Ribosomal Preparations Research Articles

The Discovery of Ribosomal Protein bL31 from Escherichia coli: A Long Story Revisited.

Ribosomal protein bL31 in Escherichia coli was initially detected as a short form (62 amino acids) using Kaltschmidt and Wittmann's two-dimensional polyacrylamide gel electrophoresis (2D PAGE), but the intact form (70 amino acids) was subsequently identified by means of Wada's improved radical-free and highly reducing (RFHR) 2D PAGE, which was consistent with the analysis of its encoding gene rpmE. Ribosomes routinely prepared from the K12 wild-type strain contained both forms of bL31. ΔompT cells, which lack protease 7, only contained intact bL31, suggesting that protease 7 cleaves intact bL31 and generates short bL31 during ribosome preparation from wild-type cells. Intact bL31 was required for subunit association, and its eight cleaved C-terminal amino acids contributed to this function. 70S ribosomes protected bL31 from cleavage by protease 7, but free 50S did not. In vitro translation was assayed using three systems. The translational activities of wild-type and ΔrpmE ribosomes were 20% and 40% lower than those of ΔompT ribosomes, which contained one copy of intact bL31. The deletion of bL31 reduces cell growth. A structural analysis predicted that bL31 spans the 30S and 50S subunits, consistent with its functions in 70S association and translation. It is important to re-analyze in vitro translation with ribosomes containing only intact bL31.

Antimicrobial Activity and 70S Ribosome Binding of Apidaecin-Derived Api805 with Increased Bacterial Uptake Rate.

In view of the global spread of multiresistant bacteria and the occurrence of panresistant bacteria, there is an urgent need for antimicrobials with novel modes of action. A promising class is antimicrobial peptides (AMPs), including them proline-rich AMPs (PrAMPs), which target the 70S ribosome to inhibit protein translation. Here, we present a new designer peptide, Api805, combining the N- and C-terminal sequences of PrAMPs Api137 and drosocin, respectively. Api805 was similarly active against two Escherichia coli B strains but was inactive against E. coli K12 strain BW25113. These different activities could not be explained by the dissociation constants measured for 70S ribosome preparations from E. coli K12 and B strains. Mutations in the SbmA transporter that PrAMPs use to pass the inner membrane or proteolytic degradation of Api805 by lysate proteases could not explain this either. Interestingly, Api805 seems not to bind to the known binding sites of PrAMPs at the 70S ribosome and inhibited in vitro protein translation, independent of release factors, most likely using a “multimodal effect”. Interestingly, Api805 entered the E. coli B strain Rosetta faster and at larger quantities than the E. coli K-12 strain BW25113, which may be related to the different LPS core structure. In conclusion, slight structural changes in PrAMPs significantly altered their binding sites and mechanisms of action, allowing for the design of different antibiotic classes.

Use of universal primers for the 18S ribosomal RNA gene and whole soil DNAs to reveal the taxonomic structures of soil nematodes by high-throughput amplicon sequencing.

Nematodes are abundant metazoans that play crucial roles in nutrient recycle in the pedosphere. Although high-throughput amplicon sequencing is a powerful tool for the taxonomic profiling of soil nematodes, polymerase chain reaction (PCR) primers for amplification of the 18S ribosomal RNA (SSU) gene and preparation of template DNAs have not been sufficiently evaluated. We investigated nematode community structure in copse soil using four nematode-specific (regions 1–4) and two universal (regions U1 and U2) primer sets for the SSU gene regions with two DNAs prepared from copse-derived mixed nematodes and whole soil. The major nematode-derived sequence variants (SVs) identified in each region was detected in both template DNAs. Order level taxonomy and feeding type of identified nematode-derived SVs were distantly related between the two DNA preparations, and the region U2 was closely related to region 4 in the non-metric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarity. Thus, the universal primers for region U2 could be used to analyze soil nematode communities. We further applied this method to analyze the nematodes living in two sampling sites of a sweet potato-cultivated field, where the plants were differently growing. The structure of nematode-derived SVs from the two sites was distantly related in the principal coordinate analysis (PCoA) with weighted unifrac distances, suggesting their distinct soil environments. The resultant ecophysiological status of the nematode communities in the copse and field on the basis of feeding behavior and maturity indices was fairly consistent with those of the copse- and the cultivated house garden-derived nematodes in prior studies. These findings will be useful for the DNA metabarcoding of soil eukaryotes, including nematodes, using soil DNAs.

