Showing papers on "Ribosomal protein published in 2012"

Reprogramming of tRNA modifications controls the oxidative stress response by codon-biased translation of proteins

Abstract: Selective translation of survival proteins is an important facet of the cellular stress response. We recently demonstrated that this translational control involves a stress-specific reprogramming of modified ribonucleosides in tRNA. Here we report the discovery of a step-wise translational control mechanism responsible for survival following oxidative stress. In yeast exposed to hydrogen peroxide, there is a Trm4 methyltransferase-dependent increase in the proportion of tRNA(Leu(CAA)) containing m(5)C at the wobble position, which causes selective translation of mRNA from genes enriched in the TTG codon. Of these genes, oxidative stress increases protein expression from the TTG-enriched ribosomal protein gene RPL22A, but not its unenriched paralogue. Loss of either TRM4 or RPL22A confers hypersensitivity to oxidative stress. Proteomic analysis reveals that oxidative stress causes a significant translational bias towards proteins coded by TTG-enriched genes. These results point to stress-induced reprogramming of tRNA modifications and consequential reprogramming of ribosomes in translational control of cell survival.

Resistance to Linezolid Caused by Modifications at Its Binding Site on the Ribosome

Abstract: Linezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S ribosomal subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little evidence has been presented to confirm this. Furthermore, recent findings on the Cfr methyltransferase underscore the modification of 23S rRNA as a highly effective and transferable form of linezolid resistance. On a positive note, detailed knowledge of the linezolid binding site has facilitated the design of a new generation of oxazolidinones that show improved properties against the known resistance mechanisms.

Phylogenomics of Prokaryotic Ribosomal Proteins

Abstract: Archaeal and bacterial ribosomes contain more than 50 proteins, including 34 that are universally conserved in the three domains of cellular life (bacteria, archaea, and eukaryotes). Despite the high sequence conservation, annotation of ribosomal (r-) protein genes is often difficult because of their short lengths and biased sequence composition. We developed an automated computational pipeline for identification of r-protein genes and applied it to 995 completely sequenced bacterial and 87 archaeal genomes available in the RefSeq database. The pipeline employs curated seed alignments of r-proteins to run position-specific scoring matrix (PSSM)-based BLAST searches against six-frame genome translations, mitigating possible gene annotation errors. As a result of this analysis, we performed a census of prokaryotic r-protein complements, enumerated missing and paralogous r-proteins, and analyzed the distributions of ribosomal protein genes among chromosomal partitions. Phyletic patterns of bacterial and archaeal r-protein genes were mapped to phylogenetic trees reconstructed from concatenated alignments of r-proteins to reveal the history of likely multiple independent gains and losses. These alignments, available for download, can be used as search profiles to improve genome annotation of r-proteins and for further comparative genomics studies.

Suprainduction of p53 by disruption of 40S and 60S ribosome biogenesis leads to the activation of a novel G2/M checkpoint

Abstract: Impairment of ribosome biogenesis leads to p53 induction and cell cycle arrest, a checkpoint involved in human disease. Induction of p53 is attributed to the binding and inhibition of human double minute 2 (Hdm2) by a subset of ribosomal proteins (RPs): RPS7, RPL5, RPL11, and RPL23. However, we found that only RPL11 or RPL5, in a mutually dependent manner, elicit this response. We show that depletion of RPS7 or RPL23, like depletion of other RPs, except for RPL11 and RPL5, induces a p53 response and that the effects of RPS7 and RPL23 on p53 induction reported earlier may be ascribed to inhibition of global translation. Moreover, we made the surprising observation that codepletion of two essential RPs, one from each subunit, but not the same subunit, leads to suprainduction of p53. This led to the discovery that the previously proposed RPL11-dependent mechanism of p53 induction, thought to be caused by abrogation of 40S biogenesis and continued 60S biogenesis, is still operating, despite abrogation of 60S biogenesis. This response leads to both a G1 block and a novel G2/M block not observed when disrupting either subunit alone. Thus, induction of p53 is mediated by distinct mechanisms, with the data pointing to an essential role for ribosomal subunits beyond translation.

