How Altered Ribosome Production Can Cause or Contribute to Human Disease: The Spectrum of Ribosomopathies.

A number of different defects in the process of ribosome production can lead to a diversified spectrum of disorders that are collectively identified as ribosomopathies. The specific factors involved may either play a role only in ribosome biogenesis or have additional extra-ribosomal functions, making it difficult to ascribe the pathogenesis of the disease specifically to an altered ribosome biogenesis, even if the latter is clearly affected. We reviewed the available literature in the field from this point of view with the aim of distinguishing, among ribosomopathies, the ones due to specific alterations in the process of ribosome production from those characterized by a multifactorial pathogenesis.

Accretion history of large ribosomal subunits deduced from theoretical minimal RNA rings is congruent with histories derived from phylogenetic and structural methods.

Accretions of tRNAs presumably formed the large complex ribosomal RNA structures. Similarities of tRNA secondary structures with rRNA secondary structures increase with the integration order of their cognate amino acid in the genetic code, indicating tRNA evolution towards rRNA-like structures. Here analyses rank secondary structure subelements of three large ribosomal RNAs (Prokaryota: Archaea: Thermus thermophilus; Bacteria: Escherichia coli; Eukaryota: Saccharomyces cerevisiae) in relation to their similarities with secondary structures formed by presumed proto-tRNAs, represented by 25 theoretical minimal RNA rings. These ranks are compared to those derived from two independent methods (ranks provide a relative evolutionary age to the rRNA substructure), (a) cladistic phylogenetic analyses and (b) 3D-crystallography where core subelements are presumed ancient and peripheral ones recent. Comparisons of rRNA secondary structure subelements with RNA ring secondary structures show congruence between ranks deduced by this method and both (a) and (b) (more with (a) than (b)), especially for RNA rings with predicted ancient cognate amino acid. Reconstruction of accretion histories of large rRNAs will gain from adequately integrating information from independent methods. Theoretical minimal RNA rings, sequences deterministically designed in silico according to specific coding constraints, might produce adequate scales for prebiotic and early life molecular evolution.

The EGF/Ras pathway controls growth in Drosophila via ribosomal RNA synthesis.

The Ras small G-protein is a conserved regulator of cell and tissue growth during animal development. Studies in Drosophila have shown how Ras can stimulate a RAF-MEK-ERK signalling pathway to control cell growth and proliferation in response to Epidermal Growth Factor (EGF) stimulation. This work has also defined several transcription factors that can function as downstream growth effectors of the EGF/Ras/ERK pathway by stimulating mRNA transcription. Here we report on stimulation of RNA polymerase I (Pol I)-mediated ribosomal RNA (rRNA) synthesis as a growth effector of Ras/ERK signalling in Drosophila. We show that Ras/ERK signalling promotes an increase in nucleolar size in larval wing discs, which is indicative of increased ribosome synthesis. We also find that activation of Ras/ERK signalling promotes rRNA synthesis both in vivo and in cultured Drosophila S2 cells. We show that Ras signalling can regulate the expression of the Pol I transcription factor TIF-IA, and that this regulation requires dMyc. Finally, we find that TIF-IA-mediated rRNA synthesis is required for Ras/ERK signalling to drive proliferation in both larval and adult Drosophila tissues. These findings indicate that Ras signalling can promote ribosome synthesis in Drosophila, and that this is one mechanism that contributes to the growth effects of the Ras signalling pathway.

rRNA Mimicry in RNA Regulation of Gene Expression.

The rRNA is the largest and most abundant RNA in bacterial and archaeal cells. It is also one of the best-characterized RNAs in terms of its structural motifs and sequence variation. Production of ribosome components including >50 ribosomal proteins (r-proteins) consumes significant cellular resources. Thus, RNA cis-regulatory structures that interact with r-proteins to repress further r-protein synthesis play an important role in maintaining appropriate stoichiometry between r-proteins and rRNA. Classically, such mRNA structures were thought to directly mimic the rRNA. However, more than 30 years of research has demonstrated that a variety of different recognition and regulatory paradigms are present. This review will demonstrate how structural mimicry between the rRNA and mRNA cis-regulatory structures may take many different forms. The collection of mRNA structures that interact with r-proteins to regulate r-protein operons are best characterized in Escherichia coli, but are increasingly found within species from nearly all phyla of bacteria and several archaea. Furthermore, they represent a unique opportunity to assess the plasticity of RNA structure in the context of RNA-protein interactions. The binding determinants imposed by r-proteins to allow regulation can be fulfilled in many ways. Some r-protein-interacting mRNAs are immediately obvious as rRNA mimics from primary sequence similarity, others are identifiable only after secondary or tertiary structure determination, and some show no obvious similarity. In addition, across different bacterial species a host of different mechanisms of action have been characterized, showing that there is no simple one-size-fits-all solution.