Supplemental Material for Tamm, Kisly, and Remme, 2019

Table S1 lists the S. cerevisiae strains used in this study. Material and Methods chapter describes the Visualization of ribosome and r-protein structures, Preparation of 80S ribosomes for mass spectrometric analysis and LC-MS/MS analysis. Figure S1 shows the structures of the r-proteins eL19 and eL24. Figure S2 illustrates the strategy for strain construction. Figure S3 shows the analysis of global levels of translation of eL24 mutants. Figure S4 shows the mass spectrometric analysis of ribosomes from Archaea-like mutants. Figure S5 shows the analyzed peptides originated from the r-protein eL19. Figure S6 shows the analyzed peptides originated from the r-protein eL24.

The Inescapable Effects of Ribosomes on In-Cell NMR Spectroscopy and the Implications for Regulation of Biological Activity.

The effects of RNA on in-cell NMR spectroscopy and ribosomes on the kinetic activity of several metabolic enzymes are reviewed. Quinary interactions between labelled target proteins and RNA broaden in-cell NMR spectra yielding apparent megadalton molecular weights in-cell. The in-cell spectra can be resolved by using cross relaxation-induced polarization transfer (CRINEPT), heteronuclear multiple quantum coherence (HMQC), transverse relaxation-optimized, NMR spectroscopy (TROSY). The effect is reproduced in vitro by using reconstituted total cellular RNA and purified ribosome preparations. Furthermore, ribosomal binding antibiotics alter protein quinary structure through protein-ribosome and protein-mRNA-ribosome interactions. The quinary interactions of Adenylate kinase, Thymidylate synthase and Dihydrofolate reductase alter kinetic properties of the enzymes. The results demonstrate that ribosomes may specifically contribute to the regulation of biological activity.

Long Noncoding RNAs AC009014.3 and Newly Discovered XPLAID Differentiate Aggressive and Indolent Prostate Cancers

INTRODUCTION: The molecular mechanisms underlying aggressive versus indolent disease are not fully understood. Recent research has implicated a class of molecules known as long noncoding RNAs (lncRNAs) in tumorigenesis and progression of cancer. Our objective was to discover lncRNAs that differentiate aggressive and indolent prostate cancers. METHODS: We analyzed paired tumor and normal tissues from six aggressive Gleason score (GS) 8-10 and six indolent GS 6 prostate cancers. Extracted RNA was split for poly(A)+ and ribosomal RNA depletion library preparations, followed byRNA sequencing (RNA-Seq) using an Illumina HiSeq 2000. We developed an RNA-Seq data analysis pipeline to discover and quantify these molecules. Candidate lncRNAs were validated using RT-qPCR on 87 tumor tissue samples: 28 (GS 6), 28 (GS 3+4), 6 (GS 4+3), and 25 (GS 8-10). Statistical correlations between lncRNAs and clinicopathologic variables were tested using ANOVA. RESULTS: The 43 differentially expressed (DE) lncRNAs between aggressive and indolent prostate cancers included 12 annotated and 31 novel lncRNAs. The top six DE lncRNAs were selected based on large, consistent fold-changes in the RNA-Seq results. Three of these candidates passed RT-qPCR validation, including AC009014.3 (P < .001 in tumor tissue) and a newly discovered X-linked lncRNA named XPLAID (P = .049 in tumor tissue and P = .048 in normal tissue). XPLAID and AC009014.3 show promise as prognostic biomarkers. CONCLUSIONS: We discovered several dozen lncRNAs that distinguish aggressive and indolent prostate cancers, of which four were validated using RT-qPCR. The investigation into their biology is ongoing.