Mutual protection of ribosomal proteins L5 and L11 from degradation is essential for p53 activation upon ribosomal biogenesis stress

Abstract: Impairment of ribosomal biogenesis can activate the p53 protein independently of DNA damage. The ability of ribosomal proteins L5, L11, L23, L26, or S7 to bind Mdm2 and inhibit its ubiquitin ligase activity has been suggested as a critical step in p53 activation under these conditions. Here, we report that L5 and L11 are particularly important for this response. Whereas several other newly synthesized ribosomal proteins are degraded by proteasomes upon inhibition of Pol I activity by actinomycin D, L5 and L11 accumulate in the ribosome-free fraction where they bind to Mdm2. This selective accumulation of free L5 and L11 is due to their mutual protection from proteasomal degradation. Furthermore, the endogenous, newly synthesized L5 and L11 continue to be imported into nucleoli even after nucleolar disruption and colocalize with Mdm2, p53, and promyelocytic leukemia protein. This suggests that the disrupted nucleoli may provide a platform for L5- and L11-dependent p53 activation, implying a role for the nucleolus in p53 activation by ribosomal biogenesis stress. These findings may have important implications with respect to understanding the pathogenesis of diseases caused by impaired ribosome biogenesis.

Ribosome Deficiency Protects Against ER Stress in Saccharomyces cerevisiae

Abstract: In Saccharomyces cerevisiae, 59 of the 78 ribosomal proteins are encoded by duplicated genes that, in most cases, encode identical or very similar protein products. However, different sets of ribosomal protein genes have been identified in screens for various phenotypes, including life span, budding pattern, and drug sensitivities. Due to potential suppressors of growth rate defects among this set of strains in the ORF deletion collection, we regenerated the entire set of haploid ribosomal protein gene deletion strains in a clean genetic background. The new strains were used to create double deletions lacking both paralogs, allowing us to define a set of 14 nonessential ribosomal proteins. Replicative life-span analysis of new strains corresponding to ORF deletion collection strains that likely carried suppressors of growth defects identified 11 new yeast replicative aging genes. Treatment of the collection of ribosomal protein gene deletion strains with tunicamycin revealed a significant correlation between slow growth and resistance to ER stress that was recapitulated by reducing translation of wild-type yeast with cycloheximide. Interestingly, enhanced tunicamycin resistance in ribosomal protein gene deletion mutants was independent of the unfolded protein response transcription factor Hac1. These data support a model in which reduced translation is protective against ER stress by a mechanism distinct from the canonical ER stress response pathway and further add to the diverse yet specific phenotypes associated with ribosomal protein gene deletions.

Proteomic analysis of age-dependent changes in protein solubility identifies genes that modulate lifespan.

Abstract: Summary While it is generally recognized that misfolding of specific proteins can cause late-onset disease, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometry-based proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)-insoluble conformations during aging in Caenorhabditis elegans. SDS-insoluble proteins extracted from young and aged C. elegans were chemically labeled by isobaric tagging for relative and absolute quantification (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDS-insoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDS-insoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Gene ontology analysis revealed highly significant enrichments for energy production and translation functions. Expression of genes encoding insoluble proteins observed in aged nematodes was knocked down using RNAi, and effects on lifespan were measured. 41% of genes tested were shown to extend lifespan after RNAi treatment, compared with 18% in a control group of genes. These data indicate that genes encoding proteins that become insoluble with age are enriched for modifiers of lifespan. This demonstrates that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that the accumulation of insoluble proteins with diverse functions may be a general feature of aging.

Ribosomal History Reveals Origins of Modern Protein Synthesis

Abstract: The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world.

Oxygenase-catalyzed ribosome hydroxylation occurs in prokaryotes and humans.

