held belief that mitochondrial ribosomes (mitoribosomes) are highly similar to bacterial ones, recent experimental evidence reveals that mitoribosomes do differ significantly from their bacterial counterparts. This review is focused on plant mitoribosomes, but we also highlight the most striking similarities and differences between the plant and non-plant mitoribosomes. An analysis of the composition and structure of mitoribosomes in trypanosomes, yeast, mammals and plants uncovers numerous organism-specific features. For the plant mitoribosome, the most striking feature is the enormous size of the small subunit compared to the large one. Apart from the new structural information, possible functional peculiarities of different types of mitoribosomes are also discussed. Studies suggest that the protein composition of mitoribosomes is dynamic, especially during development, giving rise to a heterogeneous populations of ribosomes fulfilling specific functions. Moreover, convincing data shows that mitoribosomes interact with components involved in diverse mitochondrial gene expression steps, forming large expressosome-like structures.
Loading of mRNA onto the ribosomal 43S pre-initiation complex (PIC) and its subsequent scanning require the removal of the secondary structure of the by RNA helicases such as eIF4A. However, the topology and mechanics of the scanning complex bound to mRNA (48S-PIC) and the influence of its solvent-side composition on the scanning process are poorly known. Here, we found that the ES6S region of the 48S-PIC constitutes an extended binding channel for eIF4A-mediated unwinding of mRNA and scanning. Blocking ES6S inhibited the cap-dependent translation of mRNAs that have structured 5′ UTRs (including G-quadruplexes), many of which are involved in signal transduction and growth, but it did not affect IRES-driven translation. Genome-wide analysis of mRNA translation revealed a great diversity in ES6S-mediated scanning dependency. Our data suggest that mRNA threading into the ES6S region makes scanning by 48S PIC slower but more processive. Hence, we propose a topological and functional model of the scanning 48S-PIC.
Microsporidia are eukaryotic parasites that infect essentially all animal species, including many of agricultural importance1–3, and are significant opportunistic parasites of humans⁴. They are characterized by having a specialized infection apparatus, an obligate intracellular lifestyle⁵, rudimentary mitochondria and the smallest known eukaryotic genomes5–7. Extreme genome compaction led to minimal gene sizes affecting even conserved ancient complexes such as the ribosome8–10. In the present study, the cryo-electron microscopy structure of the ribosome from the microsporidium Vairimorpha necatrix is presented, which illustrates how genome compaction has resulted in the smallest known eukaryotic cytoplasmic ribosome. Selection pressure led to the loss of two ribosomal proteins and removal of essentially all eukaryote-specific ribosomal RNA (rRNA) expansion segments, reducing the rRNA to a functionally conserved core. The structure highlights how one microsporidia-specific and several repurposed existing ribosomal proteins compensate for the extensive rRNA reduction. The microsporidian ribosome is kept in an inactive state by two previously uncharacterized dormancy factors that specifically target the functionally important E-site, P-site and polypeptide exit tunnel. The present study illustrates the distinct effects of evolutionary pressure on RNA and protein-coding genes, provides a mechanism for ribosome inhibition and can serve as a structural basis for the development of inhibitors against microsporidian parasites.
The advent of RNA-sequencing (RNA-Seq) technologies has markedly improved our knowledge and expanded the compendium of small non-coding RNAs, most of which derive from the processing of longer RNA precursors. In this review article, we will present a nonexhaustive list of referenced small non-coding RNAs (ncRNAs) derived from eukaryotic ribosomal RNA (rRNA), called rRNA fragments (rRFs). We will focus on the rRFs that are experimentally verified, and discuss their origin, length, structure, biogenesis, association with known regulatory proteins, and potential role(s) as regulator of gene expression. This relatively new class of ncRNAs remained poorly investigated and underappreciated until recently, due mainly to the a priori exclusion of rRNA sequences—because of their overabundance—from RNA-Seq datasets. The situation surrounding rRFs resembles that of microRNAs (miRNAs), which used to be readily discarded from further analyses, for more than five decades, because no one could believe that RNA of such a short length could bear biological significance. As if we had not yet learned our lesson not to restrain our investigative, scientific mind from challenging widely accepted beliefs or dogmas, and from looking for the hidden treasures in the most unexpected places.