PD60-09 DISCOVERY AND VALIDATION OF NOVEL LONG NONCODING RNAS THAT DIFFERENTIATE AGGRESSIVE AND INDOLENT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Markers III1 Apr 2018PD60-09 DISCOVERY AND VALIDATION OF NOVEL LONG NONCODING RNAS THAT DIFFERENTIATE AGGRESSIVE AND INDOLENT PROSTATE CANCER Anthony J. Cesnik, Andrew V. Truong, Tyler Etheridge, Bing Yang, Michele Spiniello, Maisie I. Steinbrink, Michael R. Shortreed, Brian L. Frey, Glenn O. Allen, David F. Jarrard, and Lloyd M. Smith Anthony J. CesnikAnthony J. Cesnik More articles by this author , Andrew V. TruongAndrew V. Truong More articles by this author , Tyler EtheridgeTyler Etheridge More articles by this author , Bing YangBing Yang More articles by this author , Michele SpinielloMichele Spiniello More articles by this author , Maisie I. SteinbrinkMaisie I. Steinbrink More articles by this author , Michael R. ShortreedMichael R. Shortreed More articles by this author , Brian L. FreyBrian L. Frey More articles by this author , Glenn O. AllenGlenn O. Allen More articles by this author , David F. JarrardDavid F. Jarrard More articles by this author , and Lloyd M. SmithLloyd M. Smith More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2821AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES There is an incomplete understanding of the molecular mechanisms that determine aggressive prostate cancer (PCa). Recent research has implicated long noncoding RNAs (lncRNAs), transcripts longer than 200 nucleotides that are not translated into protein, in cancer progression. Our objective was to discover novel lncRNAs that differentiate aggressive and indolent PCa using an innovative platform. METHODS Paired normal (N) and tumor prostate tissue (T) was obtained from 6 Gleason (GS) 8-10 and 6 indolent GS 6 cancers. RNA was divided and employed for either poly(A)+ capture or ribosomal RNA depletion library preparations, and then subjected to RNA-Seq analysis on an Illumina-HiSeq 2000. Transcript isoforms were predicted using Cufflinks, and coding transcripts were filtered with slncky (version 1.0). Significantly differentially expressed lncRNAs between high and low grade cancer and N associated tissue were evaluated using DESeq2. Further validation for the lncRNA expression using qPCR for the top 6 candidates with highest fold change was performed on 85 patients: 26 (GS 6), 28 (GS 3+4), 6 (GS 4+3), and 25 (GS 8-10). Statistical correlation between lncRNA and clinicopathologic variables was performed using two-tailed t-test, ANOVA, and Chi-squared tests. RESULTS Of 42 differentially expressed lncRNAs that were identified, 11 were annotated, and 31 were novel. Many candidates were found at very low expression in tissues. Of six candidates with higher expression, significant differences of C3, C5, and C6 were validated upon comparison of qPCR results for GS ≥ 4+3 and indolent T (table). C3 also showed higher expression in N tissue from men with high grade cancer (p=0.048). C2 is a known lncRNA (SCHLAP1). C3 and C2 are associated with higher tumor volume (p = 0.041). C2 is strongly associated with higher tumor volume (p = 0.001), PSA failure (p = 0.001), and positive lymph nodes and metastasis (p = 0.04). C5 is related to higher tumor stage (p = 0.029), higher tumor volume (p = 0.021), and extracapsular extension (p = 0.025). CONCLUSIONS A novel bioinformatics approach has identified multiple new lncRNAs, many expressed at very low levels. Several of these lncRNAs distinguish aggressive and indolent PCa, and investigation into their biology is ongoing. Interestingly, some of these lncRNA associate with the field effect of PCa. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1149-e1150 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Anthony J. Cesnik More articles by this author Andrew V. Truong More articles by this author Tyler Etheridge More articles by this author Bing Yang More articles by this author Michele Spiniello More articles by this author Maisie I. Steinbrink More articles by this author Michael R. Shortreed More articles by this author Brian L. Frey More articles by this author Glenn O. Allen More articles by this author David F. Jarrard More articles by this author Lloyd M. Smith More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Ribosomal protein L31 in Escherichia coli contributes to ribosome subunit association and translation, whereas short L31 cleaved by protease 7 reduces both activities.

Ribosomes routinely prepared from Escherichia coli strain K12 contain intact (70 amino acids) and short (62 amino acids) forms of ribosomal protein L31. By contrast, ribosomes prepared from ompT mutant cells, which lack protease 7, contain only intact L31, suggesting that L31 is cleaved by protease 7 during ribosome preparation. We compared ribosomal subunit association in wild-type and ompT- strains. In sucrose density gradient centrifugation under low Mg2+ , 70S content was very high in ompT- ribosomes, but decreased in the wild-type ribosomes containing short L31. In addition, ribosomes lacking L31 failed to associate ribosomal subunits in low Mg2+ . Therefore, intact L31 is required for subunit association, and the eight C-terminal amino acids contribute to the association function. In vitro translation was assayed using three different systems. Translational activities of ribosomes lacking L31 were 40% lower than those of ompT- ribosomes with one copy of intact L31, indicating that L31 is involved in translation. Moreover, in the stationary phase, L31 was necessary for 100S formation. The strain lacking L31 grew very slowly. A structural analysis predicted that the L31 protein spans the 30S and 50S subunits, consistent with the functions of L31 in 70S association, 100S formation, and translation.

