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.

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.

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.

The key function of a conserved and modified rRNA residue in the ribosomal response to the nascent peptide.

The ribosome is able to monitor the structure of the nascent peptide and can stall in response to specific peptide sequences. Such programmed stalling is used for the regulation of gene expression. The molecular mechanisms of the nascent-peptide recognition and ribosome stalling are unknown. We identified the conserved and posttranscriptionally modified 23S rRNA nucleotide m(2)A2503 located at the entrance of the ribosome exit tunnel as a key component of the ribosomal response mechanism. A2503 mutations abolish nascent-peptide-dependent stalling at the leader cistrons of several inducible antibiotic resistance genes and at the secM regulatory gene. Remarkably, lack of the C2 methylation of A2503 significantly function induction of expression of the ermC gene, indicating that the functional role of posttranscriptional modification is to fine-tune ribosome-nascent peptide interactions. Structural and biochemical evidence suggest that m(2)A2503 may act in concert with the previously identified nascent-peptide sensor, A2062, in the ribosome exit tunnel to relay the stalling signal to the peptidyl transferase centre.

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.

Deconstructing ribosome construction.

The ribosome is an essential ribonucleoprotein enzyme, and its biogenesis is a fundamental process in all living cells. Recent X-ray crystal structures of the bacterial ribosome and new technologies have allowed a greater interrogation of in vitro ribosome assembly; however, substantially less is known about ribosome biogenesis in vivo. Ongoing investigations are focused on elucidating the cellular processes that facilitate biogenesis of the ribosomal subunits, and many extraribosomal factors, including modification enzymes, remodeling enzymes and GTPases, are being uncovered. Moreover, specific roles for ribosome biogenesis factors in subunit maturation are now being elaborated. Ultimately, such studies will reveal a more complete understanding of processes at work in in vivo ribosome biogenesis.

rRNA mutations that inhibit transfer-messenger RNA activity on stalled ribosomes.

In eubacteria, stalled ribosomes are rescued by a conserved quality-control mechanism involving transfer-messenger RNA (tmRNA) and its protein partner, SmpB. Mimicking a tRNA, tmRNA enters stalled ribosomes, adds Ala to the nascent polypeptide, and serves as a template to encode a short peptide that tags the nascent protein for destruction. To further characterize the tagging process, we developed two genetic selections that link tmRNA activity to cell death. These negative selections can be used to identify inhibitors of tagging or to identify mutations in key residues essential for ribosome rescue. Little is known about which ribosomal elements are specifically required for tmRNA activity. Using these selections, we isolated rRNA mutations that block the rescue of ribosomes stalled at rare Arg codons or at the inefficient termination signal Pro-opal. We found that deletion of A1150 in the 16S rRNA blocked tagging regardless of the stalling sequence, suggesting that it inhibits tmRNA activity directly. The C889U mutation in 23S rRNA, however, lowered tagging levels at Pro-opal and rare Arg codons, but not at the 3' end of an mRNA lacking a stop codon. We concluded that the C889U mutation does not inhibit tmRNA activity per se but interferes with an upstream step intermediate between stalling and tagging. C889 is found in the A-site finger, where it interacts with the S13 protein in the small subunit (forming intersubunit bridge B1a).

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.

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.

Nucleolus, ribosomes, and cancer.

The complex aspects linking the nucleolus and ribosome biogenesis to cancer are reviewed here. The available evidence indicates that the morphological and functional changes in the nucleolus, widely observed in cancer tissues, are a consequence of both the increased demand for ribosome biogenesis, which characterizes proliferating cells, and the changes in the mechanisms controlling cell proliferation. In fact, the loss or functional changes in the two major tumor suppressor proteins pRB and p53 cause an up-regulation of ribosome biogenesis in cancer tissues. In this context, the association in human carcinomas of nucleolar hypertrophy with bad prognoses is worthy of note. Further, an increasing amount of data coming from studies on both hepatitis virus-induced chronic liver diseases and a subset of rare inherited disorders, including X-linked dyskeratosis congenita, suggests an active role of the nucleolus in tumorigenesis. Both an up-regulation of ribosome production and changes in the ribosome structure might causally contribute to neoplastic transformation, by affecting the balance of protein translation, thus altering the synthesis of proteins that play an important role in the genesis of cancer.

A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells.

A novel 5S RNA-protein (RNP) complex in human and mouse cells has been analyzed using patient autoantibodies. The RNP is small (approximately 7S) and contains most of the nonribosome-associated 5S RNA molecules in HeLa cells. The 5S RNA in the particle is matured at its 3' end, consistent with the results of in vivo pulse-chase experiments which indicate that this RNP represents a later step in 5S biogenesis than a previously described 5S*/La protein complex. The protein moiety of the 5S RNP has been identified as ribosomal protein L5, which is known to be released from ribosomes in a complex with 5S after various treatments of the 60S subunit. Indirect immunofluorescence indicates that the L5/5S complex is concentrated in the nucleolus. L5 may therefore play a role in delivering 5S rRNA to the nucleolus for assembly into ribosomes.

A specific amino acid binding site composed of RNA.

A specific, reversible binding site for a free amino acid is detectable on the intron of the Tetrahymena self-splicing ribosomal precursor RNA. The site selects arginine among the natural amino acids, and prefers the L- to the D-amino acid. The dissociation constant is in the millimolar range, and amino acid binding is at or in the catalytic rG splicing substrate site. Occupation of the G site by L-arginine therefore inhibits splicing by inhibiting the binding of rG, without inhibition of later reactions in the splicing reaction sequence. Arginine binding specificity seems to be directed at the side chain and the guanidino radical, and the alpha-amino and carboxyl groups are dispensable for binding. The arginine site can be placed within the G site by structural homology, with consequent implications for RNA-amino acid interaction, for the origin of the genetic code, for control of RNA activities, and for further catalytic capabilities for RNA.

Genetic expression in the developing brain.

The adult mouse brain contains complex populations of polyadenylated [poly(A)+] and nonpolyadenylated [poly(A)-] messenger RNA's (mRNA's). These mRNA's are separate sequence populations, similar in complexity, and in combination are equivalent to approximately 150,000 different mRNA sequences, of average length. Essentially all of the "adult" poly(A)+ mRNA's are present in the brain at birth. In contrast, most of the poly(A)- mRNA's are absent. Brain poly(A)- mRNA's begin to appear soon after birth, but the full adult complement is not reached until young adulthood. This suggests that these poly(A)- mRNA's specify proteins required for the biological capabilities of the brain that emerge during the course of postnatal development.

Historical review: the discovery of 'giant' RNA and RNA processing: 40 years of enigma.

RNA processing is a primordial paradigm of gene expression. Iconoclastic when discovered, after 40 years there is still no general rationale for this apparent 'wasting' of up to 90% of RNA transcripts. This article tells the story of the discovery of RNA in the laboratory of J.E. Darnell. The discovery of 'giant' RNA and its conversion into rRNA revealed the phenomenon of RNA processing and pre-rRNA. Genuine mRNA was also identified, but the majority of DNA-like nuclear RNA was also found to be giant and unstable. In spite of early evidence, pre-mRNA processing was only accepted in 1977 when the discovery of gene fragmentation in DNA made it obvious.

At the crossroads of growth control; making ribosomal RNA.

Although the mechanisms of cell cycle control are well established, the factors controlling cell growth and target size are still poorly understood. Much evidence suggests that ribosome biogenesis, and in particular the synthesis of the rRNAs, plays a central role not only in permitting growth, but also in regulating it. In the past few years we have begun to penetrate the network linking rRNA gene transcription to growth.