Mitochondria are responsible for energy production through aerobic respiration, and represent the powerhouse of eukaryotic cells. Their metabolism and gene expression processes combine bacterial-like features and traits that evolved in eukaryotes. Among mitochondrial gene expression processes, translation remains the most elusive. In plants, while numerous pentatricopeptide repeat (PPR) proteins are involved in all steps of gene expression, their function in mitochondrial translation remains unclear. Here we present the biochemical characterization of Arabidopsis mitochondrial ribosomes and identify their protein subunit composition. Complementary biochemical approaches identified 19 plant-specific mitoribosome proteins, of which ten are PPR proteins. The knockout mutations of ribosomal PPR (rPPR) genes result in distinct macroscopic phenotypes, including lethality and severe growth delay. The molecular analysis of rppr1 mutants using ribosome profiling, as well as the analysis of mitochondrial protein levels, demonstrate rPPR1 to be a generic translation factor that is a novel function for PPR proteins. Finally, single-particle cryo-electron microscopy (cryo-EM) reveals the unique structural architecture of Arabidopsis mitoribosomes, characterized by a very large small ribosomal subunit, larger than the large subunit, bearing an additional RNA domain grafted onto the head. Overall, our results show that Arabidopsis mitoribosomes are substantially divergent from bacterial and other eukaryote mitoribosomes, in terms of both structure and protein content.
The majority of eukaryotic proteins are N-terminally α-acetylated by N-terminal acetyltransferases (NATs). Acetylation usually occurs co-translationally and defects have severe consequences. Nevertheless, it is unclear how these enzymes act in concert with the translating ribosome. Here, we report the structure of a native ribosome-NatA complex from Saccharomyces cerevisiae. NatA (comprising Naa10, Naa15 and Naa50) displays a unique mode of ribosome interaction by contacting eukaryotic-specific ribosomal RNA expansion segments in three out of four binding patches. Thereby, NatA is dynamically positioned directly underneath the ribosomal exit tunnel to facilitate modification of the emerging nascent peptide chain. Methionine amino peptidases, but not chaperones or signal recognition particle, would be able to bind concomitantly. This work assigns a function to the hitherto enigmatic ribosomal RNA expansion segments and provides mechanistic insights into co-translational protein maturation by N-terminal acetylation.
Background:
The size of eukaryotic 25-28S rRNAs shows a progressive phylogenetically linked increase which is pronounced in mammals, and especially in hominids. The increase is confined to specific expansion segments, inserted at points that are highly conserved from yeast to man. These segments also show a progressive increase in nucleotide bias, mostly the GC bias. Substantial parts of the large expansion segments 7, 15 and 27 of 28S rRNA are known to be exposed at the ribosome surface, with no clear association with ribosomal proteins. These segments could bind extraneous RNAs and proteins to support regulatory events.
Methods:
This study examined the possible canonical matching of human 28S rRNA and 18S rRNA segments with 2586 human microRNAs. This was compared with matching of the microRNAs to sectors of 18810 human mRNAs.
Results:
The overall matching was rather similar across 18S rRNA segments and core segments of 28S rRNA. However, the expansion segments of 28S rRNA (abbreviated ESL) collectively have a much higher (up to two-fold) capacity for the canonical association with microRNAs. This is pronounced in large ESL, and is found to strongly relate to the GC content of microRNAs.
Conclusion:
Oligonucleotides and microRNAs of high GC content through a strong canonical hydrogen bonding could have large activity in regulation of subcellular RNAs. In view of the considerable abundance of ribosomal RNAs in many mammalian tissues, ESL could constitute an important component of microRNA balance, possibly serving to lower the availability of GC-rich microRNAs (and thereby help conservation of GC-rich mRNAs).
Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. It is unknown how the highly intertwined structure of 60S large ribosomal subunits is established. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 Å resolution, revealing a half-assembled subunit. Domains I, II and VI of 25S/5.8S rRNA pack tightly into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to establishing the global architecture of 60S subunits.
Electronic supplementary material
The online version of this article (10.1007/s13238-018-0526-7) contains supplementary material, which is available to authorized users.
Ribosomal proteins are indispensable components of a living cell, and yet their structures are remarkably diverse in different species. Here we use manually curated structural alignments to provide a comprehensive catalog of structural variations in homologous ribosomal proteins from bacteria, archaea, eukaryotes and eukaryotic organelles. By resolving numerous ambiguities and errors of automated structural and sequence alignments, we uncover a whole new class of structural variations, which reside within seemingly conserved segments of ribosomal proteins. We then illustrate that these variations reflect an apparent adaptation of ribosomal proteins to the specific environments and lifestyles of living species. Finally, we show that most of these structural variations reside within non-globular extensions of ribosomal proteins - protein segments that are thought to promote ribosome biogenesis by stabilizing the proper folding of ribosomal RNA. We show that, even though the extensions are thought to be the most ancient peptides on our planet, they are in fact the most rapidly evolving and most structurally and functionally diverse segments of ribosomal proteins. Overall, our work illustrates that, despite being long considered as slowly evolving and highly conserved, ribosomal proteins are more complex and more specialized than is generally recognized.
Eukaryote ribosomal RNAs (rRNAs) have expanded in the course of phylogeny by addition of nucleotides in specific insertion areas, the expansion segments. These number about 40 in the larger (25–28S) rRNA (up to 2,400 nucleotides), and about 12 in the smaller (18S) rRNA (<700 nucleotides). Expansion of the larger rRNA shows a clear phylogenetic increase, with a dramatic rise in mammals and especially in hominids. Substantial portions of expansion segments in this RNA are not bound to ribosomal proteins, and may engage extraneous interactants, including messenger RNAs (mRNAs). Studies on the ribosome-mRNA interaction have focused on proteins of the smaller ribosomal subunit, with some examination of 18S rRNA. However, the expansion segments of human 28S rRNA show much higher density and numbers of mRNA matches than those of 18S rRNA, and also a higher density and match numbers than its own core parts. We have studied that with frequent and potentially stable matches containing 7–15 nucleotides. The expansion segments of 28S rRNA average more than 50 matches per mRNA even assuming only 5% of their sequence as available for such interaction. Large expansion segments 7, 15, and 27 of 28S rRNA also have copious long (≥10-nucleotide) matches to most human mRNAs, with frequencies much higher than in other 28S rRNA parts. Expansion segments 7 and 27 and especially segment 15 of 28S rRNA show large size increase in mammals compared to other metazoans, which could reflect a gain of function related to interaction with non-ribosomal partners. The 28S rRNA expansion segment 15 shows very high increments in size, guanosine, and cytidine nucleotide content and mRNA matching in mammals, and especially in hominids. With these segments (but not with other 28S rRNA or any 18S rRNA expansion segments) the density and number of matches are much higher in 5′-terminal than in 3′-terminal untranslated mRNA regions, which may relate to mRNA mobilization via 5′ termini. Matches in the expansion segments 7, 15, and 27 of human 28S rRNA appear as candidates for general interaction with mRNAs, especially those associated with intracellular matrices such as the endoplasmic reticulum.