Abstract: The finding that oxygenase-catalyzed protein hydroxylation regulates animal transcription raises questions as to whether the translation machinery and prokaryotic proteins are analogously modified. Escherichia coli ycfD is a growth-regulating 2-oxoglutarate oxygenase catalyzing arginyl hydroxylation of the ribosomal protein Rpl16. Human ycfD homologs, Myc-induced nuclear antigen (MINA53) and NO66, are also linked to growth and catalyze histidyl hydroxylation of Rpl27a and Rpl8, respectively. This work reveals new therapeutic possibilities via oxygenase inhibition and by targeting modified over unmodified ribosomes.

CAG expansion induces nucleolar stress in polyglutamine diseases

Abstract: The cell nucleus is a major site for polyglutamine (polyQ) toxicity, but the underlying mechanisms involved have yet been fully elucidated. Here, we report that mutant RNAs that carry an expanded CAG repeat (expanded CAG RNAs) induce apoptosis by activating the nucleolar stress pathway in both polyQ patients and transgenic animal disease models. We showed that expanded CAG RNAs interacted directly with nucleolin (NCL), a protein that regulates rRNA transcription. Such RNA-protein interaction deprived NCL of binding to upstream control element (UCE) of the rRNA promoter, which resulted in UCE DNA hypermethylation and subsequently perturbation of rRNA transcription. The down-regulation of rRNA transcription induced nucleolar stress and provoked apoptosis by promoting physical interaction between ribosomal proteins and MDM2. Consequently, p53 protein was found to be stabilized in cells and became concentrated in the mitochondria. Finally, we showed that mitochondrial p53 disrupted the interaction between the antiapoptotic protein, Bcl-xL, and the proapoptotic protein, Bak, which then caused cytochrome c release and caspase activation. Our work provides in vivo evidence that expanded CAG RNAs trigger nucleolar stress and induce apoptosis via p53 and describes a polyQ pathogenic mechanism that involves the nucleolus.

Evidence for compensatory evolution of ribosomal proteins in response to rapid divergence of mitochondrial rRNA

Abstract: Rapid evolution of mitochondrial DNA (mtDNA) places intrinsic selective pressures on many nuclear genes involved in mitochondrial functions. Mitochondrial ribosomes, for example, are composed of mtDNA-encoded ribosomal RNAs (rRNAs) and a set of more than 60 nuclear-encoded ribosomal proteins (mRP) distinct from the cytosolic RPs (cRP). We hypothesized that the rapid divergence of mt-rRNA would result in rapid evolution of mRPs relative to cRPs, which respond to slowly evolving nuclear-encoded rRNA. In comparisons of rates of nonsynonymous and synonymous substitutions between a pair of divergent populations of the copepod Tigriopus californicus, we found that mRPs showed elevated levels of amino acid changes relative to cRPs. This pattern was equally strong at the interspecific level, between three pairs of sister species (Nasonia vitripennis vs. N. longicornis, Drosophila melanogaster vs. D. simulans, and Saccharomyces cerevisae vs. S. paradoxus). This high rate of mRP evolution may result in intergenomic incompatibilities between taxonomic lineages, and such incompatibilities could lead to dysfunction of mitochondrial ribosomes and the loss of fitness observed among interpopulation hybrids in T. californicus and interspecific hybrids in other species.

A TFIIIA-type zinc finger protein confers multiple abiotic stress tolerances in transgenic rice (Oryza sativa L.).