MP07-02 POLYA TAG LIBRARY PREPARATION FOR NEW GENERATION SEQUENCING (NGS) IN HUMAN TESTIS FAILS TO DETECT NON-CODING AND TRANSLATED RNAS IMPORTANT IN TESTICULAR FUNCTION AS COMPARED TO RIBOSOMAL RNA DEPLETION METHOD.

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology I1 Apr 2017MP07-02 POLYA TAG LIBRARY PREPARATION FOR NEW GENERATION SEQUENCING (NGS) IN HUMAN TESTIS FAILS TO DETECT NON-CODING AND TRANSLATED RNAS IMPORTANT IN TESTICULAR FUNCTION AS COMPARED TO RIBOSOMAL RNA DEPLETION METHOD. Ryan Flannigan, Anna Mielnik, Alex Bolyakov, Phil V. Bach, Peter Schlegel, and Darius Paduch Ryan FlanniganRyan Flannigan More articles by this author , Anna MielnikAnna Mielnik More articles by this author , Alex BolyakovAlex Bolyakov More articles by this author , Phil V. BachPhil V. Bach More articles by this author , Peter SchlegelPeter Schlegel More articles by this author , and Darius PaduchDarius Paduch More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.268AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Non-coding RNAs (ncRNAs) are emerging as important but poorly understood regulators of mRNA transcription and translation. However, common library preparation techniques for RNA sequencing selects for coding mRNAs by the presence of a poly-A tail; thus, by excluding non-PolyA ncRNAs, many biologically significant transcripts may be overlooked by using this method. The objective of this study was to evaluate differences in testicular RNA identification using 2 different methods of library preparation: polyA and ribosomal RNA depletion (RibZero) using Illumina kits. METHODS Total RNA was extracted from 3 human testis samples and processed using two different methods of library preparations: one based on polyA tags, and the other on depletion of ribosomal RNAs. The cDNA libraries were then sequenced at the same depth and annotated to known published databases. Identified transcripts were divided into two groups based on presence in one of the library preparation methods but not the other. Clinical and biological significance of identified genes was examined using the DAVID. Failure of detection of RibZero-only genes by RNAseq using polyA preparation was then confirmed by analyzing results of RNAseq in 64 testicular samples from normal patients, men with sertoli cell only (SCO), early and late maturation arrest and hypospermatogenesis. RESULTS 61 genes were detected only using polyA method with no detection in RibZero group (p=0.05): 17 of them belonged to small nuclear RNAs (snRNAs), 12 were snRNAs hosting genes, 3 humanin like proteins, and 3 were miRNA hosting genes: MIR137HG, MIR17HG, MIRLET7DHG. Deletion of MIR17HG leads to Feingold syndrome in humans and animal models. 74 genes were identified exclusively in RibZero group and not identified in the polyA group. The top 4 genes identified exclusively by RibZero were TAS2R50, MAGI1-AS, HIST1H3I, HIST1H4K. HIST1H4K was then further analyzed and its expression was highly abundant and specific to pachytene spermatocytes. Important components of the miRNA processing complex: AGO1,2, and 3 were expressed at >2x higher level (p=0.03) in ribosomal RNA library depletion preparation then polyA prep. CONCLUSIONS PolyA tail RNA enrichment method fails to adequately detect at least 7% of important RNAs in the human testis. Including ribosomal depletion RNA library preparation in addition to polyA tags enrichment is an important step to more comprehensively evaluate ncRNAs and testicular function. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e82 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Ryan Flannigan More articles by this author Anna Mielnik More articles by this author Alex Bolyakov More articles by this author Phil V. Bach More articles by this author Peter Schlegel More articles by this author Darius Paduch More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Preparation of ribosomes for smFRET studies: A simplified approach

During the past decade, single-molecule studies of the ribosome have significantly advanced our understanding of protein synthesis. The broadest application of these methods has been towards the investigation of ribosome conformational dynamics using single-molecule Förster resonance energy transfer (smFRET). The recent advances in fluorescently labeled ribosomes and translation components have resulted in success of smFRET experiments. Various methods have been employed to target fluorescent dyes to specific locations within the ribosome. Primarily, these methods have involved additional steps including subunit dissociation and/or full reconstitution, which could result in ribosomes of reduced activity and translation efficiency. In addition, substantial time and effort are required to produce limited quantities of material. To enable rapid and large-scale production of highly active, fluorescently labeled ribosomes, we have developed a procedure that combines partial reconstitution with His-tag purification. This allows for a homogeneous single-step purification of mutant ribosomes and subsequent integration of labeled proteins. Ribosomes produced with this method are shown to be as active as ribosomes purified using classical methods. While we have focused on two labeling sites in this report, the method is generalizable and can in principle be extended to any non-essential ribosomal protein.