The ribosome, the integration point for protein synthesis in the cell, is conventionally considered a homogeneous molecular assembly that only passively contributes to gene expression. Yet, epigenetic features of the ribosomal DNA (rDNA) operon and changes in the ribosome’s molecular composition have been associated with disease phenotypes, suggesting that the ribosome itself may possess inherent regulatory capacity. Analyzing whole-genome sequencing data from the 1000 Genomes Project and the Mouse Genomes Project, we find that rDNA copy number varies widely across individuals, and we identify pervasive intra- and interindividual nucleotide variation in the 5S, 5.8S, 18S, and 28S ribosomal RNA (rRNA) genes of both human and mouse. Conserved rRNA sequence heterogeneities map to functional centers of the assembled ribosome, variant rRNA alleles exhibit tissue-specific expression, and ribosomes bearing variant rRNA alleles are present in the actively translating ribosome pool. These findings provide a critical framework for exploring the possibility that the expression of genomically encoded variant rRNA alleles gives rise to physically and functionally heterogeneous ribosomes that contribute to mammalian physiology and human disease.
Eukaryotic 60S ribosomal subunits are comprised of three rRNAs and ∼50 ribosomal proteins. The initial steps of their formation take place in the nucleolus, but, owing to a lack of structural information, this process is poorly understood. Using cryo-EM, we solved structures of early 60S biogenesis intermediates at 3.3 Å to 4.5 Å resolution, thereby providing insights into their sequential folding and assembly pathway. Besides revealing distinct immature rRNA conformations, we map 25 assembly factors in six different assembly states. Notably, the Nsa1-Rrp1-Rpf1-Mak16 module stabilizes the solvent side of the 60S subunit, and the Erb1-Ytm1-Nop7 complex organizes and connects through Erb1's meandering N-terminal extension, eight assembly factors, three ribosomal proteins, and three 25S rRNA domains. Our structural snapshots reveal the order of integration and compaction of the six major 60S domains within early nucleolar 60S particles developing stepwise from the solvent side around the exit tunnel to the central protuberance.
RNA has the intrinsic propensity to form base pairs, leading to complex intramolecular and intermolecular helices. Direct measurement of base pairing interactions in living cells is critical to solving transcriptome structure and interactions, and investigating their functions (Lu and Chang, Curr Opin Struct Biol 36:142–148, 2016). Toward this goal, we developed an experimental method, PARIS (Psoralen Analysis of RNA Interactions and Structures), to directly determine transcriptome-wide base pairing interactions (Lu et al., Cell 165(5):1267–1279, 2016). PARIS combines four critical steps, in vivo cross-linking, 2D gel purification, proximity ligation, and high-throughput sequencing to achieve high-throughput and near-base pair resolution determination of the RNA structurome and interactome in living cells. In this chapter, we aim to provide a comprehensive discussion on the principles behind the experimental and computational strategies, and a step-by-step description of the experiment and analysis.
There is mounting evidence that the ribosome is not a static translation machinery, but a cell-specific, adaptive system. Ribosomal variations have mostly been studied at the protein level, even though the essential transcriptional functions are primarily performed by rRNAs. At the RNA level, oocyte-specific 5S rRNAs are long known for Xenopus. Recently, we described for zebrafish a similar system in which the sole maternal-type 5S rRNA present in eggs, is replaced completely during embryonic development by a somatic-type. Here, we report the discovery of an analogous system for the 45S rDNA elements; 5.8S, 18S, and 28S. The maternal-type 5.8S, 18S and 28S rRNA sequences differ substantially from those of the somatic-type, plus the maternal-type rRNAs are also replaced by the somatic-type rRNAs during embryogenesis. We discuss the structural and functional implications of the observed sequence differences with respect to the translational functions of the 5.8S, 18S, and 28S rRNA elements. Finally, in silico evidence suggests that expansion segments (ES) in 18S rRNA, previously implicated in ribosome-mRNA interaction, may have a preference for interacting with specific mRNA genes. Taken together, our findings indicate that two distinct types of ribosomes exist in zebrafish during development, each likely conducting the translation machinery in a unique way.