A systematic computational analysis of the rRNA-3' UTR sequence complementarity suggests a regulatory mechanism influencing post-termination events in metazoan translation.

Nucleic acid sequence complementarity underlies many fundamental biological processes. Although first noticed a long time ago, sequence complementarity between mRNAs and ribosomal RNAs still lacks a meaningful biological interpretation. Here we used statistical analysis of large-scale sequence data sets and high-throughput computing to explore complementarity between 18S and 28S rRNAs and mRNA 3' UTR sequences. By the analysis of 27,646 full-length 3' UTR sequences from 14 species covering both protozoans and metazoans, we show that the computed 18S rRNA complementarity creates an evolutionarily conserved localization pattern centered around the ribosomal mRNA entry channel, suggesting its biological relevance and functionality. Based on this specific pattern and earlier data showing that post-termination 80S ribosomes are not stably anchored at the stop codon and can migrate in both directions to codons that are cognate to the P-site deacylated tRNA, we propose that the 18S rRNA-mRNA complementarity selectively stabilizes post-termination ribosomal complexes to facilitate ribosome recycling. We thus demonstrate that the complementarity between 18S rRNA and 3' UTRs has a non-random nature and very likely carries information with a regulatory potential for translational control.

Functional Importance of Mobile Ribosomal Proteins.

Although the dynamic motions and peptidyl transferase activity seem to be embedded in the rRNAs, the ribosome contains more than 50 ribosomal proteins (r-proteins), whose functions remain largely elusive. Also, the precise forms of some of these r-proteins, as being part of the ribosome, are not structurally solved due to their high flexibility, which hinders the efforts in their functional elucidation. Owing to recent advances in cryo-electron microscopy, single-molecule techniques, and theoretical modeling, much has been learned about the dynamics of these r-proteins. Surprisingly, allosteric regulations have been found in between spatially separated components as distant as those in the opposite sides of the ribosome. Here, we focus on the functional roles and intricate regulations of the mobile L1 and L12 stalks and L9 and S1 proteins. Conformational flexibility also enables versatile functions for r-proteins beyond translation. The arrangement of r-proteins may be under evolutionary pressure that fine-tunes mass distributions for optimal structural dynamics and catalytic activity of the ribosome.

Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan.

Several pathways modulating longevity and stress resistance converge on translation by targeting ribosomal proteins or initiation factors, but whether this involves modifications of ribosomal RNA is unclear. Here, we show that reduced levels of the conserved RNA methyltransferase NSUN5 increase the lifespan and stress resistance in yeast, worms and flies. Rcm1, the yeast homologue of NSUN5, methylates C2278 within a conserved region of 25S rRNA. Loss of Rcm1 alters the structural conformation of the ribosome in close proximity to C2278, as well as translational fidelity, and favours recruitment of a distinct subset of oxidative stress-responsive mRNAs into polysomes. Thus, rather than merely being a static molecular machine executing translation, the ribosome exhibits functional diversity by modification of just a single rRNA nucleotide, resulting in an alteration of organismal physiological behaviour, and linking rRNA-mediated translational regulation to modulation of lifespan, and differential stress response.

Heterogeneity of the translational machinery: Variations on a common theme.

In all organisms the universal process of protein synthesis is performed by the ribosome, a complex multi-component assembly composed of RNA and protein elements. Although ribosome heterogeneity was observed already more than 40 years ago, the ribosome is still traditionally viewed as an unchangeable entity that has to be equipped with all ribosomal components and translation factors in order to precisely accomplish all steps in protein synthesis. In the recent years this concept was challenged by several studies highlighting a broad variation in the composition of the translational machinery in response to environmental signals, which leads to its adaptation and functional specialization. Here, we summarize recent reports on the variability of the protein synthesis apparatus in diverse organisms and discuss the multiple mechanisms and possibilities that can lead to functional ribosome heterogeneity. Collectively, these results indicate that all cells are equipped with a remarkable toolbox to fine tune gene expression at the level of translation and emphasize the physiological importance of ribosome heterogeneity for the immediate implementation of environmental information.