Abstract: The TFIIIA-type zinc finger transcription factors are involved in plant development and abiotic stress responses. Most TFIIIA-type zinc finger proteins are transcription repressors due to existence of an EAR-motif in their amino acid sequences. In this work, we found that ZFP182, a TFIIIA-type zinc finger protein, forms a homodimer in the nucleus and exhibits trans-activation activity in yeast cells. The deletion analysis indicated that a Leu-rich region at C-terminus is required for the trans-activation. Overexpression of ZFP182 significantly enhanced multiple abiotic stress tolerances, including salt, cold and drought tolerances in transgenic rice. Overexpression of ZFP182 promotes accumulation of compatible osmolytes, such as free proline and soluble sugars, in transgenic rice. ZFP182 activates the expression of OsP5CS encoding pyrroline-5-carboxylate synthetase and OsLEA3 under stress conditions, while OsDREB1A and OsDREB1B were regulated by ZFP182 under both normal and stress conditions. Interestingly, site-directed mutagenesis assay showed that DRE-like elements in ZFP182 promoter are involved in dehydration-induced expression of ZFP182. The yeast two-hybrid assay revealed that ZFP182 interacted with several ribosomal proteins including ZIURP1, an ubiquitin fused to ribosomal protein L40. The in vivo and in vitro interactions of ZFP182 and ZIURP1 were further confirmed by bimolecular fluorescence complementation and His pull-down assays. Our studies provide new clues in the understanding of the mechanisms for TFIIIA-type zinc finger transcription factor mediated stress tolerance and a candidate gene for improving stress tolerance in crops.

The plastid-specific ribosomal proteins of Arabidopsis thaliana can be divided into non-essential proteins and genuine ribosomal proteins

Abstract: Summary Plastid translation occurs on bacterial-type 70S ribosomes consisting of a large (50S) subunit and a small (30S) subunit. The vast majority of plastid ribosomal proteins have orthologs in bacteria. In addition, plastids also possess a small set of unique ribosomal proteins, so-called plastid-specific ribosomal proteins (PSRPs). The functions of these PSRPs are unknown, but, based on structural studies, it has been proposed that they may represent accessory proteins involved in translational regulation. Here we have investigated the functions of five PSRPs using reverse genetics in the model plant Arabidopsis thaliana. By analyzing T-DNA insertion mutants and RNAi lines, we show that three PSRPs display characteristics of genuine ribosomal proteins, in that down-regulation of their expression led to decreased accumulation of the 30S or 50S subunit of the plastid ribosomes, resulting in plastid translational deficiency. In contrast, two other PSRPs can be knocked out without visible or measurable phenotypic consequences. Our data suggest that PSRPs fall into two types: (i) PSRPs that have a structural role in the ribosome and are bona fide ribosomal proteins, and (ii) non-essential PSRPs that are not required for stable ribosome accumulation and translation under standard greenhouse conditions.

Ribosomal Route to Small Molecule Diversity

Abstract: The cyanobactin ribosomal peptide (RP) natural product pathway was manipulated to incorporate multiple tandem mutations and non-proteinogenic amino acids, using eight heterologous components simultaneously expressed in Escherichia coli. These studies reveal the potential of RPs for the rational synthesis of complex new small molecules over multiple-step biosynthetic pathways using simple genetic engineering.

Synchronizing nuclear import of ribosomal proteins with ribosome assembly.

Abstract: Ribosomal proteins are synthesized in the cytoplasm, before nuclear import and assembly with ribosomal RNA (rRNA). Little is known about coordination of nucleocytoplasmic transport with ribosome assembly. Here, we identify a transport adaptor, symportin 1 (Syo1), that facilitates synchronized coimport of the two 5S-rRNA binding proteins Rpl5 and Rpl11. In vitro studies revealed that Syo1 concomitantly binds Rpl5-Rpl11 and furthermore recruits the import receptor Kap104. The Syo1-Rpl5-Rpl11 import complex is released from Kap104 by RanGTP and can be directly transferred onto the 5S rRNA. Syo1 can shuttle back to the cytoplasm by interaction with phenylalanine-glycine nucleoporins. X-ray crystallography uncovered how the α-solenoid symportin accommodates the Rpl5 amino terminus, normally bound to 5S rRNA, in an extended groove. Symportin-mediated coimport of Rpl5-Rpl11 could ensure coordinated and stoichiometric incorporation of these proteins into pre-60S ribosomes.