Chloroplasts are cellular organelles of plants and algae that are responsible for energy conversion and carbon fixation by the photosynthetic reaction. As a consequence of their endosymbiotic origin, they still contain their own genome and the machinery for protein biosynthesis. Here, we present the atomic structure of the chloroplast 70S ribosome prepared from spinach leaves and resolved by cryo-EM at 3.4 Å resolution. The complete structure reveals the features of the 4.5S rRNA, which probably evolved by the fragmentation of the 23S rRNA, and all five plastid-specific ribosomal proteins. These proteins, required for proper assembly and function of the chloroplast translation machinery, bind and stabilize rRNA including regions that only exist in the chloroplast ribosome. Furthermore, the structure reveals plastid-specific extensions of ribosomal proteins that extensively remodel the mRNA entry and exit site on the small subunit as well as the polypeptide tunnel exit and the putative binding site of the signal recognition particle on the large subunit. The translation factor pY, involved in light- and temperature-dependent control of protein synthesis, is bound to the mRNA channel of the small subunit and interacts with 16S rRNA nucleotides at the A-site and P-site, where it protects the decoding centre and inhibits translation by preventing tRNA binding. The small subunit is locked by pY in a non-rotated state, in which the intersubunit bridges to the large subunit are stabilized.
Ribosomes are the protein synthesizing machines of the cell. Recent advances in cryo-EM have led to the determination of structures from a variety of species, including bacterial 70S and eukaryotic 80S ribosomes as well as mitoribosomes from eukaryotic mitochondria, however, to date high resolution structures of plastid 70S ribosomes have been lacking. Here we present a cryo-EM structure of the spinach chloroplast 70S ribosome, with an average resolution of 5.4 Å for the small 30S subunit and 3.6 Å for the large 50S ribosomal subunit. The structure reveals the location of the plastid-specific ribosomal proteins (RPs) PSRP1, PSRP4, PSRP5 and PSRP6 as well as the numerous plastid-specific extensions of the RPs. We discover many features by which the plastid-specific extensions stabilize the ribosome via establishing additional interactions with surrounding ribosomal RNA and RPs. Moreover, we identify a large conglomerate of plastid-specific protein mass adjacent to the tunnel exit site that could facilitate interaction of the chloroplast ribosome with the thylakoid membrane and the protein-targeting machinery. Comparing the Escherichia coli 70S ribosome with that of the spinach chloroplast ribosome provides detailed insight into the co-evolution of RP and rRNA.
The secondary structure of ribosomal RNA (rRNA) is largely conserved across all kingdoms of life. However, eukaryotes have evolved extra blocks of rRNA sequences, relative to those of prokaryotes, called expansion segments (ES). A thorough characterization of the potential roles of ES remains to be done, possibly because of limitations in the availability of robust systems to study rRNA mutants. We sought to systematically investigate the potential functions, if any, of the ES in 25S rRNA of Saccharomyces cerevisiae by deletion mutagenesis. We deleted 14 of the 16 different eukaryote-specific ES in yeast 25S rRNA individually and assayed their phenotypes. Our results show that all but two of the ES tested are necessary for optimal growth and are required for production of 25S rRNA, suggesting that ES play roles in ribosome biogenesis. Further, we classified expansion segments into groups that participate in early nucleolar, middle, and late nucleoplasmic steps of ribosome biogenesis, by assaying their pre-rRNA processing phenotypes. This study is the first of its kind to systematically identify the functions of eukaryote-specific expansion segments by showing that they play roles in specific steps of ribosome biogenesis. The catalog of phenotypes we identified, combined with previous investigations of the roles ribosomal proteins in large subunit biogenesis, leads us to infer that assembling ribosomes are composed of distinct RNA and protein structural neighborhood clusters that participate in specific steps of ribosome biogenesis.
Cellular production of ribosomes involves the formation of highly defined interactions between ribosomal proteins (r-proteins) and ribosomal RNAs (rRNAs). Moreover in eukaryotic cells, efficient ribosome maturation requires the transient association of a large number of ribosome biogenesis factors (RBFs) with newly forming ribosomal subunits. Here, we investigated how r-protein assembly events in the large ribosomal subunit (LSU) rRNA domain II are coordinated with each other and with the association of RBFs in early LSU precursors of the yeast Saccharomyces cerevisiae. Specific effects on the pre-ribosomal association of RBFs could be observed in yeast mutants blocked in LSU rRNA domain II assembly. Moreover, formation of a cluster of r-proteins was identified as a downstream event in LSU rRNA domain II assembly. We analyzed in more detail the functional relevance of eukaryote specific bridges established by this r-protein cluster between LSU rRNA domain II and VI and discuss how they can support the stabilization and efficient processing of yeast early LSU precursor RNAs.