On the expansion of ribosomal proteins and RNAs in eukaryotes.

While the ribosome constitution is similar in all biota, there is a considerable increase in size of both ribosomal proteins (RPs) and RNAs in eukaryotes as compared to archaea and bacteria. This is pronounced in the large (60S) ribosomal subunit (LSU). In addition to enlargement (apparently maximized already in lower eukarya), the RP changes include increases in fraction, segregation and clustering of basic residues, and decrease in hydrophobicity. The acidic fraction is lower in eukaryote as compared to prokaryote RPs. In all eukaryote groups tested, the LSU RPs have significantly higher content of basic residues and homobasic segments than the SSU RPs. The vertebrate LSU RPs have much higher sequestration of basic residues than those of bacteria, archaea and even of the lower eukarya. The basic clusters are highly aligned in the vertebrate, but less in the lower eukarya, and only within families in archaea and bacteria. Increase in the basicity of RPs, besides helping transport to the nucleus, should promote stability of the assembled ribosome as well as the association with translocons and other intracellular matrix proteins. The size and GC nucleotide bias of the expansion segments of large LSU rRNAs also culminate in the vertebrate, and should support ribosome association with the endoplasmic reticulum and other intracellular networks. However, the expansion and nucleotide bias of eukaryote LSU rRNAs do not clearly correlate with changes in ionic parameters of LSU ribosomal proteins.

Quality control mechanisms during ribosome maturation.

Protein synthesis on ribosomes is carefully quality-controlled to ensure the faithful transmission of genetic information from mRNA to protein. Many of these mechanisms rely on communication between distant sites on the ribosomes, and thus on the integrity of the ribosome structure. Furthermore, haploinsufficiency of ribosomal proteins, which increases the chances of forming incompletely assembled ribosomes, can predispose to cancer. Finally, release of inactive ribosomes into the translating pool will lead to their degradation together with the degradation of the bound mRNA. Together, these findings suggest that quality control mechanisms must be in place to survey nascent ribosomes and ensure their functionality. This review gives an account of these mechanisms as currently known.

The tumor suppressive role of eIF3f and its function in translation inhibition and rRNA degradation.

Deregulated translation plays an important role in human cancer. We previously reported decreased eukaryotic initiation factor 3 subunit f (eIF3f) expression in pancreatic cancer. Whether decreased eIF3f expression can transform normal epithelial cells is not known. In our current study, we found evidence that stable knockdown of eIF3f in normal human pancreatic ductal epithelial cells increased cell size, nuclear pleomorphism, cytokinesis defects, cell proliferation, clonogenicity, apoptotic resistance, migration, and formation of 3-dimensional irregular masses. Our findings support the tumor suppressive role of eIF3f in pancreatic cancer. Mechanistically, we found that eIF3f inhibited both cap-dependent and cap-independent translation. An increase in the ribosomal RNA (rRNA) level was suggested to promote the generation of cancer. The regulatory mechanism of rRNA degradation in mammals is not well understood. We demonstrated here that eIF3f promotes rRNA degradation through direct interaction with heterogeneous nuclear ribonucleoprotein (hnRNP) K. We showed that hnRNP K is required for maintaining rRNA stability: under stress conditions, eIF3f dissociates hnRNP K from rRNA, thereby preventing it from protecting rRNA from degradation. We also demonstrated that rRNA degradation occurred in non-P body, non-stress granule cytoplasmic foci that contain eIF3f. Our findings established a new mechanism of rRNA decay regulation mediated by hnRNP K/eIF3f and suggest that the tumor suppressive function of eIF3f may link to impaired rRNA degradation and translation.

Immobilization of proteins in the nucleolus by ribosomal intergenic spacer noncoding RNA.