Central dogma alchemy.

Central dogma alchemy.

Isolation of ribosomes and polysomes.

Here we describe a preparative differential centrifugation protocol for the isolation of ribosomes from a crude cell homogenate. The subcellular fraction obtained is enriched in ribosome monomers and polysomes. The protocol has been optimized for the homogenization and collection of the ribosomal fraction from prokaryotic cells, mammalian and plant tissues, reticulocytes, and chloroplasts. The quality of the ribosomal preparation is enhanced by the removal of the remaining cellular components and adsorbed proteins by pelleting through a sucrose cushion with a high concentration of monovalent salts, NH4Cl or KCl. The different components of the ribosomal fraction isolated using this protocol can be further purified by sucrose gradient centrifugation.

Identical RNA-Protein Interactions in Vivo and in Vitro and a Scheme of Folding the Newly Synthesized Proteins by Ribosomes

A distinct three-dimensional shape of rRNA inside the ribosome is required for the peptidyl transfer activity of its peptidyltransferase center (PTC). In contrast, even the in vitro transcribed PTC RNA interacts with unfolded protein(s) at about five sites to let them attain their native states. We found that the same set of conserved nucleotides in the PTC interact identically with nascent and chemically unfolded proteins in vivo and in vitro, respectively. The time course of this interaction, difficult to follow in vivo, was observed in vitro. It suggested nucleation of folding of cytosolic globular proteins vectorially from hydrophilic N to hydrophobic C termini, consistent with our discovery of a regular arrangement of cumulative hydrophobic indices of the peptide segments of cytosolic proteins from N to C termini. Based on this observation, we propose a model here for the nucleation of folding of the nascent protein chain by the PTC.

Single-Molecule Fluorescence Measurements of Ribosomal Translocation Dynamics

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G⋅GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA⋅EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.

A systematic study of 50S ribosomal subunit purification enabling robust crystallization

A systematic analysis was undertaken to seek correlations between the integrity, purity and activity of 50S ribosomal subunit preparations from Deinococcus radiodurans and their ability to crystallize. Conditions of fermentation, purification and crystallization were varied in a search for crystals that could reliably supply an industrial X-ray crystallography program for the structure-based design of ribosomal antibiotics. A robust protocol was obtained to routinely obtain crystals that gave diffraction patterns extending to 2.9 A resolution and that were large enough to yield a complete data set from a single crystal. To our knowledge, this is the most systematic study of this challenging area so far undertaken. Ribosome crystallization is a complex multi-factorial problem and although a clear correlation of crystallization with subunit properties was not obtained, the search for key factors that potentiate crystallization has been greatly narrowed and promising areas for further inquiry are suggested.

Rationale for the clinical use of a ribosome-component immune modulator

Vaccines have long been used to boost the immune system and to confer protection against microbial infections. In contrast, immunotherapy based on ribosomal preparations has been proposed to enhance both specific and nonspecific immune responses (in particular, the mucosal immune defense system) against common respiratory tract pathogens. The aim of this review is to summarize current knowledge on the ability of a combination of four immunogenic ribosomal fractions from different bacteria species and of the extracted fraction from the membrane of Klebsiella pneumoniae to modulate several immune functions. The immunomodulatory activity of the ribosome component is attributed to the presence of highly purified epitopes from cellular fractions obtained by bacterial lysis. The ribosomal preparation is hypothesized to induce a T-dependent immune response consequent network with proliferation of B lymphocytes and production of secretory high-affinity antibodies (in particular, IgA), as well as the creation of a specific immune memory. Indeed, this agent stimulates the activity of macrophages, polymorphonuclear cells, and natural killer cells. The "ideal" vaccine is a highly purified protein bound to a carrier and an adjuvant that enhances the vaccination effect.