Subnuclear fractionation and coprecipitation by antibodies against the nucleolar protein NOP1 demonstrate that the essential Saccharomyces cerevisiae RNA snR30 is localized to the nucleolus. By using aminomethyl trimethyl-psoralen, snR30 can be cross-linked in vivo to 35S pre-rRNA. To determine whether snR30 has a role in rRNA processing, a conditional allele was constructed by replacing the authentic SNR30 promoter with the GAL10 promoter. Repression of snR30 synthesis results in a rapid depletion of snR30 and a progressive increase in cell doubling time. rRNA processing is disrupted during the depletion of snR30; mature 18S rRNA and its 20S precursor underaccumulate, and an aberrant 23S pre-rRNA intermediate can be detected. Initial results indicate that this 23S pre-rRNA is the same as the species detected on depletion of the small nucleolar RNA-associated proteins NOP1 and GAR1 and in an snr10 mutant strain. It was found that the 3' end of 23S pre-rRNA is located in the 3' region of ITS1 between cleavage sites A2 and B1 and not, as previously suggested, at the B1 site, snR30 is the fourth small nucleolar RNA shown to play a role in rRNA processing.
Regions extremely variable in size and sequence occur at conserved locations in eukaryotic rRNAs. The functional importance of one such region was determined by gene reconstruction and replacement in Tetrahymena thermophila. Deletion of the D8 region of the large-subunit rRNA inactivates T. thermophila rRNA genes (rDNA): transformants containing only this type of rDNA are unable to grow. Replacement with an unrelated sequence of similar size or a variable region from a different position in the rRNA also inactivated the rDNA. Mutant rRNAs resulting from such constructs were present only in precursor forms, suggesting that these rRNAs are deficient in either processing or stabilization of the mature form. Replacement with D8 regions from three other organisms restored function, even though the sequences are very different. Thus, these D8 regions share an essential functional feature that is not reflected in their primary sequences. Similar tertiary structures may be the quality these sequences share that allows them to function interchangeably.
Ribosomes have been suggested to directly control gene regulation, but regulatory roles for ribosomal RNA (rRNA) remain largely unexplored. Expansion segments (ESs) consist of multitudes of tentacle-like rRNA structures extending from the core ribosome in eukaryotes. ESs are remarkably variable in sequence and size across eukaryotic evolution with largely unknown functions. In characterizing ribosome binding to a regulatory element within a Homeobox (Hox) 5′ UTR, we identify a modular stem-loop within this element that binds to a single ES, ES9S. Engineering chimeric, “humanized” yeast ribosomes for ES9S reveals that an evolutionary change in the sequence of ES9S endows species-specific binding of Hoxa9 mRNA to the ribosome. Genome editing to site-specifically disrupt the Hoxa9-ES9S interaction demonstrates the functional importance for such selective mRNA-rRNA binding in translation control. Together, these studies unravel unexpected gene regulation directly mediated by rRNA and how ribosome evolution drives translation of critical developmental regulators.