Cellular pathways are established and maintained by stochastic interactions of highly mobile molecules. The nucleolus plays a central role in the regulation of these molecular networks by capturing and immobilizing proteins. Here, we report a function for noncoding RNA (ncRNA) in the regulation of protein dynamics of key cellular factors, including VHL, Hsp70 and MDM2/PML. Stimuli-specific loci of the nucleolar intergenic spacer produce ncRNA capable of capturing and immobilizing proteins that encode a discrete peptidic code referred to as the nucleolar detention sequence (NoDS). Disruption of the NoDS/intergenic RNA interaction enables proteins to evade nucleolar sequestration and retain their dynamic profiles. Mislocalization of intergenic ncRNA triggers protein immobilization outside of the nucleolus, demonstrating that these ncRNA species can operate independently from the nucleolar architecture. We propose a model whereby protein immobilization by ncRNA is a posttranslational regulatory mechanism.

Overexpression of ribosomal RNA in prostate cancer is common but not linked to rDNA promoter hypomethylation.

Alterations in nucleoli, including increased numbers, increased size, altered architecture and increased function are hallmarks of prostate cancer cells. The mechanisms that result in increased nucleolar size, number and function in prostate cancer have not been fully elucidated. The nucleolus is formed around repeats of a transcriptional unit encoding a 45S ribosomal RNA (rRNA) precursor that is then processed to yield the mature 18S, 5.8S and 28S RNA species. Although it has been generally accepted that tumor cells overexpress rRNA species, this has not been examined in clinical prostate cancer. We find that indeed levels of the 45S rRNA, 28S, 18S and 5.8S are overexpressed in the majority of human primary prostate cancer specimens as compared with matched benign tissues. One mechanism that can alter nucleolar function and structure in cancer cells is hypomethylation of CpG dinucleotides of the upstream rDNA promoter region. However, this mechanism has not been examined in prostate cancer. To determine whether rRNA overexpression could be explained by hypomethylation of these CpG sites, we also evaluated the DNA methylation status of the rDNA promoter in prostate cancer cell lines and the clinical specimens. Bisulfite sequencing of genomic DNA revealed two roughly equal populations of loci in cell lines consisting of those that contained densely methylated deoxycytidine residues within CpGs and those that were largely unmethylated. All clinical specimens also contained two populations with no marked changes in methylation of this region in cancer as compared with normal. We recently reported that MYC can regulate rRNA levels in human prostate cancer; here we show that MYC mRNA levels are correlated with 45S, 18S and 5.8S rRNA levels. Further, as a surrogate for nucleolar size and number, we examined the expression of fibrillarin, which did not correlate with rRNA levels. We conclude that rRNA levels are increased in human prostate cancer, but that hypomethylation of the rDNA promoter does not explain this increase, nor does hypomethylation explain alterations in nucleolar number and structure in prostate cancer cells. Rather, rRNA levels and nucleolar size and number relate more closely to MYC overexpression.

Asymmetric segregation: the shape of things to come?

Yeast mother cells pay a sacrifice during budding: they keep the extrachromosomal rDNA circles (ERCs) so that their buds have a full lifespan ahead. Two new studies indicate that retention of ERCs in mother cells occurs not by tethering to the nuclear periphery but rather by the simple rules of diffusion in a time-limited and complex landscape.

The roles of RNA in the synthesis of protein.

The crystal structures of ribosomes that have been obtained since 2000 have transformed our understanding of protein synthesis. In addition to proving that RNA is responsible for catalyzing peptide bond formation, these structures have provided important insights into the mechanistic details of how the ribosome functions. This review emphasizes what has been learned about the mechanism of peptide bond formation, the antibiotics that inhibit ribosome function, and the fidelity of decoding.

Role of the mitochondrial mutations, m.827A>G and the novel m.7462C>T, in the origin of hearing loss.

Samples from 30 deaf probands exhibiting features suggestive of syndromic mitochondrial deafness or from families with maternal transmission of deafness were selected for investigation of mutations in the mitochondrial genes MT-RNR1 and MT-TS1. Patients with mutation m.1555A>G had been previously excluded from this sample. In the MT-RNR1 gene, five probands presented the m.827A>G sequence variant, of uncertain pathogenicity. This change was also detected in 66 subjects of an unaffected control sample of 306 Brazilian individuals from various ethnic backgrounds. Given its high frequency, we consider it unlikely to have a pathogenic role on hereditary deafness. As to the MT-TS1 gene, one proband presented the previously known pathogenic m.7472insC mutation and three probands presented a novel variant, m.7462C>T, which was absent from the same control sample of 306 individuals. Because of its absence in control samples and association with a family history of hearing impairment, we suggest it might be a novel pathogenic mutation.