Arabidopsis ribosomal proteins control developmental programs through translational regulation of auxin response factors

Abstract: Upstream ORFs are elements found in the 5′-leader sequences of specific mRNAs that modulate the translation of downstream ORFs encoding major gene products. In Arabidopsis, the translational control of auxin response factors (ARFs) by upstream ORFs has been proposed as a regulatory mechanism required to respond properly to complex auxin-signaling inputs. In this study, we identify and characterize the aberrant auxin responses in specific ribosomal protein mutants in which multiple ARF transcription factors are simultaneously repressed at the translational level. This characteristic lends itself to the use of these mutants as genetic tools to bypass the genetic redundancy among members of the ARF family in Arabidopsis. Using this approach, we were able to assign unique functions for ARF2, ARF3, and ARF6 in plant development.

The kinase activity of human Rio1 is required for final steps of cytoplasmic maturation of 40S subunits

Abstract: RIO proteins form a conserved family of atypical protein kinases. Humans possess three distinct RIO kinases—hRio1, hRio2, and hRio3, of which only hRio2 has been characterized with respect to its role in ribosomal biogenesis. Here we show that both hRio1 and hRio3, like hRio2, are associated with precursors of 40S ribosomal subunits in human cells. Furthermore, we demonstrate that depletion of hRio1 by RNA interference affects the last step of 18S rRNA maturation and causes defects in the recycling of several trans-acting factors (hEnp1, hRio2, hLtv1, hDim2/PNO1, and hNob1) from pre-40S subunits in the cytoplasm. Although the effects of hRio1 and hRio2 depletion are similar, we show that the two kinases are not fully interchangeable. Moreover, rescue experiments with a kinase-dead mutant of hRio1 revealed that the kinase activity of hRio1 is essential for the recycling of the endonuclease hNob1 and its binding partner hDim2 from cytoplasmic pre-40S. Kinase-dead hRio1 is trapped on pre-40S particles containing hDim2 and hNob1 but devoid of hEnp1, hLtv1, and hRio2. These data reveal a role of hRio1 in the final stages of cytoplasmic pre-40S maturation.

Multiple‐Site Labeling of Proteins with Unnatural Amino Acids

Abstract: The advent of efficient systems using genetic encoding for the site-specific incorporation of unnatural amino acids (UAA) into proteins has opened countless new possibilities for studying the structure, dynamics, and interactions of proteins. In particular, orthogonal pairs of amber suppressor tRNA (MjtRNA) and tyrosyl-tRNA synthetase (MjTyrRS) of Methanocaldococcus jannaschii have been evolved that specifically recognize amber stop codons for the incorporation of over 40 different UAAs. Although these systems can produce mutant protein with yields as high as for the wildtype, the protein yields are strongly context dependent. Thus, the presence of a single amber stop codon in the gene of a target protein can lead to unacceptably poor expression yields. Many efforts have been directed at biasing the competition between the Escherichia coli release factor RF1 that recognizes the amber stop codon and the suppressor tRNA in favor of the production of full-length protein. Thus, the incorporation of UAAs can be enhanced by omission of RF1 from a cell-free (CF) synthesis system reconstituted from the individually purified enzyme components. CF systems were also used to eliminate RF1 with anti-RF1 antibodies, or by deploying an RNA aptamer against RF1. Unfortunately, all these approaches are affordable only for small-scale sample preparations, and protein yields can be compromised by antibodies, while aptamers still yield truncated protein as the predominant product. Heat-shock inactivation of a thermosensitive mutant of RF1 in a low-yield E. coli CF system increased the incorporation efficiencies of UAAs to at most 75% (< 50% in most cases), while the fidelity of translation was compromised by prolonged heat treatment. Depletion of tagged RF1 from a cell-free S30 extract by affinity chromatography was reported, but the identity of the tag was not revealed and anti-RF1 antibodies were required for complete elimination. In a different approach, truncation of the ribosomal protein L11 was shown to weaken the binding of RF1, but the resulting protein yields with UAAs were only moderately enhanced. Initial efforts to produce RF1-deficient E. coli strains led to compromised strains that depended on unnatural amino acids for growth (making protein expression expensive) or strains that were not entirely independent of RF1. Recently, an enhanced version of release factor RF2 enabled the production of an RF1-free E. coli DH10b strain and the incorporation of UAAs at multiple sites in vivo, but the protein yields obtained with UAAs were significantly reduced compared with those obtained with the natural amino acid (tyrosine). In view of the cost of many of the most attractive UAAs and the difficulty to tailor the concentrations of the aminoacyl-tRNA synthetase (RS) and suppressor tRNA in in vivo expression systems to the requirements of different UAAs and incorporation sites, we developed a continuous exchange cell-free (CECF) system that allows facile, inexpensive, and complete removal of the release factor RF1 from an S30 extract derived from the widely used high-yielding and protease-deficient E. coli strain BL21 Star (DE3) (Invitrogen). The approach relies on replacing wild-type RF1 by a mutant with a C-terminal affinity tag consisting of three consecutive chitin-binding domains (RF1-CBD3) for selective removal by filtration through a chitin column after production of an S30 extract in the usual way. The chitin-binding-domain tag allows the removal of RF1 under conditions that maintain the full activity of the S30 extract and at the same time delivers dramatically improved incorporation yields of difficult UAAs at difficult positions, suppresses the production of truncated protein, and allows the incorporation of UAAs at multiple sites in the same protein. Conveniently, the modified strain is fully compatible with protein expression from pET vectors, which are the most frequently used vectors for protein production in structural biology. The yields of wild-type protein obtained with chitintreated (S30 ) and untreated (S30) extracts were indistinguishable from the yields obtained with S30 extracts prepared from the original BL21 Star (DE3) strain (Figure S3 in the Supporting Information). To assess and optimize the expression yields with UAAs without having to purify the proteins, we used the MjtRNA/MjTyrRS pair evolved for incorporation of the fluorescent UAA l-(7-hydroxy-coumarin-4-yl)ethylglycine (Hco). Optimization employed the West Nile virus NS2B-NS3 protease (WNVpro), a 27 kDa protein that is also an established drug target. Optimization of the concentration of aminoacyl-tRNA syn[*] Dr. K. V. Loscha, A. J. Herlt, Dr. R. Qi, Dr. T. Huber, Dr. K. Ozawa, Prof. G. Otting Research School of Chemistry, The Australian National University Canberra ACT 0200 (Australia) E-mail: gottfried.otting@anu.edu.au Homepage: http://rsc.anu.edu.au/~go/

Inactivation of Ribosomal Protein Genes in Bacillus subtilis Reveals Importance of Each Ribosomal Protein for Cell Proliferation and Cell Differentiation

Abstract: Among the 57 genes that encode ribosomal proteins in the genome of Bacillus subtilis, a Gram-positive bacterium, 50 genes were targeted by systematic inactivation Individual deletion mutants of 16 ribosomal proteins (L1, L9, L15, L22, L23, L28, L29, L32, L331, L332, L34, L35, L36, S6, S20, and S21) were obtained successfully In conjunction with previous reports, 22 ribosomal proteins have been shown to be nonessential in B subtilis, at least for cell proliferation Although several mutants that harbored a deletion of a ribosomal protein gene did not show any significant differences in any of the phenotypes that were tested, various mutants showed a reduced growth rate and reduced levels of 70S ribosomes compared with the wild type In addition, severe defects in the sporulation frequency of the ΔrplA (L1) mutant and the motility of the ΔrpsU (S21) mutant were observed These data provide the first evidence in B subtilis that L1 and S21 are required for the progression of cellular differentiation

Cryo-EM structures of Arx1 and maturation factors Rei1 and Jjj1 bound to the 60S ribosomal subunit

Abstract: Cryo-EM studies of the 60S ribosomal subunit reconstituted with biogenesis factor Arx1 and Rei1 and Jji1 suggest that, in addition to its role during pre-60S nuclear export, Arx1 shields the polypeptide tunnel-exit region and inhibits the premature association of nascent chain–processing factors, whereas Rei1 and Jjj1, which have been implicated in Arx1 recycling, may function in Arx1 release from the tunnel exit.

mTOR signaling regulates the processing of pre-rRNA in human cells

Abstract: Signaling through the mammalian target of rapamycin, complex 1 (mTORC1), positively regulates the transcription of ribosomal RNA (rRNA) and the synthesis of ribosomal proteins, thereby promoting the complex process of ribosome biogenesis. The major rRNAs are transcribed as a single precursor, which must be processed to create the 5.8S, 18S and 28S rRNAs. We used a new non-radioactive labeling approach to study the effects of rapamycin, an inhibitor of mTORC1, on rRNA synthesis. Rapamycin not only impaired synthesis of new 18S, 28S or 5S rRNA but also induced their decay. This prompted us to examine the effects of rapamycin on rRNA processing. We show that rapamycin also interferes with the processing events that generate 18S and 28S rRNA. rRNA transcription and processing occur in regions of the nucleus known as nucleoli. We find that the mTORC1 components raptor and mTOR are both present in nucleoli, where they may regulate rRNA maturation events. While rapamycin has no effect on overall nucleolar morphology or its proteome, it does induce loss of mTOR and raptor from them. These data show that mTORC1 is located in nucleoli where it acts to regulate events involved in ribosome biogenesis including the maturation of rRNA molecules.

p53 -Dependent and -Independent Nucleolar Stress Responses.

Abstract: The nucleolus has emerged as a cellular stress sensor and key regulator of p53-dependent and -independent stress responses. A variety of abnormal metabolic conditions, cytotoxic compounds, and physical insults induce alterations in nucleolar structure and function, a situation known as nucleolar or ribosomal stress. Ribosomal proteins, including RPL11 and RPL5, become increasingly bound to the p53 regulatory protein MDM2 following nucleolar stress. Ribosomal protein binding to MDM2 blocks its E3 ligase function leading to stabilization and activation of p53. In this review we focus on a number of novel regulators of the RPL5/RPL11-MDM2-p53 complex including PICT1 (GLTSCR2), MYBBP1A, PML and NEDD8. p53-independent pathways mediating the nucleolar stress response are also emerging and in particular the negative control that RPL11 exerts on Myc oncoprotein is of importance, given the role of Myc as a master regulator of ribosome biogenesis. We also briefly discuss the potential of chemotherapeutic drugs that specifically target RNA polymerase I to induce nucleolar stress.

An Essential Pentatricopeptide Repeat Protein Facilitates 5′ Maturation and Translation Initiation of rps3 mRNA in Maize Mitochondria

Abstract: Pentatricopeptide repeat (PPR) proteins are members of one of the largest nucleus-encoded protein families in plants. Here, we describe the previously uncharacterized maize (Zea mays) PPR gene, MPPR6, which was isolated from a Mutator-induced collection of maize kernel mutants by a cDNA-based forward genetic approach. Identification of a second mutant allele and cosegregation analysis confirmed correlation with the mutant phenotype. Histological investigations revealed that the mutation coincides with abnormities in the transfer cell layer, retardation of embryo development, and a considerable reduction of starch level. The function of MPPR6 is conserved across a wide phylogenetic distance as revealed by heterologous complementation of the Arabidopsis thaliana mutant in the orthologous APPR6 gene. MPPR6 appeared to be exclusively present in mitochondria. RNA coimmunoprecipitation and in vitro binding studies revealed a specific physical interaction of MPPR6 with the 5′ untranslated region of ribosomal protein S3 (rps3) mRNA. Mapping of transcript termini showed specifically extended rps3 5′ ends in the mppr6 mutant. Considerable reduction of mitochondrial translation was observed, indicating loss of RPS3 function. This is consistent with the appearance of truncated RPS3 protein lacking the N terminus in mppr6. Our results suggest that MPPR6 is directly involved in 5′ maturation and translation initiation of rps3 mRNA.

RsfA (YbeB) Proteins Are Conserved Ribosomal Silencing Factors

Abstract: The YbeB (DUF143) family of uncharacterized proteins is encoded by almost all bacterial and eukaryotic genomes but not archaea. While they have been shown to be associated with ribosomes, their molecular function remains unclear. Here we show that YbeB is a ribosomal silencing factor (RsfA) in the stationary growth phase and during the transition from rich to poor media. A knock-out of the rsfA gene shows two strong phenotypes: (i) the viability of the mutant cells are sharply impaired during stationary phase (as shown by viability competition assays), and (ii) during transition from rich to poor media the mutant cells adapt slowly and show a growth block of more than 10 hours (as shown by growth competition assays). RsfA silences translation by binding to the L14 protein of the large ribosomal subunit and, as a consequence, impairs subunit joining (as shown by molecular modeling, reporter gene analysis, in vitro translation assays, and sucrose gradient analysis). This particular interaction is conserved in all species tested, including Escherichia coli, Treponema pallidum, Streptococcus pneumoniae, Synechocystis PCC 6803, as well as human mitochondria and maize chloroplasts (as demonstrated by yeast two-hybrid tests, pull-downs, and mutagenesis). RsfA is unrelated to the eukaryotic ribosomal anti-association/60S-assembly factor eIF6, which also binds to L14, and is the first such factor in bacteria and organelles. RsfA helps cells to adapt to slow-growth/stationary phase conditions by down-regulating protein synthesis, one of the most energy-consuming processes in both bacterial and eukaryotic cells.

Ter-dependent stress response systems: novel pathways related to metal sensing, production of a nucleoside-like metabolite, and DNA-processing

Abstract: The mode of action of the bacterial ter cluster and TelA genes, implicated in natural resistance to tellurite and other xenobiotic toxic compounds, pore-forming colicins and several bacteriophages, has remained enigmatic for almost two decades. Using comparative genomics, sequence-profile searches and structural analysis we present evidence that the ter gene products and their functional partners constitute previously underappreciated, chemical stress response and anti-viral defense systems of bacteria. Based on contextual information from conserved gene neighborhoods and domain architectures, we show that the ter gene products and TelA lie at the center of membrane-linked metal recognition complexes with regulatory ramifications encompassing phosphorylation-dependent signal transduction, RNA-dependent regulation, biosynthesis of nucleoside-like metabolites and DNA processing. Our analysis suggests that the multiple metal-binding and non-binding TerD paralogs and TerC are likely to constitute a membrane-associated complex, which might also include TerB and TerY, and feature several, distinct metal-binding sites. Versions of the TerB domain might also bind small molecule ligands and link the TerD paralog-TerC complex to biosynthetic modules comprising phosphoribosyltransferases (PRTases), ATP grasp amidoligases, TIM-barrel carbon-carbon lyases, and HAD phosphoesterases, which are predicted to synthesize novel nucleoside-like molecules. One of the PRTases is also likely to interact with RNA by means of its Pelota/Ribosomal protein L7AE-like domain. The von Willebrand factor A domain protein, TerY, is predicted to be part of a distinct phosphorylation switch, coupling a protein kinase and a PP2C phosphatase. We show, based on the evidence from numerous conserved gene neighborhoods and domain architectures, that both the TerB and TelA domains have been linked to diverse lipid-interaction domains, such as two novel PH-like and the Coq4 domains, in different bacteria, and are likely to comprise membrane-associated sensory complexes that might additionally contain periplasmic binding-protein-II and OmpA domains. We also show that the TerD and TerB domains and the TerY-associated phosphorylation system are functionally linked to many distinct DNA-processing complexes, which feature proteins with SWI2/SNF2 and RecQ-like helicases, multiple AAA+ ATPases, McrC-N-terminal domain proteins, several restriction endonuclease fold DNases, DNA-binding domains and a type-VII/Esx-like system, which is at the center of a predicted DNA transfer apparatus. These DNA-processing modules and associated genes are predicted to be involved in restriction or suicidal action in response to phages and possibly repairing xenobiotic-induced DNA damage. In some eukaryotes, certain components of the ter system appear to be recruited to function in conjunction with the ubiquitin system and calcium-signaling pathways.