The ribosome is an ancient molecular fossil that provides a telescope to the origins of life. Made from RNA and protein, the ribosome translates mRNA to coded protein in all living systems. Universality, economy, centrality and antiquity are ingrained in translation. The translation machinery dominates the set of genes that are shared as orthologues across the tree of life. The lineage of the translation system defines the universal tree of life. The function of a ribosome is to build ribosomes; to accomplish this task, ribosomes make ribosomal proteins, polymerases, enzymes, and signaling proteins. Every coded protein ever produced by life on Earth has passed through the exit tunnel, which is the birth canal of biology. During the root phase of the tree of life, before the last common ancestor of life (LUCA), exit tunnel evolution is dominant and unremitting. Protein folding coevolved with evolution of the exit tunnel. The ribosome shows that protein folding initiated with intrinsic disorder, supported through a short, primitive exit tunnel. Folding progressed to thermodynamically stable β-structures and then to kinetically trapped α-structures. The latter were enabled by a long, mature exit tunnel that partially offset the general thermodynamic tendency of all polypeptides to form β-sheets. RNA chaperoned the evolution of protein folding from the very beginning. The universal common core of the ribosome, with a mass of nearly 2 million Daltons, was finalized by LUCA. The ribosome entered stasis after LUCA and remained in that state for billions of years. Bacterial ribosomes never left stasis. Archaeal ribosomes have remained near stasis, except for the superphylum Asgard, which has accreted rRNA post LUCA. Eukaryotic ribosomes in some lineages appear to be logarithmically accreting rRNA over the last billion years. Ribosomal expansion in Asgard and Eukarya has been incremental and iterative, without substantial remodeling of pre-existing basal structures. The ribosome preserves information on its history.
Protein synthesis in all the living cells is mediated by a large protein-RNA complex called the ribosome. These macromolecular complexes can range from 2.5 (prokaryotes) to 4.2 MDa.
(eukaryotes) in size and undergo various conformational transitions during protein synthesis to translate the genetic code into the nascent polypeptide chains. Recent advances in cryo-electron microscopy (cryo-EM) and image processing methods have provided numerous detailed structures of ribosomes from diverse sources and in different conformational states resolved to near-atomic resolutions. These structures have not only helped in better understanding of the translational mechanism but also revealed species-specific variations or adaptations in the ribosome structures. Structural investigations of the ribosomes from Mycobacterium smegmatis (Msm) and its closely related pathogenic Mycobacterium tuberculosis (Mtb) lead to the identification of two additional ribosomal proteins named as bS22 and bL37 and several unique extensions in ribosomal-protein and ribosomal-RNA. Hibernation Promoting Factor (HPF) bound structure of Msm ribosome, termed as the hibernating ribosome, possibly indicates a new mechanism of ribosome protection during dormancy. These studies enabled the identification of the mycobacteria-specific ribosomal features and provides an opportunity to understand their function and target them for further drug-discovery purposes. Here we review the unique structural features identified in Msm ribosome and their possible implications in comparison to a well-studied Escherichia coli (Ec) ribosome.
Expansion segments (ESs) are enigmatic insertions within the eukaryotic ribosome, the longest of which resemble tentacle-like extensions that vary in length and sequence across evolution, with a largely unknown function. By selectively engineering rRNA in yeast, we find that one of the largest ESs, ES27L, has an unexpected function in translation fidelity. Ribosomes harboring a deletion in the distal portion of ES27L have increased amino acid misincorporation, as well as readthrough and frameshifting errors. By employing quantitative mass spectrometry, we further find that ES27L acts as an RNA scaffold to facilitate binding of a conserved enzyme, methionine amino peptidase (MetAP). We show that MetAP unexpectedly controls the accuracy of ribosome decoding, which is coupled to an increase in its enzymatic function through its interaction with ES27L. These findings reveal that variable ESs of the ribosome serve important functional roles and act as platforms for the binding of proteins that modulate translation across evolution.
Molecular mechanisms behind ribosomopathies
Ribosomopathies are t issuespecific disorders that result from mutations in ribosomal proteins or ribosome biogenesis factors. Such disorders include Diamond-Blackfan anemia, isolated congenital asplenia, and Treacher Collins syndrome. Mills and Green review the underlying mechanisms of tissue-specific defects in these and related disorders. Because ribosomes are central to all cellular life, it is puzzling why mutations in components of the ribosome disproportionately affect certain tissues. The authors suggest that ribosome homeostasis is an overarching and simplifying principle that governs the sensitivity of specific cells and tissue types to mutation in components of the translational machinery.