Safety and tolerability of ribosome-component immune modulator in adults and children

Ribosomal preparations (ribosome-component immune modulators [RCIMs]) do not contain attenuated bacteria and, in contrast to live bacterial extracts, which may induce severe side effects, retain immune stimulating activity without infectious capability. This study was designed to profile the tolerability of RCIM by reviewing narratively all randomized, double-blind, placebo-controlled clinical trials and open-label studies as well as data from postmarketing surveillance studies representing >30 million prescriptions. In the various clinical trials, RCIM tolerability in terms of clinical and laboratory parameters was good. There were no significant differences between patients receiving active treatment or placebo in a survey of tolerability results from randomized, double-blind, placebo-controlled studies. Pharmacovigilance analysis does not show a change in the risk profile of RCIM. The only contraindication was correlated with known hypersensitivity to any of the product components. RCIM should not be used in case of acute streptococcal glomerulonephritis, acquired immune deficiency syndrome, severe viral disease, or severe autoimmune disease. Risk associated with the use of RCIM is negligible in recurrent upper and lower airway infections in selected populations, such as children and elderly people.

Visualization of the Hybrid State of tRNA Binding Promoted by Spontaneous Ratcheting of the Ribosome

A crucial step in translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called hybrid states. At this stage, the small subunit is rotated with respect to the large subunit, and the anticodon stem loops reside in the A and P sites of the small subunit, while the acceptor ends interact with the P and E sites of the large subunit. In this work, by means of cryo-EM and particle classification procedures, we visualize the hybrid state of both A/P and P/E tRNAs in an authentic factor-free ribosome complex during translocation. In addition, we show how the repositioning of the tRNAs goes hand in hand with the change in the interplay between S13, L1 stalk, L5, H68, H69, and H38 that is caused by the ratcheting of the small subunit.

Richard V. Eck (1922–2006): Bioinformatics: In the beginning

Richard V. Eck (1922–2006): Bioinformatics: In the beginning

NPM-ALK Fusion Tyrosine Kinase of Anaplastic Large Cell Lymphoma Exerts Its Transforming Potential by Increasing Translation of JUNB through mTOR and S6K1.

The nucleophosmin (NPM) - anaplastic lymphoma kinase (ALK) fusion protein, which is the product of the balanced chromosomal rearrangement t(2;5)(p23;q35), occurs in about 50% of nodal anaplastic large cell lymphoma (ALCL). Expression of this fusion kinase results in neoplastic transformation by modulating multiple intracellular signaling molecules, such as the PI3-kinase and the ERK1/2 kinases.Here we show high activation of JunB in various human ALCL cell lines. Moreover, using human embryonic kidney (HEK293) and murine hematopoietic Ba/F3 cells ectopically expressing NPM-ALK, we demonstrate that NPM-ALK is the trigger for JunB activation.Knock down of JUNB in NPM-ALK expressing cells using RNA interference results in upregulation of p16INK4a and downregulation of Cyclin D1, causing G1/S cell cycle arrest. Thus, JunB plays a critical oncogenic role in NPM-ALK transformed cells.Using the PI3-kinase inhibitor LY294002 and the MEK-inhibitor UO126, we demonstrate that NPM-ALK-induced JunB activity as well as cellular proliferation are dependent on both PI3-kinase and MEK-ERK signaling. Moreover, we illustrate that NPM-ALK and subsequently PI3-kinase activate AKT and mTOR/S6K1 which are implicated in the translational control of specific mRNA molecules containing a polypyrimidine motif (see pathway diagram). Since JUNB mRNA harbors such a motif we confirmed that JUNB mRNA is translated in large polysomes using ribosomal gradient preparations. Selective block of mTOR with the immunosuppressant rapamycin decreases proliferation and reduces protein but not mRNA levels of JunB in human and murine NPM-ALK positive cell lines. A similar effect is seen when inhibiting the upstream activator of mTOR, PI3-kinase. In contrast, a significant decrease of JUNB mRNA levels is shown in cells treated by the MEK-inhibitor UO126.An analogous effect of NPM-ALK is observed in ALCL patient samples. Hyperphosphorylation of S6K1 at Thr389 indicating activated mTOR/S6K1 is seen in 9/10 NPM-ALK positive ALCL cases in contrast to only 1/15 of NPM-ALK negative ALCL samples (P < 0,001). Hence, these findings suggest that the signaling cascade NPM-ALK→PI3K→AKT→mTOR/S6K1 is crucial to induce JUNB translation in human NPM-ALK positive ALCL.In conclusion, we reveal that JunB acts as an oncogene in NPM-ALK positive neoplastic cells and therefore represents a valid therapeutic target. Our data indicate a novel mechanism for regulation of AP-1 activity in general and suggest a new therapeutic approach to specifically modulate translation of an oncogene. [Display omitted]