Large Ribosomal Subunit Protein Research Articles

A ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mRNAs

Initiation is the primary target of translational control for all organisms. Regulation of eukaryotic translation is traditionally thought to occur through initiation factors and RNA structures. Here, we characterize a transcript-specific translation initiation mechanism that is mediated by the ribosome. By studying vesicular stomatitis virus (VSV), we identify the large ribosomal subunit protein rpL40 as requisite for VSV cap-dependent translation but not bulk cellular or internal ribosome entry site-driven translation. This requirement is conserved among members of the order Mononegavirales, including measles virus and rabies virus. Polysome analyses and in vitro reconstitution of initiation demonstrate that rpL40 is required for 80S formation on VSV mRNAs through a cis-regulatory element. Using deep sequencing, we further uncover a subset of cellular transcripts that are selectively sensitive to rpL40 depletion, suggesting VSV may have usurped an endogenous translation pathway. Together, these findings demonstrate that the ribosome acts as a translational regulator outside of its catalytic role during protein synthesis.

Insights Into Diagnosis and Etiology of Diamond Blackfan Anemia by Analysis of Pre-rRNA Processing

AbstractAbstract 3476Most cases of Diamond Blackfan anemia are caused by haploinsufficiency for genes encoding proteins of the large or small ribosomal subunit. All of the ribosomal proteins affected in DBA are essential components of the ribosome required for the assembly of their respective subunits, including processing of the primary pre-rRNA transcript to mature 18S, 5.8S, and 28S rRNAs. Pre-rRNA processing signatures associated with ribosomal protein haploinsufficiency demonstrate a role for individual proteins in subunit assembly and can differ depending on which protein is affected. A facile pre-rRNA processing assay that can discriminate between loss of function alleles for different ribosomal protein genes would be an invaluable aide to DBA diagnosis and gene discovery efforts. Such an assay could also provide insight into different aspects of DBA pathophysiology. [Display omitted] We have developed a robust procedure to assess pre-rRNA processing patterns in activated lymphocytes from the peripheral blood of patients with known or suspected Diamond Blackfan anemia. This assay typically involves the electrophoretic separation of total RNA from activated lymphocytes followed by Northern blotting with various hybridization probes to different rRNA precursors. Using this assay, we have found a common 32S pre-rRNA processing intermediate present in RNA from DBA patients with mutations in virtually all known large ribosomal subunit genes. This 32S pre-rRNA can be visualized in situ in gels stained with ethidium bromide (see accompanying figure) greatly simplifying the identification of large subunit ribosomal protein genes harboring loss of function mutations. As more and more ribosomal protein genes are identified within the DBA population, it has become increasingly important to distinguish between variants that affect ribosomal protein function and benign polymorphisms. Therefore, an analysis of pre-rRNA processing can be used to identify causative genes in patients with complex genotypes where sequence variants are found in more than one ribosomal protein gene. We analyzed a patient with variants in genes encoding RPS19 and RPL11, two known DBA genes, plus a deletion containing the RPL31 gene that has not been previously linked to DBA. Data analyses overwhelmingly support the deletion of RPL31 as the causative lesion in this patient and identify RPL31 as a new DBA gene. Analysis of pre-rRNA processing can also guide additional gene discovery efforts. We performed pre-rRNA processing studies on two patients lacking mutations in known DBA genes. In one case, we observed a clear defect in the18S rRNA pathway, implicating a gene involved in the biogenesis of the small ribosomal subunit, whereas in a second patient there is no evidence of a ribosome biogenesis defect suggesting that the underlying mutation may not affect ribosome synthesis. These results will help guide further efforts to identify causative genes in this patient cohort. Finally, we have used pre-rRNA processing patterns to begin to examine the mechanisms underlying remission in DBA patients. To date, we have examined two samples from DBA patients in remission and showed the ribosome synthesis defect is retained even while in remission for one of these patients, whereas the pre-rRNA processing defect has resolved in the other patient. Disclosures:No relevant conflicts of interest to declare.

Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication

Arenaviruses are responsible for acute hemorrhagic fevers with high mortality and pose significant threats to public health and biodefense. These enveloped negative-sense RNA viruses replicate in the cell cytoplasm and express four proteins. To better understand how these proteins insinuate themselves into cellular processes to orchestrate productive viral replication, we have identified and characterized novel cytosolic structures involved in arenavirus replication and transcription. In cells infected with the nonpathogenic Tacaribe virus or the attenuated Candid#1 strain of Junín virus, we find that newly synthesized viral RNAs localize to cytosolic puncta containing the nucleoprotein (N) of the virus. Density gradient centrifugation studies reveal that these replication-transcription complexes (RTCs) are associated with cellular membranes and contain full-length genomic- and antigenomic-sense RNAs. Viral mRNAs segregate at a higher buoyant density and are likewise scant in immunopurified RTCs, consistent with their translation on bulk cellular ribosomes. In addition, confocal microscopy analysis reveals that RTCs contain the lipid phosphatidylinositol-4-phosphate and proteins involved in cellular mRNA metabolism, including the large and small ribosomal subunit proteins L10a and S6, the stress granule protein G3BP1, and a subset of translation initiation factors. Elucidating the structure and function of RTCs will enhance our understanding of virus-cell interactions that promote arenavirus replication and mitigate against host cell immunity. This knowledge may lead to novel intervention strategies to limit viral virulence and pathogenesis.

Use of the novel technique of analytical ultracentrifugation with fluorescence detection system identifies a 77S monosomal translation complex

A fundamental problem in proteomics is the identification of protein complexes and their components. We have used analytical ultracentrifugation with a fluorescence detection system (AU-FDS) to precisely and rapidly identify translation complexes in the yeast Saccharomyces cerevisiae. Following a one-step affinity purification of either poly(A)-binding protein (PAB1) or the large ribosomal subunit protein RPL25A in conjunction with GFP-tagged yeast proteins/RNAs, we have detected a 77S translation complex that contains the 80S ribosome, mRNA, and components of the closed-loop structure, eIF4E, eIF4G, and PAB1. This 77S structure, not readily observed previously, is consistent with the monosomal translation complex. The 77S complex abundance decreased with translational defects and following the stress of glucose deprivation that causes translational stoppage. By quantitating the abundance of the 77S complex in response to different stress conditions that block translation initiation, we observed that the stress of glucose deprivation affected translation initiation primarily by operating through a pathway involving the mRNA cap binding protein eIF4E whereas amino acid deprivation, as previously known, acted through the 43S complex. High salt conditions (1M KCl) and robust heat shock acted at other steps. The presumed sites of translational blockage caused by these stresses coincided with the types of stress granules, if any, which are subsequently formed.

Berberine enhances defects in the establishment of leaf polarity in asymmetric leaves1 and asymmetric leaves2 of Arabidopsis thaliana

Leaves develop as flat lateral organs from the indeterminate shoot apical meristem. The establishment of polarity along three-dimensional axes, proximal–distal, medial–lateral, and adaxial–abaxial axes, is crucial for the growth of normal leaves. The mutations of ASYMMETRIC LEAVES1 (AS1) and AS2 of Arabidopsis thaliana cause defects in repression of the indeterminate state and the establishment of axis formation in leaves. Although many mutations have been identified that enhance the adaxial–abaxial polarity defects of as1 and as2 mutants, the roles of the causative genes in leaf development are still unknown. In this study, we found that wild-type plants treated with berberine produced pointed leaves, which are often observed in the single mutants that enhance phenotypes of as1 and as2 mutants. The berberine-treated as1 and as2 mutants formed abaxialized filamentous leaves. Berberine, an isoquinoline alkaloid compound naturally produced in various plant sources, has a growth inhibitory effect on plants that do not produce berberine. We further showed that transcript levels of meristem-specific class 1 KNOX homeobox genes and abaxial determinant genes were increased in berberine-treated as1 and as2. Berberine treated plants carrying double mutations of AS2 and the large subunit ribosomal protein gene RPL5B showed more severe defects in polarity than did the as2 single mutant plants. We suggest that berberine inhibits (a) factor(s) that might be required for leaf adaxial cell differentiation through a pathway independent of AS1 and AS2. Multiple pathways might play important roles in the formation of flat symmetric leaves.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-012-9929-7) contains supplementary material, which is available to authorized users.

Ribosome Distribution in HeLa Cells during the Cell Cycle

In this study, we employed a surface-specific antibody against the large ribosome subunit to investigate the distribution of ribosomes in cells during the cell cycle. The antibody, anti-L7n, was raised against an expansion segment (ES) peptide from the large subunit ribosomal protein L7, and its ribosome-surface specificity was evident from the positive immuno-reactivity of ribosome particles and the detection of 60 S immune-complex formation by an immuno-electron microscopy. Using immunofluorescent staining, we have microscopically revealed that ribosomes are dispersed in the cytoplasm of cells throughout all phases of the cell cycle, except at the G2 phase where ribosomes show a tendency to gather toward the nuclear envelope. The finding in G2 cells was confirmed by electron microscopy using a morphometric assay and paired t test. Furthermore, further observations have shown that ribosomes are not distributed immune-fluorescently with nuclear envelope markers including the nuclear pore complex, the integral membrane protein gp210, the inner membrane protein lamin B2, and the endoplasm reticulum membrane during cell division we propose that the mechanism associated with ribosome segregation into daughter cells could be independent of the processes of disassembly and reassembly of the nuclear envelope.

Histone Deacetylase 6 Associates With Ribosomes and Regulates De Novo Protein Translation During Arsenite Stress

Histone deacetylase 6 (HDAC6) is known as a cytoplasmic enzyme that regulates cell migration, cell adhesion, and degradation of misfolded proteins by deacetylating substrates such as α-tubulin and Hsp90. When HaCaT keratinocytes were exposed to 1-200μM sodium arsenite, we observed perinuclear localization of HDAC6 within 30 min. Although the overall level of HDAC6 protein did not change, sodium arsenite caused an increase of HDAC6 in ribosomal fractions. Separation of ribosomal subunits versus intact ribosomes or polysomes indicated that HDAC6 was mainly detected in 40/43S fractions containing the small ribosomal subunit in untreated cells but was associated with 40/43S and 60/80S ribosomal fractions in arsenite-treated cells. Immunocytochemistry studies revealed that arsenite caused colocalization of HDAC6 with the ribosomal large and small subunit protein L36a and S6. Both L36a and S6 were detected in the immunocomplex of HDAC6 isolated from arsenite-treated cells. The observed physical interaction of HDAC6 with ribosomes pointed to a role of HDAC6 in stress-induced protein translation. Among arsenite stress-induced proteins, de novo Nrf2 protein translation was inhibited by Tubastatin A. These data demonstrate that HDAC6 was recruited to ribosomes, physically interacted with ribosomal proteins, and regulated de novo protein translation in keratinocytes responding to arsenite stress.

Quantum mechanical studies of lincosamides.

Lincosamides are a class of antibiotics used both in clinical and veterinary practice for a wide range of pathogens. This group of drugs inhibits the activity of the bacterial ribosome by binding to the 23S RNA of the large ribosomal subunit and blocking protein synthesis. Currently, three X-ray structures of the ribosome in complex with clindamycin are available in the Protein Data Bank, which reveal that there are two distinct conformations of the pyrrolidinyl propyl group of the bound clindamycin. In this work, we used quantum mechanical methods to investigate the probable conformations of clindamycin in order to explain the two binding modes in the ribosomal 23S RNA. We studied three lincosamide antibiotics: clindamycin, lincomycin, and pirlimycin at the B3LYP level with the 6-31G** basis set. The focus of our work was to connect the conformational landscape and electron densities of the two clindamycin conformers found experimentally with their physicochemical properties. For both functional conformers, we applied natural bond orbital (NBO) analysis and the atoms in molecules (AIM) theory, and calculated the NMR parameters. Based on the results obtained, we were able to show that the structure with the intramolecular hydrogen bond C=O…H–O is the most stable conformer of clindamycin. The charge transfer between the pyrrolidine-derivative ring and the six-atom sugar (methylthiolincosamide), which are linked via an amide bond, was found to be the dominant factor influencing the high stability of this conformer. FigureMolecular graph of more stable conformer of clindamycin.

Knockdown of p53 Improves Hematopoietic and Erythroid Differentiation Defects in Mouse Embryonic Stem Cell Models of Diamond Blackfan Anemia

Abstract 728 Background:Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by red blood cell hypoplasia, congenital anomalies and cancer predisposition. The disease has been shown to result from haploinsufficiency of large or small ribosomal subunit proteins. The p53 pathway, known to be activated by abortive ribosome assembly, may play a role in the pathogenesis of DBA. Previously, we described murine embryonic stem (ES) cell models of DBA and reported hematopoietic and erythroid defects common to Rps19- and Rpl5-deficient cell lines, as well as a primitive erythropoiesis defect unique to an Rpl5-deficient cell line [Blood 116(21), 877, 2010]. Methods:We studied the effects of p53 knockdown on hematopoiesis in our Rps19- and Rpl5-mutant murine ES cell lines created by gene trap technology. Small interfering RNA (siRNA) targeting p53 was transfected into mutant cell lines at the ES cell stage. A non-targeting siRNA served as a negative control. After 24 hours, cells were plated into methylcellulose medium with fetal bovine serum and stem cell factor (SCF) to generate embryoid bodies (EBs). On day 7, EBs were fed with medium containing SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (epo). EBs were scored on day 12 for total quantity and hematopoietic percentage. For secondary differentiation into primitive erythroid colonies, day 5 EBs were disrupted, and individual cells were suspended in a methylcellulose medium containing fetal bovine plasma-derived serum and epo. Primitive erythroid colonies were counted on day 7 of culture. Definitive hematopoiesis assays were performed by disruption of day 7 EBs, followed by suspension of cells in methylcellulose medium containing SCF, IL-3, IL-6 and epo. Definitive hematopoietic colonies were counted on day 10. In an independent set of experiments, we created an isogenic pair of wild-type and mutant DBA ES cells by electroporation of another Rps19- mutant line with a plasmid vector expressing wild-type Rps19 cDNA (wild-type) or an empty vector (mutant). Results:By immunoblot assays, we detected an increased amount of p53 protein in our Rps19-and Rpl5- mutant cell lines. Following p53 siRNA transfection, we confirmed 82–95% reduction in p53 expression by quantitative PCR, whereas ES cells transfected with non-targeting siRNA did not alter p53 expression. For both Rps19- and Rpl5- mutants, previously shown to have EB formation defects in comparison to parental controls, p53 knockdown significantly improved EB formation, especially hematopoietic-type EBs, compared to mutants treated with non-targeting siRNA. In addition, p53 knockdown in both mutants reversed the definitive hematopoiesis defect by increasing the ratio of erythroid colony to myeloid colony formation. Furthermore, p53 siRNA transfection of the Rpl5- mutant rescued the primitive erythropoiesis defect previously shown by us.To further explore the mechanistic basis of our findings, we additionally tested the effects of Rpl11 knockdown in our DBA models. The presence of free RPL11 secondary to abortive ribosome assembly has been hypothesized to be responsible for increased p53 in DBA by binding to and inhibiting the p53 inhibitor HDM2 (Mdm2 in mice). Transfection of Rpl11 siRNA into both Rps19- and Rpl5-mutant cell lines at the ES cell stage led to a marked reduction in EB formation, compared to cells transfected with non-targeting siRNA.Finally, we also extended our analysis to an isogenic pair of Rps19- wild-type and mutant cells. In the mutant line, we confirmed a 5–8 fold rescue of EB formation with siRNA targeting p53 when compared to the non-targeting siRNA. In order to clarify the role of two major downstream effectors of p53, siRNA targeting either Bax or p21 was transfected into the mutant cell line. Surprisingly, neither siRNA was able to rescue the EB formation defect of the mutant cells. Conclusions:(1) Knockdown of p53 markedly improves erythroid defects of Rps19- and Rpl5-deficient murine ES cell models of DBA, while inhibition of the upstream target Rpl11 causes significant toxicity to cells already haploinsufficient for Rps19 or Rpl5. (2) Knockdown of either Bax or p21 does not recapitulate knockdown of p53, suggesting that neither plays a significant individual role in downstream signaling from p53 in this model. (3) Further exploration of the p53 pathway may provide insights into the pathogenesis of DBA and identify new targets for therapy. Disclosures:No relevant conflicts of interest to declare.

The Ribosomal L1 Protuberance in Yeast Is Methylated on a Lysine Residue Catalyzed by a Seven-β-strand Methyltransferase

Modification of proteins of the translational apparatus is common in many organisms. In the yeast Saccharomyces cerevisiae, we provide evidence for the methylation of Rpl1ab, a well conserved protein forming the ribosomal L1 protuberance of the large subunit that functions in the release of tRNA from the exit site. We show that the intact mass of Rpl1ab is 14 Da larger than its calculated mass with the previously described loss of the initiator methionine residue and N-terminal acetylation. We determined that the increase in mass of yeast Rpl1ab is consistent with the addition of a methyl group to lysine 46 using top-down mass spectrometry. Lysine modification was confirmed by detecting (3)H-N-ε-monomethyllysine in hydrolysates of Rpl1ab purified from yeast cells radiolabeled in vivo with S-adenosyl-l-[methyl-(3)H]methionine. Mass spectrometric analysis of intact Rpl1ab purified from 37 deletion strains of known and putative yeast methyltransferases revealed that only the deletion of the YLR137W gene, encoding a seven-β-strand methyltransferase, results in the loss of the +14-Da modification. We expressed the YLR137W gene as a His-tagged protein in Escherichia coli and showed that it catalyzes N-ε-monomethyllysine formation within Rpl1ab on ribosomes from the ΔYLR137W mutant strain lacking the methyltransferase activity but not from wild-type ribosomes. We also showed that the His-tagged protein could catalyze monomethyllysine formation on a 16-residue peptide corresponding to residues 38-53 of Rpl1ab. We propose that the YLR137W gene be given the standard name RKM5 (ribosomal lysine (K) methyltransferase 5). Orthologs of RKM5 are found only in fungal species, suggesting a role unique to their survival.

Untangling the Phenotypic Heterogeneity of Diamond Blackfan Anemia

Diamond Blackfan anemia (DBA) is a lineage-selective inherited bone marrow failure syndrome characterized primarily by anemia and physical malformations. Recent advances in identifying the genetic abnormalities underlying DBA have demonstrated involvement of genes encoding both large (RPL) and small (RPS) ribosomal subunit proteins, including mutations of RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26 in 50% to 60% of affected patients. Despite significant progress, identification of gene abnormalities in the remaining patients remains an important question since present data suggest that mutations in other members of the ribosomal protein gene complement do not explain those cases without an identified genetic lesion in these genes. Genetic studies have also raised new questions with the recognition of substantial variability in the manifestations of DBA, ranging from ribosomal protein mutations in otherwise asymptomatic individuals to those with classic severe red blood cell aplasia with characteristic malformations, at times within the same kindred. In this review, we summarize the genetic basis of DBA and discuss mechanisms by which the phenotype of DBA might be modified.

Formation of Antiviral Cytoplasmic Granules during Orthopoxvirus Infection

Vaccinia virus (VV) mutants lacking the double-stranded RNA (dsRNA)-binding E3L protein (ΔE3L mutant VV) show restricted replication in most cell types, as dsRNA produced by VV activates protein kinase R (PKR), leading to eIF2α phosphorylation and impaired translation initiation. Here we show that cells infected with ΔE3L mutant VV assemble cytoplasmic granular structures which surround the VV replication factories at an early stage of the nonproductive infection. These structures contain the stress granule-associated proteins G3BP, TIA-1, and USP10, as well as poly(A)-containing RNA. These structures lack large ribosomal subunit proteins, suggesting that they are translationally inactive. Formation of these punctate structures correlates with restricted replication, as they occur in >80% of cells infected with ΔE3L mutant VV but in only 10% of cells infected with wild-type VV. We therefore refer to these structures as antiviral granules (AVGs). Formation of AVGs requires PKR and phosphorylated eIF2α, as mouse embryonic fibroblasts (MEFs) lacking PKR displayed reduced granule formation and MEFs lacking phosphorylatable eIF2α showed no granule formation. In both cases, these decreased levels of AVG formation correlated with increased ΔE3L mutant VV replication. Surprisingly, MEFs lacking the AVG component protein TIA-1 supported increased replication of ΔE3L mutant VV, despite increased eIF2α phosphorylation and the assembly of AVGs that lacked TIA-1. These data indicate that the effective PKR-mediated restriction of ΔE3L mutant VV replication requires AVG formation subsequent to eIF2α phosphorylation. This is a novel finding that supports the hypothesis that the formation of subcellular protein aggregates is an important component of the successful cellular antiviral response.

The role of polymorphisms in the spliced leader addition domain in determining promoter activity in Brugia malayi

Previous studies of Brugia malayi promoters have suggested that they are unusual in that they lack the CAAT or TATAA boxes that are often emblematic of eucaryotic core promoter domains. Instead, the region surrounding the spliced leader (SL) addition site appears to function as the core promoter domain in B. malayi. To test the hypothesis that polymorphisms in this SL addition domain are important determinants of promoter activity, a series of domain swap mutants were prepared replacing the SL addition domain of the B. malayi 13kDa large subunit ribosomal protein (BmRPL13) with those of other ribosomal protein (RP) promoters exhibiting a wide range of activities. These constructs were then tested for promoter activity in a homologous transient transfection system. On average, polymorphisms in the SL addition domain were found to be responsible for 80% of the variation in promoter activity exhibited by the RP promoters tested. Essentially all of this effect could be attributable to polymorphisms in the 10nt located directly upstream of the SL addition site. A comparison of the sequence of this domain to the promoter activity exhibited by the domain swap mutants suggested that promoter activity was related to the number of T residues present in the coding strand of the upstream domain. Confirming this, mutation of the upstream domain of the promoter of the BmRPS4 gene to a homogeneous stretch of 10 T residues resulted in a significant increase in promoter activity.

MTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide

The mechanisms that couple translation and protein processing are poorly understood in higher eukaryotes. Although mammalian target of rapamycin (mTOR) complex 1 (mTORC1) controls translation initiation, the function of mTORC2 in protein synthesis remains to be defined. In this study, we find that mTORC2 can colocalize with actively translating ribosomes and can stably interact with rpL23a, a large ribosomal subunit protein present at the tunnel exit. Exclusively during translation of Akt, mTORC2 mediates phosphorylation of the nascent polypeptide at the turn motif (TM) site, Thr450, to avoid cotranslational Akt ubiquitination. Constitutive TM phosphorylation occurs because the TM site is accessible, whereas the hydrophobic motif (Ser473) site is concealed in the ribosomal tunnel. Thus, mTORC2 can function cotranslationally by phosphorylating residues in nascent chains that are critical to attain proper conformation. Our findings reveal that mTOR links protein production with quality control.

RRP1B Targets PP1 to Mammalian Cell Nucleoli and Is Associated with Pre-60S Ribosomal Subunits

A pool of protein phosphatase 1 (PP1) accumulates within nucleoli and accounts for a large fraction of the serine/threonine protein phosphatase activity in this subnuclear structure. Using a combination of fluorescence imaging with quantitative proteomics, we mapped the subnuclear localization of the three mammalian PP1 isoforms stably expressed as GFP-fusions in live cells and identified RRP1B as a novel nucleolar targeting subunit that shows a specificity for PP1β and PP1γ. RRP1B, one of two mammalian orthologues of the yeast Rrp1p protein, shows an RNAse-dependent localization to the granular component of the nucleolus and distributes in a similar manner throughout the cell cycle to proteins involved in later steps of rRNA processing. Quantitative proteomic analysis of complexes containing both RRP1B and PP1γ revealed enrichment of an overlapping subset of large (60S) ribosomal subunit proteins and pre-60S nonribosomal proteins involved in mid-late processing. Targeting of PP1 to this complex by RRP1B in mammalian cells is likely to contribute to modulation of ribosome biogenesis by mechanisms involving reversible phosphorylation events, thus playing a role in the rapid transduction of cellular signals that call for regulation of ribosome production in response to cellular stress and/or changes in growth conditions.

Inferring the Ancient History of the Translation Machinery and Genetic Code via Recapitulation of Ribosomal Subunit Assembly Orders

Universally conserved positions in ribosomal proteins have significant biases in amino acid usage, likely indicating the expansion of the genetic code at the time leading up to the most recent common ancestor(s) (MRCA). Here, we apply this principle to the evolutionary history of the ribosome before the MRCA. It has been proposed that the experimentally determined order of assembly for ribosomal subunits recapitulates their evolutionary chronology. Given this model, we produce a probabilistic evolutionary ordering of the universally conserved small subunit (SSU) and large subunit (LSU) ribosomal proteins. Optimizing the relative ordering of SSU and LSU evolutionary chronologies with respect to minimizing differences in amino acid usage bias, we find strong compositional evidence for a more ancient origin for early LSU proteins. Furthermore, we find that this ordering produces several trends in specific amino acid usages compatible with models of genetic code evolution.

RPL41, a Small Ribosomal Peptide Deregulated in Tumors, Is Essential for Mitosis and Centrosome Integrity

Ribosomal large subunit protein RPL41 is a basic (positively charged) peptide consisting of only 25 amino acids. An antisense-based functional screening revealed that the down-regulation of RPL41 led to an anchorage-independent growth of NIH3T3 cells in soft agar plates. RPL41 depletion with gene-specific small interfering RNA also resulted in malignant transformation of NIH3T3 cells including increased tumor growth in mice. RPL41 deletion was detected in 59% of tumor cell lines by fluorescence in situ hybridization analyses and RPL41 down-regulation in 75% of primary breast cancers by real-time quantitative reverse transcription-polymerase chain reaction. These studies suggest a tumor suppression role for RPL41. By mass spectrometry, RPL41 was associated with several cytoskeleton components including tubulin β, γ, and myosin IIA, which was confirmed by Western blot analysis on both cellular lysis and individually in vitro-expressed proteins. RPL41 also bound directly to polymerized tubulins. Cells overexpressing a GFP-RPL41 were resistant to nocodazole-induced microtubule depolymerization. A synthetic RPL41 induced cellular α-tubulin acetylation and G2/M cell cycle arrest. These results indicate a stabilizing role of RPL41 on microtubule. Microtubule spindles are essential for chromosome segregation during mitosis. Cells with RPL41 knock-down showed abnormal spindles, frequent failure of cytokinesis, and formation of polynuclear cells. In interphase cells, RPL41-depleted cells had premature splitting of centrosome. Our results provide evidence that RPL41 is a microtubule-associated protein essential for functional spindles and for the integrity of centrosome and that the abnormal mitosis and disrupted centrosome associated with the RPL41 down-regulation may be related to malignant transformation.

Cell‐type specific analysis of translating RNAs in developing flowers reveals new levels of control

Determining both the expression levels of mRNA and the regulation of its translation is important in understanding specialized cell functions. In this study, we describe both the expression profiles of cells within spatiotemporal domains of the Arabidopsis thaliana flower and the post-transcriptional regulation of these mRNAs, at nucleotide resolution. We express a tagged ribosomal protein under the promoters of three master regulators of flower development. By precipitating tagged polysomes, we isolated cell type-specific mRNAs that are probably translating, and quantified those mRNAs through deep sequencing. Cell type comparisons identified known cell-specific transcripts and uncovered many new ones, from which we inferred cell type-specific hormone responses, promoter motifs and coexpressed cognate binding factor candidates, and splicing isoforms. By comparing translating mRNAs with steady-state overall transcripts, we found evidence for widespread post-transcriptional regulation at both the intron splicing and translational stages. Sequence analyses identified structural features associated with each step. Finally, we identified a new class of noncoding RNAs associated with polysomes. Findings from our profiling lead to new hypotheses in the understanding of flower development.

RRNA Maturation in Yeast Cells Depleted of Large Ribosomal Subunit Proteins

The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins). They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU) proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein – rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i) how individual r-proteins control the productive processing of the major 5′ end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii) the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.

Mutations in Ribosomal Protein Genes of Diamond-Blackfan Anemia Patients in Japan.

Abstract 3204Poster Board III-141Diamond-Blackfan anemia (DBA) is an inherited congenital bone marrow failure syndrome, characterized by red blood cell aplasia, macrocytic anemia, and increased risk of malignancy. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, which occur in about 40% of patients. Approximately 90% of patients present during the first year of life or in early childhood. Recent studies have shown that the disease is associated with heterozygous mutations in the ribosomal protein (RP) genes RPS19, RPS24, and RPS17, encoding small ribosomal subunit proteins, and in RPL5, RPL11 and RPL35a, encoding large ribosomal subunit proteins, in about 50% of patients with DBA in Western countries. There have been no studies to determine the incidence of these mutations in Asian patients with DBA. In this study, 44 probands (46 patients) with DBA in Japan were screened for mutations of the 6 known DBA genes RPS19, RPS24, RPS17, RPL5, RPL11, and RPL35a, in addition to RPS14, which is implicated in the 5q- syndrome, a subtype of myelodysplastic syndrome characterized by a defect in erythroid differentiation. Mutations in RPS19, which have been found in 25% of patients in Western countries, were detected in 6 probands (13.6%). Missense mutations were noted in 5 of these probands, and a frameshift mutation caused by a single-nucleotide insertion was found in 1 case. Three of 7 patients had multiple malformations. Novel mutations in RPL5 were identified in 3 probands (6.8%). Insertion of 2 nucleotides was found in 1 case, affecting the reading frame. Two cases had point mutations, which resulted in a loss of the first initiation codon. All 3 patients with RPL5 mutations had multiple physical anomalies. Remarkably, 2 of 3 patients with RPL5 mutations had cleft palate, whereas no other DBA patients presented with cleft palate. Mutations in RPL11 were identified in 2 patients (4.5%). Deletion of 1 or 2 nucleotides was found in each case, leading to a shift in the reading frame. In contrast to previous reports on patients with RPL11 mutations, thumb anomalies were not seen. Deletion of 1 nucleotide in RPS17 was identified in 1 patient (2.3%), resulting in introduction of a premature stop codon. RPS17 mutations are rare and have been only reported in 2 patients with DBA. Anomalies were not seen in our patient. In summary, RP gene mutations were identified in 27.3% of DBA index cases in Japan. No mutations were detected in RPS14, RPS24 and RPL35a. In Japan, the frequency of mutations in the RP genes appears to be lower than in Western countries. Mutations in RPL5 are associated with multiple physical abnormalities, including cleft palate. DisclosuresNo relevant conflicts of interest to declare.