Science , this issue p. eaan2755
Diversity in eukaryotic rRNA structure and function offers possibilities of novel therapeutic targets. Unlike ribosomes of prokaryotes, eukaryotic ribosomes contain species-specific rRNA expansion segments (ESs) with idiosyncratic structures and functions that are essential and specific to some organisms. Here we investigate expansion segment 7 (ES7), one of the largest and most variable expansions of the eukaryotic ribosome. We hypothesize that ES7 of the pathogenic fungi Candida albicans (ES7CA) could be a prototypic drug target. We show that isolated ES7CA folds reversibly to a native-like state. We developed a fluorescent displacement (FD) assay using an RNA binding fluorescent probe, F-neo. F-neo binds tightly to ES7CA with a Kd of 2.5 x 10-9 M but binds weakly to ES7 of humans (ES7HS) with a Kd estimated to be greater than 10 µM. The FD assay was used to investigate the affinities of a library of peptidic aminosugar conjugates (PAs) for ES7CA. For conjugates with highest affinities for ES7CA (NeoRH, NeoFH and NeoYH), the lowest dose needed to induce mortality in C. albicans (minimum inhibitory concentration, MIC) was determined. PAs with the lowest MIC values were tested for cytotoxicity in HEK293T cells. Molecules with high affinity for ES7CAin vitro induce mortality in C. albicans but not in HEK293T cells. The results are consistent with the hypothesis that ESs represent useful targets for chemotherapeutics directed against eukaryotic pathogens.
Mitochondria maintain their own genome and its corresponding protein synthesis machine, the mitochondrial ribosome (mitoribosome). Mitoribosomes primarily synthesize highly hydrophobic proteins of the inner mitochondrial membrane. Recent studies revealed the complete structure of the isolated mammalian mitoribosome, but its mode of membrane association remained hypothetical. In this study, we used cryoelectron tomography to visualize human mitoribosomes in isolated mitochondria. The subtomogram average of the membrane-associated human mitoribosome reveals a single major contact site with the inner membrane, mediated by the mitochondria-specific protein mL45. A second rRNA-mediated contact site that is present in yeast is absent in humans, resulting in a more variable association of the human mitoribosome with the inner membrane. Despite extensive structural differences of mammalian and fungal mitoribosomal structure, the principal organization of peptide exit tunnel and the mL45 homolog remains invariant, presumably to align the mitoribosome with the membrane-embedded insertion machinery.
Sequences totaling 3,500 bases from the 28S rRNA gene and from one of the ribosomal internal transcribed spacers (ITS1) have been determined for human, chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus). Analyses of the rRNA alignments show (1) a clustering of substitutions in the "variable regions" of the 28S gene, (2) a 1.5-3-fold increase in divergence in the transcribed spacer over that in the exon, and (3) that human and chimpanzee are the most closely related pair, in agreement with the results of Miyamoto et al., Sibley and Ahlquist, and Caccone and Powell.
The yeast mitoribosome
Mitochondria are eukaryotic organelles that produce ATP, the energy source of the cell. They have dedicated ribosomes (mitoribosomes) that encode some of the membrane proteins that are essential to ATP production. Desai et al. present a high-resolution structure of the 75-component yeast mitoribosome, determined by electron cryomicroscopy. Mitoribosomes share an ancestor with modern bacterial ribosomes. Comparing the structure of the yeast mitoribosome with mammalian mitoribosomes suggests how they have evolved differently to perform species-specific functions.
Science , this issue p. 528
Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate production in eukaryotic cells. Throughout evolution, mitoribosomes have become functionally specialized for synthesizing mitochondrial membrane proteins, and this has been accompanied by large changes to their structure and composition. We review recent high-resolution structural data that have provided unprecedented insight into the structure and function of mitoribosomes in mammals and fungi.
Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm(1,2). Hundreds of assembly factors, organized into sequential functional groups(3,4), facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly.