Sequence and structure determinants of Drosophila Hsp70 mRNA translation: 5'UTR secondary structure specifically inhibits heat shock protein mRNA translation.

PubMed Central

Hess, M A; Duncan, R F

1996-01-01

Preferential translation of Drosophila heat shock protein 70 (Hsp70) mRNA requires only the 5'-untranslated region (5'-UTR). The sequence of this region suggests that it has relatively little secondary structure, which may facilitate efficient protein synthesis initiation. To determine whether minimal 5'-UTR secondary structure is required for preferential translation during heat shock, the effect of introducing stem-loops into the Hsp70 mRNA 5'-UTR was measured. Stem-loops of -11 kcal/mol abolished translation during heat shock, but did not reduce translation in non-heat shocked cells. A -22 kcal/mol stem-loop was required to comparably inhibit translation during growth at normal temperatures. To investigate whether specific sequence elements are also required for efficient preferential translation, deletion and mutation analyses were conducted in a truncated Hsp70 5'-UTR containing only the cap-proximal and AUG-proximal segments. Linker-scanner mutations in the cap-proximal segment (+1 to +37) did not impair translation. Re-ordering the segments reduced mRNA translational efficiency by 50%. Deleting the AUG-proximal segment severely inhibited translation. A 5-extension of the full-length leader specifically impaired heat shock translation. These results indicate that heat shock reduces the capacity to unwind 5-UTR secondary structure, allowing only mRNAs with minimal 5'-UTR secondary structure to be efficiently translated. A function for specific sequences is also suggested. PMID:8710519

Translational autocontrol of the Escherichia coli hfq RNA chaperone gene.

PubMed

Vecerek, Branislav; Moll, Isabella; Bläsi, Udo

2005-06-01

The conserved bacterial RNA chaperone Hfq has been shown to play an important role in post-transcriptional regulation. Here, we demonstrate that Hfq synthesis is autoregulated at the translational level. We have mapped two Hfq binding sites in the 5'-untranslated region of hfq mRNA and show that Hfq binding inhibits formation of the translation initiation complex. In vitro translation and in vivo studies further revealed that Hfq binding to both sites is required for efficient translational repression of hfq mRNA.

Directed Evolution of a Cyclized Peptoid-Peptide Chimera against a Cell-Free Expressed Protein and Proteomic Profiling of the Interacting Proteins to Create a Protein-Protein Interaction Inhibitor.

PubMed

Kawakami, Takashi; Ogawa, Koji; Hatta, Tomohisa; Goshima, Naoki; Natsume, Tohru

2016-06-17

N-alkyl amino acids are useful building blocks for the in vitro display evolution of ribosomally synthesized peptides because they can increase the proteolytic stability and cell permeability of these peptides. However, the translation initiation substrate specificity of nonproteinogenic N-alkyl amino acids has not been investigated. In this study, we screened various N-alkyl amino acids and nonamino carboxylic acids for translation initiation with an Escherichia coli reconstituted cell-free translation system (PURE system) and identified those that efficiently initiated translation. Using seven of these efficiently initiating acids, we next performed in vitro display evolution of cyclized peptidomimetics against an arbitrarily chosen model human protein (β-catenin) cell-free expressed from its cloned cDNA (HUPEX) and identified a novel β-catenin-binding cyclized peptoid-peptide chimera. Furthermore, by a proteomic approach using direct nanoflow liquid chromatography-tandem mass spectrometry (DNLC-MS/MS), we successfully identified which protein-β-catenin interaction is inhibited by the chimera. The combination of in vitro display evolution of cyclized N-alkyl peptidomimetics and in vitro expression of human proteins would be a powerful approach for the high-speed discovery of diverse human protein-targeted cyclized N-alkyl peptidomimetics.

Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms.

PubMed

Kahvejian, Avak; Svitkin, Yuri V; Sukarieh, Rami; M'Boutchou, Marie-Noël; Sonenberg, Nahum

2005-01-01

Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5'-end of the mRNA to promote the recruitment of the ribosome. Although the 3' poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (approximately 65% vs. approximately 35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5'-end of mRNA.

Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms

PubMed Central

Kahvejian, Avak; Svitkin, Yuri V.; Sukarieh, Rami; M'Boutchou, Marie-Noël; Sonenberg, Nahum

2005-01-01

Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5′-end of the mRNA to promote the recruitment of the ribosome. Although the 3′ poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (∼65% vs. ∼35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5′-end of mRNA. PMID:15630022

Leveraging genome-wide datasets to quantify the functional role of the anti-Shine-Dalgarno sequence in regulating translation efficiency.

PubMed

Hockenberry, Adam J; Pah, Adam R; Jewett, Michael C; Amaral, Luís A N

2017-01-01

Studies dating back to the 1970s established that sequence complementarity between the anti-Shine-Dalgarno (aSD) sequence on prokaryotic ribosomes and the 5' untranslated region of mRNAs helps to facilitate translation initiation. The optimal location of aSD sequence binding relative to the start codon, the full extents of the aSD sequence and the functional form of the relationship between aSD sequence complementarity and translation efficiency have not been fully resolved. Here, we investigate these relationships by leveraging the sequence diversity of endogenous genes and recently available genome-wide estimates of translation efficiency. We show that-after accounting for predicted mRNA structure-aSD sequence complementarity increases the translation of endogenous mRNAs by roughly 50%. Further, we observe that this relationship is nonlinear, with translation efficiency maximized for mRNAs with intermediate levels of aSD sequence complementarity. The mechanistic insights that we observe are highly robust: we find nearly identical results in multiple datasets spanning three distantly related bacteria. Further, we verify our main conclusions by re-analysing a controlled experimental dataset. © 2017 The Authors.

Development of an Agile Knowledge Engineering Framework in Support of Multi-Disciplinary Translational Research

PubMed Central

Borlawsky, Tara B.; Dhaval, Rakesh; Hastings, Shannon L.; Payne, Philip R. O.

2009-01-01

In October 2006, the National Institutes of Health launched a new national consortium, funded through Clinical and Translational Science Awards (CTSA), with the primary objective of improving the conduct and efficiency of the inherently multi-disciplinary field of translational research. To help meet this goal, the Ohio State University Center for Clinical and Translational Science has launched a knowledge management initiative that is focused on facilitating widespread semantic interoperability among administrative, basic science, clinical and research computing systems, both internally and among the translational research community at-large, through the integration of domain-specific standard terminologies and ontologies with local annotations. This manuscript describes an agile framework that builds upon prevailing knowledge engineering and semantic interoperability methods, and will be implemented as part this initiative. PMID:21347164

Development of an agile knowledge engineering framework in support of multi-disciplinary translational research.

PubMed

Borlawsky, Tara B; Dhaval, Rakesh; Hastings, Shannon L; Payne, Philip R O

2009-03-01

In October 2006, the National Institutes of Health launched a new national consortium, funded through Clinical and Translational Science Awards (CTSA), with the primary objective of improving the conduct and efficiency of the inherently multi-disciplinary field of translational research. To help meet this goal, the Ohio State University Center for Clinical and Translational Science has launched a knowledge management initiative that is focused on facilitating widespread semantic interoperability among administrative, basic science, clinical and research computing systems, both internally and among the translational research community at-large, through the integration of domain-specific standard terminologies and ontologies with local annotations. This manuscript describes an agile framework that builds upon prevailing knowledge engineering and semantic interoperability methods, and will be implemented as part this initiative.

Translational autocontrol of the Escherichia coli hfq RNA chaperone gene

PubMed Central

VEČEREK, BRANISLAV; MOLL, ISABELLA; BLÄSI, UDO

2005-01-01

The conserved bacterial RNA chaperone Hfq has been shown to play an important role in post-transcriptional regulation. Here, we demonstrate that Hfq synthesis is autoregulated at the translational level. We have mapped two Hfq binding sites in the 5′-untranslated region of hfq mRNA and show that Hfq binding inhibits formation of the translation initiation complex. In vitro translation and in vivo studies further revealed that Hfq binding to both sites is required for efficient translational repression of hfq mRNA. PMID:15872186

A high-level prokaryotic expression system: synthesis of human interleukin 1 alpha and its receptor antagonist.

PubMed

Birikh, K R; Lebedenko, E N; Boni, I V; Berlin, Y A

1995-10-27

Synthetic intronless genes, coding for human interleukin 1 alpha (IL 1 alpha) and interleukin 1 receptor antagonist (IL1ra), have been expressed efficiently in a specially designed prokaryotic vector, pGMCE (a pGEM1 derivative), where the target gene forms the second part of a two-cistron system. The first part of the system is a translation enhancer-containing mini-cistron, whose termination codon overlaps the start codon of the target gene. In the case of the IL1 alpha gene, the high expression level is largely due to the direct efficient translation initiation at the second cistron, whereas with the IL1ra gene in the same system, the proximal translation initiation region (TIR) provides a high level of coupled expression of the target gene. Thus, pGMCE is a potentially versatile vector for direct prokaryotic expression.

eIF4B stimulates translation of long mRNAs with structured 5′ UTRs and low closed-loop potential but weak dependence on eIF4G

PubMed Central

Sen, Neelam Dabas; Zhou, Fujun; Harris, Michael S.; Ingolia, Nicholas T.

2016-01-01

DEAD-box RNA helicases eukaryotic translation initiation factor 4A (eIF4A) and Ded1 promote translation by resolving mRNA secondary structures that impede preinitiation complex (PIC) attachment to mRNA or scanning. Eukaryotic translation initiation factor 4B (eIF4B) is a cofactor for eIF4A but also might function independently of eIF4A. Ribosome profiling of mutants lacking eIF4B or with impaired eIF4A or Ded1 activity revealed that eliminating eIF4B reduces the relative translational efficiencies of many more genes than does inactivation of eIF4A, despite comparable reductions in bulk translation, and few genes display unusually strong requirements for both factors. However, either eliminating eIF4B or inactivating eIF4A preferentially impacts mRNAs with longer, more structured 5′ untranslated regions (UTRs). These findings reveal an eIF4A-independent role for eIF4B in addition to its function as eIF4A cofactor in promoting PIC attachment or scanning on structured mRNAs. eIF4B, eIF4A, and Ded1 mutations also preferentially impair translation of longer mRNAs in a fashion mitigated by the ability to form closed-loop messenger ribonucleoprotein particles (mRNPs) via eIF4F–poly(A)-binding protein 1 (Pab1) association, suggesting cooperation between closed-loop assembly and eIF4B/helicase functions. Remarkably, depleting eukaryotic translation initiation factor 4G (eIF4G), the scaffold subunit of eukaryotic translation initiation factor 4F (eIF4F), preferentially impacts short mRNAs with strong closed-loop potential and unstructured 5′ UTRs, exactly the opposite features associated with hyperdependence on the eIF4B/helicases. We propose that short, highly efficient mRNAs preferentially depend on the stimulatory effects of eIF4G-dependent closed-loop assembly. PMID:27601676

uAUG-mediated translational initiations are responsible for human mu opioid receptor gene expression

PubMed Central

Song, Kyu Young; Kim, Chun Sung; Hwang, Cheol Kyu; Choi, Hack Sun; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H

2010-01-01

Abstract Mu opioid receptor (MOR) is the main site of interaction for major clinical analgesics, particularly morphine. MOR expression is regulated at the transcriptional and post-transcriptional levels. However, the protein expression of the MOR gene is relatively low and the translational control of MOR gene has not been well studied. The 5′-untranslated region (UTR) of the human MOR (OPRM1) mRNA contains four upstream AUG codons (uAUG) preceding the main translation initiation site. We mutated the four uAUGs individually and in combination. Mutations of the third uAUG, containing the same open reading frame, had the strongest inhibitory effect. The inhibitory effect caused by the third in-frame uAUG was confirmed by in vitro translation and receptor-binding assays. Toeprinting results showed that OPRM1 ribosomes initiated efficiently at the first uAUG, and subsequently re-initiated at the in-frame #3 uAUG and the physiological AUG site. This re-initiation resulted in negative expression of OPRM1 under normal conditions. These results indicate that re-initiation in MOR gene expression could play an important role in OPRM1 regulation. PMID:19438807

Targeting the eIF4F translation initiation complex: a critical nexus for cancer development.

PubMed

Pelletier, Jerry; Graff, Jeremy; Ruggero, Davide; Sonenberg, Nahum

2015-01-15

Elevated protein synthesis is an important feature of many cancer cells and often arises as a consequence of increased signaling flux channeled to eukaryotic initiation factor 4F (eIF4F), the key regulator of the mRNA-ribosome recruitment phase of translation initiation. In many cellular and preclinical models of cancer, eIF4F deregulation results in changes in translational efficiency of specific mRNA classes. Importantly, many of these mRNAs code for proteins that potently regulate critical cellular processes, such as cell growth and proliferation, enhanced cell survival and cell migration that ultimately impinge on several hallmarks of cancer, including increased angiogenesis, deregulated growth control, enhanced cellular survival, epithelial-to-mesenchymal transition, invasion, and metastasis. By being positioned as the molecular nexus downstream of key oncogenic signaling pathways (e.g., Ras, PI3K/AKT/TOR, and MYC), eIF4F serves as a direct link between important steps in cancer development and translation initiation. Identification of mRNAs particularly responsive to elevated eIF4F activity that typifies tumorigenesis underscores the critical role of eIF4F in cancer and raises the exciting possibility of developing new-in-class small molecules targeting translation initiation as antineoplastic agents. ©2014 American Association for Cancer Research.

Effect of the nucleotides surrounding the start codon on the translation of foot-and-mouth disease virus RNA.

PubMed

Ma, X X; Feng, Y P; Gu, Y X; Zhou, J H; Ma, Z R

2016-06-01

As for the alternative AUGs in foot-and-mouth disease virus (FMDV), nucleotide bias of the context flanking the AUG(2nd) could be used as a strong signal to initiate translation. To determine the role of the specific nucleotide context, dicistronic reporter constructs were engineered to contain different versions of nucleotide context linking between internal ribosome entry site (IRES) and downstream gene. The results indicate that under FMDV IRES-dependent mechanism, the nucleotide contexts flanking start codon can influence the translation initiation efficiencies. The most optimal sequences for both start codons have proved to be UUU AUG(1st) AAC and AAG AUG(2nd) GAA.

The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

PubMed Central

Williams, N P; Mueller, P P; Hinnebusch, A G

1988-01-01

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function. Images PMID:3065626

Short ORF-Dependent Ribosome Shunting Operates in an RNA Picorna-Like Virus and a DNA Pararetrovirus that Cause Rice Tungro Disease

PubMed Central

Pooggin, Mikhail M.; Rajeswaran, Rajendran; Schepetilnikov, Mikhail V.; Ryabova, Lyubov A.

2012-01-01

Rice tungro disease is caused by synergistic interaction of an RNA picorna-like virus Rice tungro spherical virus (RTSV) and a DNA pararetrovirus Rice tungro bacilliform virus (RTBV). It is spread by insects owing to an RTSV-encoded transmission factor. RTBV has evolved a ribosome shunt mechanism to initiate translation of its pregenomic RNA having a long and highly structured leader. We found that a long leader of RTSV genomic RNA remarkably resembles the RTBV leader: both contain several short ORFs (sORFs) and potentially fold into a large stem-loop structure with the first sORF terminating in front of the stem basal helix. Using translation assays in rice protoplasts and wheat germ extracts, we show that, like in RTBV, both initiation and proper termination of the first sORF translation in front of the stem are required for shunt-mediated translation of a reporter ORF placed downstream of the RTSV leader. The base pairing that forms the basal helix is required for shunting, but its sequence can be varied. Shunt efficiency in RTSV is lower than in RTBV. But in addition to shunting the RTSV leader sequence allows relatively efficient linear ribosome migration, which also contributes to translation initiation downstream of the leader. We conclude that RTSV and RTBV have developed a similar, sORF-dependent shunt mechanism possibly to adapt to the host translation system and/or coordinate their life cycles. Given that sORF-dependent shunting also operates in a pararetrovirus Cauliflower mosaic virus and likely in other pararetroviruses that possess a conserved shunt configuration in their leaders it is tempting to propose that RTSV may have acquired shunt cis-elements from RTBV during their co-existence. PMID:22396650

Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G.

PubMed

Papadopoulos, Evangelos; Jenni, Simon; Kabha, Eihab; Takrouri, Khuloud J; Yi, Tingfang; Salvi, Nicola; Luna, Rafael E; Gavathiotis, Evripidis; Mahalingam, Poornachandran; Arthanari, Haribabu; Rodriguez-Mias, Ricard; Yefidoff-Freedman, Revital; Aktas, Bertal H; Chorev, Michael; Halperin, Jose A; Wagner, Gerhard

2014-08-05

The interaction of the eukaryotic translation initiation factor eIF4E with the initiation factor eIF4G recruits the 40S ribosomal particle to the 5' end of mRNAs, facilitates scanning to the AUG start codon, and is crucial for eukaryotic translation of nearly all genes. Efficient recruitment of the 40S particle is particularly important for translation of mRNAs encoding oncoproteins and growth-promoting factors, which often harbor complex 5' UTRs and require efficient initiation. Thus, inhibiting the eIF4E/eIF4G interaction has emerged as a previously unpursued route for developing anticancer agents. Indeed, we discovered small-molecule inhibitors of this eIF4E/eIF4G interaction (4EGIs) that inhibit translation initiation both in vitro and in vivo and were used successfully in numerous cancer-biology and neurobiology studies. However, their detailed molecular mechanism of action has remained elusive. Here, we show that the eIF4E/eIF4G inhibitor 4EGI-1 acts allosterically by binding to a site on eIF4E distant from the eIF4G binding epitope. Data from NMR mapping and high-resolution crystal structures are congruent with this mechanism, where 4EGI-1 attaches to a hydrophobic pocket of eIF4E between β-sheet2 (L60-T68) and α-helix1 (E69-N77), causing localized conformational changes mainly in the H78-L85 region. It acts by unfolding a short 310-helix (S82-L85) while extending α-helix1 by one turn (H78-S82). This unusual helix rearrangement has not been seen in any previous eIF4E structure and reveals elements of an allosteric inhibition mechanism leading to the dislocation of eIF4G from eIF4E.

What determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame?

PubMed Central

Pöyry, Tuija A.A.; Kaminski, Ann; Jackson, Richard J.

2004-01-01

If the 5′-proximal AUG triplet in a mammalian mRNA is followed by a short open reading frame (sORF), a significant fraction of ribosomes resume scanning after termination of sORF translation, and reinitiate at a downstream AUG. To examine the underlying mechanism, we examined reinitiation in vitro using a series of mRNAs that differed only in the 5′-untranslated region (UTR). Efficient reinitiation was found to occur only if the eIF4F complex, or at a minimum the central one-third fragment of eIF4G, participated in the primary initiation event at the sORF initiation codon. It did not occur, however, when sORF translation was driven by the classical swine fever virus or cricket paralysis virus internal ribosome entry sites (IRESs), which do not use eIF4A, 4B, 4E, or 4G. A critical test was provided by an mRNA with an unstructured 5′-UTR, which is translated by scanning but does not absolutely need eIF4G and eIF4A: There was efficient reinitiation in a standard reticulocyte lysate, when initiation would be largely driven by eIF4F, but no reinitiation in an eIF4G-depleted lysate. These results suggest that resumption of scanning may depend on the interaction between eIF4F (or the eIF4G central domain) and the ribosome being maintained while the ribosome translates the sORF. PMID:14701882

Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites

PubMed Central

Sterk, Maaike; Romilly, Cédric; Wagner, E Gerhart H

2018-01-01

Abstract Initiation is the rate-limiting step in translation. It is well-known that stable structure at a ribosome binding site (RBS) impedes initiation. The ribosome standby model of de Smit and van Duin, based on studies of the MS2 phage coat cistron, proposed how high translation rates can be reconciled with stable, inhibitory structures at an RBS. Here, we revisited the coat protein system and assessed the translation efficiency from its sequestered RBS by introducing standby mutations. Further experiments with gfp reporter constructs assessed the effects of 5′-tails—as standby sites—with respect to length and sequence contributions. In particular, combining in vivo and in vitro assays, we can show that tails of CA-dinucleotide repeats—and to a lesser extent, AU-repeats—dramatically increase translation rates. Tails of increasing length reach maximal rate-enhancing effects at 16–18 nucleotides. These standby tails are single-stranded and do not exert their effect by structure changes in the neighboring RBS stem–loop. In vitro translation and toeprinting assays furthermore demonstrate that standby effects are exerted at the level of translation initiation. Finally, as expected, destabilizing mutations within the coat RBS indicate an interplay with the effects of standby tails. PMID:29420821

Sensitivity of mRNA Translation

PubMed Central

Poker, Gilad; Margaliot, Michael; Tuller, Tamir

2015-01-01

Using the dynamic mean-field approximation of the totally asymmetric simple exclusion process (TASEP), we investigate the effect of small changes in the initiation, elongation, and termination rates along the mRNA strand on the steady-state protein translation rate. We show that the sensitivity of mRNA translation is equal to the sensitivity of the maximal eigenvalue of a symmetric, nonnegative, tridiagonal, and irreducible matrix. This leads to new analytical results as well as efficient numerical schemes that are applicable for large-scale models. Our results show that in the usual endogenous case, when initiation is more rate-limiting than elongation, the sensitivity of the translation rate to small mutations rapidly increases towards the 5′ end of the ORF. When the initiation rate is high, as may be the case for highly expressed and/or heterologous optimized genes, the maximal sensitivity is with respect to the elongation rates at the middle of the mRNA strand. We also show that the maximal possible effect of a small increase/decrease in any of the rates along the mRNA is an increase/decrease of the same magnitude in the translation rate. These results are in agreement with previous molecular evolutionary and synthetic biology experimental studies. PMID:26238363

Biological insights into the expression of translation initiation factors from recombinant CHOK1SV cell lines and their relationship to enhanced productivity.

PubMed

Mead, Emma J; Masterton, Rosalyn J; Feary, Marc; Obrezanova, Olga; Zhang, Lin; Young, Robert; Smales, C Mark

2015-12-15

Translation initiation is on the critical pathway for the production of monoclonal antibodies (mAbs) by mammalian cells. Formation of a closed loop structure comprised of mRNA, a number of eukaryotic initiation factors (eIFs) and ribosomal proteins has been proposed to aid re-initiation of translation and therefore increase global translational efficiency. We have determined mRNA and protein levels of the key components of the closed loop, eIFs (eIF3a, eIF3b, eIF3c, eIF3h, eIF3i and eIF4G1), poly(A)-binding protein (PABP) 1 and PABP-interacting protein 1 (PAIP1), across a panel of 30 recombinant mAb-producing GS-CHOK1SV cell lines with a broad range of growth characteristics and production levels of a model recombinant mAb. We have used a multi-level statistical approach to investigate the relationship between key performance indicators (cell growth and recombinant antibody productivity) and the intracellular amounts of target translation initiation factor proteins and the mRNAs encoding them. We show that high-producing cell lines maintain amounts of the translation initiation factors involved in the formation of the closed loop mRNA, maintaining these proteins at appropriate levels to deliver enhanced recombinant protein production. We then utilize knowledge of the amounts of these factors to build predictive models for and use cluster analysis to identify, high-producing cell lines. The present study therefore defines the translation initiation factor amounts that are associated with highly productive recombinant GS-CHOK1SV cell lines that may be targets for screening highly productive cell lines or to engineer new host cell lines with the potential for enhanced recombinant antibody productivity. © 2015 Authors; published by Portland Press Limited.

AtaT blocks translation initiation by N-acetylation of the initiator tRNAfMet.

PubMed

Jurėnas, Dukas; Chatterjee, Sneha; Konijnenberg, Albert; Sobott, Frank; Droogmans, Louis; Garcia-Pino, Abel; Van Melderen, Laurence

2017-06-01

Toxin-antitoxin (TA) loci are prevalent in bacterial genomes. They are suggested to play a central role in dormancy and persister states. Under normal growth conditions, TA toxins are neutralized by their cognate antitoxins, and under stress conditions, toxins are freed and inhibit essential cellular processes using a variety of mechanisms. Here we characterize ataR-ataT, a novel TA system, from enterohemorrhagic Escherichia coli. We show that the toxin AtaT is a GNAT family enzyme that transfers an acetyl group from acetyl coenzyme A to the amine group of the methionyl aminoacyl moiety of initiator tRNA. AtaT specifically modifies Met-tRNA fMet , but no other aminoacyl-tRNAs, including the elongator Met-tRNA Met . We demonstrate that once acetylated, AcMet-tRNA fMet fails to interact with initiation factor-2 (IF2), resulting in disruption of the translation initiation complex. This work reveals a new mechanism of translation inhibition and confirms Met-tRNA fMet as a prime target to efficiently block cell growth.

New insights into the interplay between the translation machinery and nonsense-mediated mRNA decay factors.

PubMed

Raimondeau, Etienne; Bufton, Joshua C; Schaffitzel, Christiane

2018-06-19

Faulty mRNAs with a premature stop codon (PTC) are recognized and degraded by nonsense-mediated mRNA decay (NMD). Recognition of a nonsense mRNA depends on translation and on the presence of NMD-enhancing or the absence of NMD-inhibiting factors in the 3'-untranslated region. Our review summarizes our current understanding of the molecular function of the conserved NMD factors UPF3B and UPF1, and of the anti-NMD factor Poly(A)-binding protein, and their interactions with ribosomes translating PTC-containing mRNAs. Our recent discovery that UPF3B interferes with human translation termination and enhances ribosome dissociation in vitro , whereas UPF1 is inactive in these assays, suggests a re-interpretation of previous experiments and modification of prevalent NMD models. Moreover, we discuss recent work suggesting new functions of the key NMD factor UPF1 in ribosome recycling, inhibition of translation re-initiation and nascent chain ubiquitylation. These new findings suggest that the interplay of UPF proteins with the translation machinery is more intricate than previously appreciated, and that this interplay quality-controls the efficiency of termination, ribosome recycling and translation re-initiation. © 2018 The Author(s).

Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression

PubMed Central

Fahey, Ciara; Kenny, Elaine M; Terenin, Ilya M; Dmitriev, Sergey E; Cormican, Paul; Morris, Derek W; Shatsky, Ivan N; Baranov, Pavel V

2015-01-01

Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated upstream open reading frame (uORF) that represses translation of the main coding ORF under normal conditions. Site-specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products. DOI: http://dx.doi.org/10.7554/eLife.03971.001 PMID:25621764

Suppressed translation as a mechanism of initiation of CASP8 (caspase 8)-dependent apoptosis in autophagy-deficient NSCLC cells under nutrient limitation.

PubMed

Allavena, Giulia; Cuomo, Francesca; Baumgartner, Georg; Bele, Tadeja; Sellgren, Alexander Yarar; Oo, Kyaw Soe; Johnson, Kaylee; Gogvadze, Vladimir; Zhivotovsky, Boris; Kaminskyy, Vitaliy O

2018-01-01

Macroautophagy/autophagy inhibition under stress conditions is often associated with increased cell death. We found that under nutrient limitation, activation of CASP8/caspase-8 was significantly increased in autophagy-deficient lung cancer cells, which precedes mitochondria outer membrane permeabilization (MOMP), CYCS/cytochrome c release, and activation of CASP9/caspase-9, indicating that under such conditions the activation of CASP8 is a primary event in the initiation of apoptosis as well as essential to reduce clonogenic survival of autophagy-deficient cells. Starvation leads to suppression of CFLAR proteosynthesis and accumulation of CASP8 in SQSTM1 puncta. Overexpression of CFLARs reduces CASP8 activation and apoptosis during starvation, while its silencing promotes efficient activation of CASP8 and apoptosis in autophagy-deficient U1810 lung cancer cells even under nutrient-rich conditions. Similar to starvation, inhibition of protein translation leads to efficient activation of CASP8 and cell death in autophagy-deficient lung cancer cells. Thus, here for the first time we report that suppressed translation leads to activation of CASP8-dependent apoptosis in autophagy-deficient NSCLC cells under conditions of nutrient limitation. Our data suggest that targeting translational machinery can be beneficial for elimination of autophagy-deficient cells via the CASP8-dependent apoptotic pathway.

Back to basics: the untreated rabbit reticulocyte lysate as a competitive system to recapitulate cap/poly(A) synergy and the selective advantage of IRES-driven translation.

PubMed

Soto Rifo, Ricardo; Ricci, Emiliano P; Décimo, Didier; Moncorgé, Olivier; Ohlmann, Théophile

2007-01-01

Translation of most eukaryotic mRNAs involves the synergistic action between the 5' cap structure and the 3' poly(A) tail at the initiation step. The poly(A) tail has also been shown to stimulate translation of picornavirus internal ribosome entry sites (IRES)-directed translation. These effects have been attributed principally to interactions between eIF4G and poly(A)-binding protein (PABP) but also to the participation of PABP in other steps during translation initiation. As the rabbit reticulocyte lysate (RRL) does not recapitulate this cap/poly(A) synergy, several systems based on cellular cell-free extracts have been developed to study the effects of poly(A) tail in vitro but they generally exhibit low translational efficiency. Here, we describe that the non-nuclease-treated RRL (untreated RRL) is able to recapitulate the effects of poly(A) tail on translation in vitro. In this system, translation of a capped/polyadenylated RNA was specifically inhibited by either Paip2 or poly(rA), whereas translation directed by HCV IRES remained unaffected. Moreover, cleavage of eIF4G by FMDV L protease strongly stimulated translation directed by the EMCV IRES, thus recapitulating the competitive advantage that the proteolytic processing of eIF4G confers to IRES-driven RNAs.

Translational efficiency of poliovirus mRNA: mapping inhibitory cis-acting elements within the 5' noncoding region.

PubMed Central

Pelletier, J; Kaplan, G; Racaniello, V R; Sonenberg, N

1988-01-01

Poliovirus mRNA contains a long 5' noncoding region of about 750 nucleotides (the exact number varies among the three virus serotypes), which contains several AUG codons upstream of the major initiator AUG. Unlike most eucaryotic mRNAs, poliovirus does not contain a m7GpppX (where X is any nucleotide) cap structure at its 5' end and is translated by a cap-independent mechanism. To study the manner by which poliovirus mRNA is expressed, we examined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. In this paper we report striking translation system-specific differences in the ability of the altered mRNAs to be translated. The results suggest the existence of an inhibitory cis-acting element(s) within the 5' noncoding region of poliovirus (between nucleotides 70 and 381) which restricts mRNA translation in reticulocyte lysate, wheat germ extract, and Xenopus oocytes, but not in HeLa cell extracts. In addition, we show that HeLa cell extracts contain a trans-acting factor(s) that overcomes this restriction. Images PMID:2836606

Engineering Translational Activators with CRISPR-Cas System.

PubMed

Du, Pei; Miao, Chensi; Lou, Qiuli; Wang, Zefeng; Lou, Chunbo

2016-01-15

RNA parts often serve as critical components in genetic engineering. Here we report a design of translational activators which is composed of an RNA endoribonuclease (Csy4) and two exchangeable RNA modules. Csy4, a member of Cas endoribonuclease, cleaves at a specific recognition site; this cleavage releases a cis-repressive RNA module (crRNA) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. Unlike small RNA as a translational activator, the endoribonuclease-based activator is able to efficiently unfold the perfect RBS-crRNA pairing. As an exchangeable module, the crRNA-RBS duplex was forwardly and reversely engineered to modulate the dynamic range of translational activity. We further showed that Csy4 and its recognition site, together as a module, can also be replaced by orthogonal endoribonuclease-recognition site homologues. These modularly structured, high-performance translational activators would endow the programming of gene expression in the translation level with higher feasibility.

Mutations That Affect the Efficiency of Translation of mRNA for the cII Gene of Coliphage Lambda

PubMed Central

Dul, Ed; Mahoney, Michael E.; Wulff, Daniel L.

1987-01-01

Starting with the λ pRE- strain λctr1 cy3008, which forms clear plaques, we have isolated two mutant strains, λdya2 ctr1 cy3008 and λ dya3 ctr1 cy3008, that form plaques with very slightly turbid centers. The dya2 and dya3 mutations lie in the region of overlap between the PRE promoter and the ribosome recognition region of the cII gene, and have nucleotide alterations at positions -1 and +5 of pRE, and alterations of cII mRNA at -16 and -21 nucleotides before the initial AUG codon of the gene. Both mutations destabilize a stem structure that may be formed by cII mRNA, and dya2 also changes the sequence on cII mRNA that is complementary to the 3'-end of 16 S rRNA from 5'-UAAGGA-3' to 5'-UGAGGA-3'.—The dya2 and dya3 mutations, along with the ctr1 mutation, which destabilizes either of two alternate stem structures which may be formed by cII mRNA (these being more stable stem structures than the one affected by dya2 and dya3), were tested for their ability to reverse two cII- mutations that are characterized by inefficient translation of cII mRNA. These are cII3088, an A → G mutation four bases before the initial AUG codon, and cII3059 , a GUU → GAU (Val2 → Asp) second codon mutation. It was found that ctr1 completely reverses the translation defects of these two mutations, while dya2 partially reverses these translation defects. The dya3 mutation has no effect on translation efficiency under any condition tested. However neither the ctr1 mutation nor the dya2 mutation has much effect on translation efficiency in an otherwise cII+ background, indicating that other factors must limit the rate of translation of cII mRNA under these conditions. PMID:2953647

Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver

PubMed Central

Atger, Florian; Gobet, Cédric; Marquis, Julien; Martin, Eva; Wang, Jingkui; Weger, Benjamin; Lefebvre, Grégory; Descombes, Patrick; Naef, Felix; Gachon, Frédéric

2015-01-01

Diurnal oscillations of gene expression are a hallmark of rhythmic physiology across most living organisms. Such oscillations are controlled by the interplay between the circadian clock and feeding rhythms. Although rhythmic mRNA accumulation has been extensively studied, comparatively less is known about their transcription and translation. Here, we quantified simultaneously temporal transcription, accumulation, and translation of mouse liver mRNAs under physiological light–dark conditions and ad libitum or night-restricted feeding in WT and brain and muscle Arnt-like 1 (Bmal1)-deficient animals. We found that rhythmic transcription predominantly drives rhythmic mRNA accumulation and translation for a majority of genes. Comparison of wild-type and Bmal1 KO mice shows that circadian clock and feeding rhythms have broad impact on rhythmic gene expression, Bmal1 deletion affecting surprisingly both transcriptional and posttranscriptional levels. Translation efficiency is differentially regulated during the diurnal cycle for genes with 5′-Terminal Oligo Pyrimidine tract (5′-TOP) sequences and for genes involved in mitochondrial activity, many harboring a Translation Initiator of Short 5′-UTR (TISU) motif. The increased translation efficiency of 5′-TOP and TISU genes is mainly driven by feeding rhythms but Bmal1 deletion also affects amplitude and phase of translation, including TISU genes. Together this study emphasizes the complex interconnections between circadian and feeding rhythms at several steps ultimately determining rhythmic gene expression and translation. PMID:26554015

Modeling timelines for translational science in cancer; the impact of technological maturation

PubMed Central

McNamee, Laura M.; Ledley, Fred D.

2017-01-01

This work examines translational science in cancer based on theories of innovation that posit a relationship between the maturation of technologies and their capacity to generate successful products. We examined the growth of technologies associated with 138 anticancer drugs using an analytical model that identifies the point of initiation of exponential growth and the point at which growth slows as the technology becomes established. Approval of targeted and biological products corresponded with technological maturation, with first approval averaging 14 years after the established point and 44 years after initiation of associated technologies. The lag in cancer drug approvals after the increases in cancer funding and dramatic scientific advances of the 1970s thus reflects predictable timelines of technology maturation. Analytical models of technological maturation may be used for technological forecasting to guide more efficient translation of scientific discoveries into cures. PMID:28346525

Factors affecting expression of the recF gene of Escherichia coli K-12.

PubMed

Sandler, S J; Clark, A J

1990-01-31

This report describes four factors which affect expression of the recF gene from strong upstream lambda promoters under temperature-sensitive cIAt2-encoded repressor control. The first factor was the long mRNA leader sequence consisting of the Escherichia coli dnaN gene and 95% of the dnaA gene and lambda bet, N (double amber) and 40% of the exo gene. When most of this DNA was deleted, RecF became detectable in maxicells. The second factor was the vector, pBEU28, a runaway replication plasmid. When we substituted pUC118 for pBEU28, RecF became detectable in whole cells by the Coomassie blue staining technique. The third factor was the efficiency of initiation of translation. We used site-directed mutagenesis to change the mRNA leader, ribosome-binding site and the 3 bp before and after the translational start codon. Monitoring the effect of these mutational changes by translational fusion to lacZ, we discovered that the efficiency of initiation of translation was increased 30-fold. Only an estimated two- or threefold increase in accumulated levels of RecF occurred, however. This led us to discover the fourth factor, namely sequences in the recF gene itself. These sequences reduce expression of the recF-lacZ fusion genes 100-fold. The sequences responsible for this decrease in expression occur in four regions in the N-terminal half of recF. Expression is reduced by some sequences at the transcriptional level and by others at the translational level.

Defects in tRNA processing and nuclear export induce GCN4 translation independently of phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2.

PubMed

Qiu, H; Hu, C; Anderson, J; Björk, G R; Sarkar, S; Hopper, A K; Hinnebusch, A G

2000-04-01

Induction of GCN4 translation in amino acid-starved cells involves the inhibition of initiator tRNA(Met) binding to eukaryotic translation initiation factor 2 (eIF2) in response to eIF2 phosphorylation by protein kinase GCN2. It was shown previously that GCN4 translation could be induced independently of GCN2 by overexpressing a mutant tRNA(AAC)(Val) (tRNA(Val*)) or the RNA component of RNase MRP encoded by NME1. Here we show that overexpression of the tRNA pseudouridine 55 synthase encoded by PUS4 also leads to translational derepression of GCN4 (Gcd(-) phenotype) independently of eIF2 phosphorylation. Surprisingly, the Gcd(-) phenotype of high-copy-number PUS4 (hcPUS4) did not require PUS4 enzymatic activity, and several lines of evidence indicate that PUS4 overexpression did not diminish functional initiator tRNA(Met) levels. The presence of hcPUS4 or hcNME1 led to the accumulation of certain tRNA precursors, and their Gcd(-) phenotypes were reversed by overexpressing the RNA component of RNase P (RPR1), responsible for 5'-end processing of all tRNAs. Consistently, overexpression of a mutant pre-tRNA(Tyr) that cannot be processed by RNase P had a Gcd(-) phenotype. Interestingly, the Gcd(-) phenotype of hcPUS4 also was reversed by overexpressing LOS1, required for efficient nuclear export of tRNA, and los1Delta cells have a Gcd(-) phenotype. Overproduced PUS4 appears to impede 5'-end processing or export of certain tRNAs in the nucleus in a manner remedied by increased expression of RNase P or LOS1, respectively. The mutant tRNA(Val*) showed nuclear accumulation in otherwise wild-type cells, suggesting a defect in export to the cytoplasm. We propose that yeast contains a nuclear surveillance system that perceives defects in processing or export of tRNA and evokes a reduction in translation initiation at the step of initiator tRNA(Met) binding to the ribosome.

Defects in tRNA Processing and Nuclear Export Induce GCN4 Translation Independently of Phosphorylation of the α Subunit of Eukaryotic Translation Initiation Factor 2

PubMed Central

Qiu, Hongfang; Hu, Cuihua; Anderson, James; Björk, Glenn R.; Sarkar, Srimonti; Hopper, Anita K.; Hinnebusch, Alan G.

2000-01-01

Induction of GCN4 translation in amino acid-starved cells involves the inhibition of initiator tRNAMet binding to eukaryotic translation initiation factor 2 (eIF2) in response to eIF2 phosphorylation by protein kinase GCN2. It was shown previously that GCN4 translation could be induced independently of GCN2 by overexpressing a mutant tRNAAACVal (tRNAVal*) or the RNA component of RNase MRP encoded by NME1. Here we show that overexpression of the tRNA pseudouridine 55 synthase encoded by PUS4 also leads to translational derepression of GCN4 (Gcd− phenotype) independently of eIF2 phosphorylation. Surprisingly, the Gcd− phenotype of high-copy-number PUS4 (hcPUS4) did not require PUS4 enzymatic activity, and several lines of evidence indicate that PUS4 overexpression did not diminish functional initiator tRNAMet levels. The presence of hcPUS4 or hcNME1 led to the accumulation of certain tRNA precursors, and their Gcd− phenotypes were reversed by overexpressing the RNA component of RNase P (RPR1), responsible for 5′-end processing of all tRNAs. Consistently, overexpression of a mutant pre-tRNATyr that cannot be processed by RNase P had a Gcd− phenotype. Interestingly, the Gcd− phenotype of hcPUS4 also was reversed by overexpressing LOS1, required for efficient nuclear export of tRNA, and los1Δ cells have a Gcd− phenotype. Overproduced PUS4 appears to impede 5′-end processing or export of certain tRNAs in the nucleus in a manner remedied by increased expression of RNase P or LOS1, respectively. The mutant tRNAVal* showed nuclear accumulation in otherwise wild-type cells, suggesting a defect in export to the cytoplasm. We propose that yeast contains a nuclear surveillance system that perceives defects in processing or export of tRNA and evokes a reduction in translation initiation at the step of initiator tRNAMet binding to the ribosome. PMID:10713174

Translational Redefinition of UGA Codons Is Regulated by Selenium Availability*

PubMed Central

Howard, Michael T.; Carlson, Bradley A.; Anderson, Christine B.; Hatfield, Dolph L.

2013-01-01

Incorporation of selenium into ∼25 mammalian selenoproteins occurs by translational recoding whereby in-frame UGA codons are redefined to encode the selenium containing amino acid, selenocysteine (Sec). Here we applied ribosome profiling to examine the effect of dietary selenium levels on the translational mechanisms controlling selenoprotein synthesis in mouse liver. Dietary selenium levels were shown to control gene-specific selenoprotein expression primarily at the translation level by differential regulation of UGA redefinition and Sec incorporation efficiency, although effects on translation initiation and mRNA abundance were also observed. Direct evidence is presented that increasing dietary selenium causes a vast increase in ribosome density downstream of UGA-Sec codons for a subset of selenoprotein mRNAs and that the selenium-dependent effects on Sec incorporation efficiency are mediated in part by the degree of Sec-tRNA[Ser]Sec Um34 methylation. Furthermore, we find evidence for translation in the 5′-UTRs for a subset of selenoproteins and for ribosome pausing near the UGA-Sec codon in those mRNAs encoding the selenoproteins most affected by selenium availability. These data illustrate how dietary levels of the trace element selenium can alter the readout of the genetic code to affect the expression of an entire class of proteins. PMID:23696641

Balanced Codon Usage Optimizes Eukaryotic Translational Efficiency

PubMed Central

Qian, Wenfeng; Yang, Jian-Rong; Pearson, Nathaniel M.; Maclean, Calum; Zhang, Jianzhi

2012-01-01

Cellular efficiency in protein translation is an important fitness determinant in rapidly growing organisms. It is widely believed that synonymous codons are translated with unequal speeds and that translational efficiency is maximized by the exclusive use of rapidly translated codons. Here we estimate the in vivo translational speeds of all sense codons from the budding yeast Saccharomyces cerevisiae. Surprisingly, preferentially used codons are not translated faster than unpreferred ones. We hypothesize that this phenomenon is a result of codon usage in proportion to cognate tRNA concentrations, the optimal strategy in enhancing translational efficiency under tRNA shortage. Our predicted codon–tRNA balance is indeed observed from all model eukaryotes examined, and its impact on translational efficiency is further validated experimentally. Our study reveals a previously unsuspected mechanism by which unequal codon usage increases translational efficiency, demonstrates widespread natural selection for translational efficiency, and offers new strategies to improve synthetic biology. PMID:22479199

TranslatomeDB: a comprehensive database and cloud-based analysis platform for translatome sequencing data

PubMed Central

Liu, Wanting; Xiang, Lunping; Zheng, Tingkai; Jin, Jingjie

2018-01-01

Abstract Translation is a key regulatory step, linking transcriptome and proteome. Two major methods of translatome investigations are RNC-seq (sequencing of translating mRNA) and Ribo-seq (ribosome profiling). To facilitate the investigation of translation, we built a comprehensive database TranslatomeDB (http://www.translatomedb.net/) which provides collection and integrated analysis of published and user-generated translatome sequencing data. The current version includes 2453 Ribo-seq, 10 RNC-seq and their 1394 corresponding mRNA-seq datasets in 13 species. The database emphasizes the analysis functions in addition to the dataset collections. Differential gene expression (DGE) analysis can be performed between any two datasets of same species and type, both on transcriptome and translatome levels. The translation indices translation ratios, elongation velocity index and translational efficiency can be calculated to quantitatively evaluate translational initiation efficiency and elongation velocity, respectively. All datasets were analyzed using a unified, robust, accurate and experimentally-verifiable pipeline based on the FANSe3 mapping algorithm and edgeR for DGE analyzes. TranslatomeDB also allows users to upload their own datasets and utilize the identical unified pipeline to analyze their data. We believe that our TranslatomeDB is a comprehensive platform and knowledgebase on translatome and proteome research, releasing the biologists from complex searching, analyzing and comparing huge sequencing data without needing local computational power. PMID:29106630

Structural and Functional Diversity of Plant Virus 3'-Cap-Independent Translation Enhancers (3'-CITEs).

PubMed

Truniger, Verónica; Miras, Manuel; Aranda, Miguel A

2017-01-01

Most of the positive-strand RNA plant viruses lack the 5'-cap and/or the poly(A)-tail that act synergistically to stimulate canonical translation of cellular mRNAs. However, they have RNA elements in the 5'- or 3'-untranslated regions of their RNAs that are required for their cap-independent translation. Cap-independent translation enhancers (CITEs) have been identified in the genomic 3'-end of viruses belonging to the family Tombusviridae and the genus Luteovirus . Seven classes of 3'-CITEs have been described to date based on their different RNA structures. They generally control the efficient formation of the translation initiation complex by varying mechanisms. Some 3'-CITEs bind eukaryotic translation initiation factors, others ribosomal subunits, bridging these to the 5'-end by different mechanisms, often long-distance RNA-RNA interactions. As previously proposed and recently found in one case in nature, 3'-CITEs are functionally independent elements that are transferable through recombination between viral genomes, leading to potential advantages for virus multiplication. In this review, the knowledge on 3'-CITEs and their functioning is updated. We also suggest that there is local structural conservation in the regions interacting with eIF4E of 3'-CITEs belonging to different classes.

Structural and Functional Diversity of Plant Virus 3′-Cap-Independent Translation Enhancers (3′-CITEs)

PubMed Central

Truniger, Verónica; Miras, Manuel; Aranda, Miguel A.

2017-01-01

Most of the positive-strand RNA plant viruses lack the 5′-cap and/or the poly(A)-tail that act synergistically to stimulate canonical translation of cellular mRNAs. However, they have RNA elements in the 5′- or 3′-untranslated regions of their RNAs that are required for their cap-independent translation. Cap-independent translation enhancers (CITEs) have been identified in the genomic 3′-end of viruses belonging to the family Tombusviridae and the genus Luteovirus. Seven classes of 3′-CITEs have been described to date based on their different RNA structures. They generally control the efficient formation of the translation initiation complex by varying mechanisms. Some 3′-CITEs bind eukaryotic translation initiation factors, others ribosomal subunits, bridging these to the 5′-end by different mechanisms, often long-distance RNA–RNA interactions. As previously proposed and recently found in one case in nature, 3′-CITEs are functionally independent elements that are transferable through recombination between viral genomes, leading to potential advantages for virus multiplication. In this review, the knowledge on 3′-CITEs and their functioning is updated. We also suggest that there is local structural conservation in the regions interacting with eIF4E of 3′-CITEs belonging to different classes. PMID:29238357

Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation

PubMed Central

Aboulhouda, Soufiane; Di Santo, Rachael; Therizols, Gabriel; Weinberg, David

2017-01-01

The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to determine the number of ribosomes associated with an mRNA molecule, normalized to the length of the coding sequence. The primary method for this analysis of individual mRNAs is sucrose gradient fractionation, which physically separates mRNAs based on the number of bound ribosomes. Here, we describe a streamlined protocol for accurate analysis of mRNA association with ribosomes. Compared to previous protocols, our method incorporates internal controls and improved buffer conditions that together reduce artifacts caused by non-specific mRNA–ribosome interactions. Moreover, our direct-from-fraction qRT-PCR protocol eliminates the need for RNA purification from gradient fractions, which greatly reduces the amount of hands-on time required and facilitates parallel analysis of multiple conditions or gene targets. Additionally, no phenol waste is generated during the procedure. We initially developed the protocol to investigate the translationally repressed state of the HAC1 mRNA in S. cerevisiae, but we also detail adapted procedures for mammalian cell lines and tissues. PMID:29170751

Absence of Internal Ribosome Entry Site-Mediated Tissue Specificity in the Translation of a Bicistronic Transgene

PubMed Central

Shaw-Jackson, Chloë; Michiels, Thomas

1999-01-01

The 5′ noncoding regions of the genomes of picornaviruses form a complex structure that directs cap-independent initiation of translation. This structure has been termed the internal ribosome entry site (IRES). The efficiency of translation initiation was shown, in vitro, to be influenced by the binding of cellular factors to the IRES. Hence, we hypothesized that the IRES might control picornavirus tropism. In order to test this possibility, we made a bicistronic construct in which translation of the luciferase gene is controlled by the IRES of Theiler’s murine encephalomyelitis virus. In vitro, we observed that the IRES functions in various cell types and in macrophages, irrespective of their activation state. In vivo, we observed that the IRES is functional in different tissues of transgenic mice. Thus, it seems that the IRES is not an essential determinant of Theiler’s virus tropism. On the other hand, the age of the mouse could be critical for IRES function. Indeed, the IRES was found to be more efficient in young mice. Picornavirus IRESs are becoming popular tools in transgenesis technology, since they allow the expression of two genes from the same transcription unit. Our results show that the Theiler’s virus IRES is functional in cells of different origins and that it is thus a broad-spectrum tool. The possible age dependency of the IRES function, however, could be a drawback for gene expression in adult mice. PMID:10074119

The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition

PubMed Central

2006-01-01

Most eukaryotic mRNAs are monocistronic and translated by cap-dependent initiation. LINE-1 RNA is exceptional because it is naturally dicistronic, encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Here, we show that sequences upstream of ORF1 and ORF2 in mouse L1 function as internal ribosome entry sites (IRESes). Deletion analysis of the ORF1 IRES indicates that RNA structure is critical for its function. Conversely, the ORF2 IRES localizes to 53 nt near the 3′ end of ORF1, and appears to depend upon sequence rather than structure. The 40 nt intergenic region (IGR) is not essential for ORF2 IRES function or retrotransposition. Because of strong cis-preference for both proteins during L1 retrotransposition, correct stoichiometry of the two proteins can only be achieved post-transcriptionally. Although the precise stoichiometry is unknown, the retrotransposition intermediate likely contains hundreds of ORF1ps for every ORF2p, together with one L1 RNA. IRES-mediated translation initiation is a well-established mechanism of message-specific regulation, hence, unique mechanisms for the recognition and control of these two IRESes in the L1 RNA could explain differences in translational efficiency of ORF1 and ORF2. In addition, translational regulation may provide an additional layer of control on L1 retrotransposition efficiency, thereby protecting the integrity of the genome. PMID:16464823

Mechanisms employed by retroviruses to exploit host factors for translational control of a complicated proteome

PubMed Central

Bolinger, Cheryl; Boris-Lawrie, Kathleen

2009-01-01

Retroviruses have evolved multiple strategies to direct the synthesis of a complex proteome from a single primary transcript. Their mechanisms are modulated by a breadth of virus-host interactions, which are of significant fundamental interest because they ultimately affect the efficiency of virus replication and disease pathogenesis. Motifs located within the untranslated region (UTR) of the retroviral RNA have established roles in transcriptional trans-activation, RNA packaging, and genome reverse transcription; and a growing literature has revealed a necessary role of the UTR in modulating the efficiency of viral protein synthesis. Examples include a 5' UTR post-transcriptional control element (PCE), present in at least eight retroviruses, that interacts with cellular RNA helicase A to facilitate cap-dependent polyribosome association; and 3' UTR constitutive transport element (CTE) of Mason-Pfizer monkey virus that interacts with Tap/NXF1 and SR protein 9G8 to facilitate RNA export and translational utilization. By contrast, nuclear protein hnRNP E1 negatively modulates HIV-1 Gag, Env, and Rev protein synthesis. Alternative initiation strategies by ribosomal frameshifting and leaky scanning enable polycistronic translation of the cap-dependent viral transcript. Other studies posit cap-independent translation initiation by internal ribosome entry at structural features of the 5' UTR of selected retroviruses. The retroviral armamentarium also commands mechanisms to counter cellular post-transcriptional innate defenses, including protein kinase R, 2',5'-oligoadenylate synthetase and the small RNA pathway. This review will discuss recent and historically-recognized insights into retrovirus translational control. The expanding knowledge of retroviral post-transcriptional control is vital to understanding the biology of the retroviral proteome. In a broad perspective, each new insight offers a prospective target for antiviral therapy and strategic improvement of gene transfer vectors. PMID:19166625

The herpes simplex virus 1 virion host shutoff protein enhances translation of viral late mRNAs by preventing mRNA overload.

PubMed

Dauber, Bianca; Saffran, Holly A; Smiley, James R

2014-09-01

We recently demonstrated that the virion host shutoff (vhs) protein, an mRNA-specific endonuclease, is required for efficient herpes simplex virus 1 (HSV-1) replication and translation of viral true-late mRNAs, but not other viral and cellular mRNAs, in many cell types (B. Dauber, J. Pelletier, and J. R. Smiley, J. Virol. 85:5363-5373, 2011, http://dx.doi.org/10.1128/JVI.00115-11). Here, we evaluated whether the structure of true-late mRNAs or the timing of their transcription is responsible for the poor translation efficiency in the absence of vhs. To test whether the highly structured 5' untranslated region (5'UTR) of the true-late gC mRNA is the primary obstacle for translation initiation, we replaced it with the less structured 5'UTR of the γ-actin mRNA. However, this mutation did not restore translation in the context of a vhs-deficient virus. We then examined whether the timing of transcription affects translation efficiency at late times. To this end, we engineered a vhs-deficient virus mutant that transcribes the true-late gene US11 with immediate-early kinetics (IEUS11-ΔSma). Interestingly, IEUS11-ΔSma showed increased translational activity on the US11 transcript at late times postinfection, and US11 protein levels were restored to wild-type levels. These results suggest that mRNAs can maintain translational activity throughout the late stage of infection if they are present before translation factors and/or ribosomes become limiting. Taken together, these results provide evidence that in the absence of the mRNA-destabilizing function of vhs, accumulation of viral mRNAs overwhelms the capacity of the host translational machinery, leading to functional exclusion of the last mRNAs that are made during infection. The process of mRNA translation accounts for a significant portion of a cell's energy consumption. To ensure efficient use of cellular resources, transcription, translation, and mRNA decay are tightly linked and highly regulated. However, during virus infection, the overall amount of mRNA may increase drastically, possibly overloading the capacity of the translation apparatus. Our results suggest that the HSV-1 vhs protein, an mRNA-specific endoribonuclease, prevents mRNA overload during infection, thereby allowing translation of late viral mRNAs. The requirement for vhs varies between cell types. Further studies of the basis for this difference likely will offer insights into how cells regulate overall mRNA levels and access to the translational apparatus. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

TranslatomeDB: a comprehensive database and cloud-based analysis platform for translatome sequencing data.

PubMed

Liu, Wanting; Xiang, Lunping; Zheng, Tingkai; Jin, Jingjie; Zhang, Gong

2018-01-04

Translation is a key regulatory step, linking transcriptome and proteome. Two major methods of translatome investigations are RNC-seq (sequencing of translating mRNA) and Ribo-seq (ribosome profiling). To facilitate the investigation of translation, we built a comprehensive database TranslatomeDB (http://www.translatomedb.net/) which provides collection and integrated analysis of published and user-generated translatome sequencing data. The current version includes 2453 Ribo-seq, 10 RNC-seq and their 1394 corresponding mRNA-seq datasets in 13 species. The database emphasizes the analysis functions in addition to the dataset collections. Differential gene expression (DGE) analysis can be performed between any two datasets of same species and type, both on transcriptome and translatome levels. The translation indices translation ratios, elongation velocity index and translational efficiency can be calculated to quantitatively evaluate translational initiation efficiency and elongation velocity, respectively. All datasets were analyzed using a unified, robust, accurate and experimentally-verifiable pipeline based on the FANSe3 mapping algorithm and edgeR for DGE analyzes. TranslatomeDB also allows users to upload their own datasets and utilize the identical unified pipeline to analyze their data. We believe that our TranslatomeDB is a comprehensive platform and knowledgebase on translatome and proteome research, releasing the biologists from complex searching, analyzing and comparing huge sequencing data without needing local computational power. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Specific Increase of Protein Levels by Enhancing Translation Using Antisense Oligonucleotides Targeting Upstream Open Frames.

PubMed

Liang, Xue-Hai; Shen, Wen; Crooke, Stanley T

2017-01-01

A number of diseases are caused by low levels of key proteins; therefore, increasing the amount of specific proteins in human bodies is of therapeutic interest. Protein expression is downregulated by some structural or sequence elements present in the 5' UTR of mRNAs, such as upstream open reading frames (uORF). Translation initiation from uORF(s) reduces translation from the downstream primary ORF encoding the main protein product in the same mRNA, leading to a less efficient protein expression. Therefore, it is possible to use antisense oligonucleotides (ASOs) to specifically inhibit translation of the uORF by base-pairing with the uAUG region of the mRNA, redirecting translation machinery to initiate from the primary AUG site. Here we review the recent findings that translation of specific mRNAs can be enhanced using ASOs targeting uORF regions. Appropriately designed and optimized ASOs are highly specific, and they act in a sequence- and position-dependent manner, with very minor off-target effects. Protein levels can be increased using this approach in different types of human and mouse cells, and, importantly, also in mice. Since uORFs are present in around half of human mRNAs, the uORF-targeting ASOs may thus have valuable potential as research tools and as therapeutics to increase the levels of proteins for a variety of genes.

Efficient Cleavage of Ribosome-Associated Poly(A)-Binding Protein by Enterovirus 3C Protease

PubMed Central

Kuyumcu-Martinez, N. Muge; Joachims, Michelle; Lloyd, Richard E.

2002-01-01

Poliovirus (PV) causes a rapid and drastic inhibition of host cell cap-dependent protein synthesis during infection while preferentially allowing cap-independent translation of its own genomic RNA via an internal ribosome entry site element. Inhibition of cap-dependent translation is partly mediated by cleavage of an essential translation initiation factor, eIF4GI, during PV infection. In addition to cleavage of eIF4GI, cleavage of eIF4GII and poly(A)-binding protein (PABP) has been recently proposed to contribute to complete host translation shutoff; however, the relative importance of eIF4GII and PABP cleavage has not been determined. At times when cap-dependent translation is first blocked during infection, only 25 to 35% of the total cellular PABP is cleaved; therefore, we hypothesized that the pool of PABP associated with polysomes may be preferentially targeted by viral proteases. We have investigated what cleavage products of PABP are produced in vivo and the substrate determinants for cleavage of PABP by 2A protease (2Apro) or 3C protease (3Cpro). Our results show that PABP in ribosome-enriched fractions is preferentially cleaved in vitro and in vivo compared to PABP in other fractions. Furthermore, we have identified four N-terminal PABP cleavage products produced during PV infection and have shown that viral 3C protease generates three of the four cleavage products. Also, 3Cpro is more efficient in cleaving PABP in ribosome-enriched fractions than 2Apro in vitro. In addition, binding of PABP to poly(A) RNA stimulates 3Cpro-mediated cleavage and inhibits 2Apro-mediated cleavage. These results suggest that 3Cpro plays a major role in processing PABP during virus infection and that the interaction of PABP with translation initiation factors, ribosomes, or poly(A) RNA may promote its cleavage by viral 2A and 3C proteases. PMID:11836384

Hydrogen as the solar energy translator. [in photochemical and photovoltaic processes

NASA Technical Reports Server (NTRS)

Kelley, J. H.

1979-01-01

Many concepts are being investigated to convert sunlight to workable energy forms with emphasis on electricity and thermal energy. The electrical alternatives include direct conversion of photons to electricity via photovoltaic solar cells and solar/thermal production of electricity via heat-energy cycles. Solar cells, when commercialized, are expected to have efficiencies of about 12 to 14 percent. The cells would be active about eight hours per day. However, solar-operated water-splitting process research, initiated through JPL, shows promise for direct production of hydrogen from sunlight with efficiencies of up to 35 to 40 percent. The hydrogen, a valuable commodity in itself, can also serve as a storable energy form, easily and efficiently converted to electricity by fuel cells and other advanced-technology devices on a 24-hour basis or on demand with an overall efficiency of 25 to 30 percent. Thus, hydrogen serves as the fundamental translator of energy from its solar form to electrical form more effectively, and possibly more efficiently, than direct conversion. Hydrogen also can produce other chemical energy forms using solar energy.

AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo.

PubMed Central

Butler, J S; Springer, M; Grunberg-Manago, M

1987-01-01

We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo. PMID:2954162

AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo.

PubMed

Butler, J S; Springer, M; Grunberg-Manago, M

1987-06-01

We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo.

Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency

PubMed Central

Barna, Maria; Pusic, Aya; Zollo, Ornella; Costa, Maria; Kondrashov, Nadya; Rego, Eduardo; Rao, Pulivarthi H; Ruggero, Davide

2008-01-01

The Myc oncogene regulates the expression of multiple components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, Pol III, and rDNA1,2. An outstanding question is whether and how increasing the cellular protein synthesis capacity can affect the multi-step process leading to cancer. We utilized ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Eμ–Myc/+ transgenic mice to normal levels and show that in this context Myc's oncogenic potential is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a novel paradigm that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation employed to regulate the expression of selective mRNAs. We show that an aberrant increase in cap-dependent translation downstream Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (p58-PITSLRE)3-5, which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Eμ–Myc/+ mice. When accurate translational control is re-established in Eμ–Myc/+ mice, genome instability is suppressed. Our findings reveal how perturbations in translational control provide a highly specific outcome on gene expression, genome stability, and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level. PMID:19011615

Codon influence on protein expression in E. coli correlates with mRNA levels

PubMed Central

Boël, Grégory; Wong, Kam-Ho; Su, Min; Luff, Jon; Valecha, Mayank; Everett, John K.; Acton, Thomas B.; Xiao, Rong; Montelione, Gaetano T.; Aalberts, Daniel P.; Hunt, John F.

2016-01-01

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyze the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli. PMID:26760206

Translating an AI application from Lisp to Ada: A case study

NASA Technical Reports Server (NTRS)

Davis, Gloria J.

1991-01-01

A set of benchmarks was developed to test the performance of a newly designed computer executing both Lisp and Ada. Among these was AutoClassII -- a large Artificial Intelligence (AI) application written in Common Lisp. The extraction of a representative subset of this complex application was aided by a Lisp Code Analyzer (LCA). The LCA enabled rapid analysis of the code, putting it in a concise and functionally readable form. An equivalent benchmark was created in Ada through manual translation of the Lisp version. A comparison of the execution results of both programs across a variety of compiler-machine combinations indicate that line-by-line translation coupled with analysis of the initial code can produce relatively efficient and reusable target code.

Translation efficiencies of synonymous codons are not always correlated with codon usage in tobacco chloroplasts.

PubMed

Nakamura, Masayuki; Sugiura, Masahiro

2007-01-01

Codon usage in chloroplasts is different from that in prokaryotic and eukaryotic nuclear genomes. However, no experimental approach has been made to analyse the translation efficiency of individual codons in chloroplasts. We devised an in vitro assay for translation efficiencies using synthetic mRNAs, and measured the translation efficiencies of five synonymous codon groups in tobacco chloroplasts. Among four alanine codons (GCN, where N is U, C, A or G), GCU was the most efficient for translation, whereas the chloroplast genome lacks tRNA genes corresponding to GCU. Phenylalanine and tyrosine are each encoded by two codons (UUU/C and UAU/C, respectively). Phenylalanine UUC and tyrosine UAC were translated more than twice as efficiently than UUU and UAU, respectively, contrary to their codon usage, whereas translation efficiencies of synonymous codons for alanine, aspartic acid and asparagine were parallel to their codon usage. These observations indicate that translation efficiencies of individual codons are not always correlated with codon usage in vitro in chloroplasts. This raises an important issue for foreign gene expression in chloroplasts.

Detection of signals in mRNAs that influence translation.

PubMed

Brown, Chris M; Jacobs, Grant; Stockwell, Peter; Schreiber, Mark

2003-01-01

Genome sequencing efforts mean that we now have extensive data from a wide range of organisms to study. Understanding the differing natures of the biology of these organisms is an important aim of genome analysis. We are interested in signals that affect translation of mRNAs. Some signals in the mRNA influence how efficiently it is translated into protein. Previous studies have indicated that many important signals are located around the initiation and termination codons. We have developed tools described here to extract the relevant sequence regions from GenBank. To create databases organised by species, or higher taxonomic groupings (eg planta), a program was developed to dynamically view and edit the taxonomy database. Data from relevant species were then extracted using our Genbank feature table parser. We analysed all available sequences, particularly those from complete genomes. Patterns were then identified using information theory. The software is available from http://transterm.otago.ac.nz. Patterns around the initiation codons for most of the organisms fall into two groups, containing the previously known Shine-Dalgarno and Kozaks efficiency signals. However, we have identified several organisms that appear to utilise novel systems. Our analysis indicates that some organisms with extremely high GC% genomes do not have a strong dependence on base pairing ribosome binding sites, as the complementary sequence is absent from many genes.

Translational profiling of B cells infected with the Epstein-Barr virus reveals 5' leader ribosome recruitment through upstream open reading frames.

PubMed

Bencun, Maja; Klinke, Olaf; Hotz-Wagenblatt, Agnes; Klaus, Severina; Tsai, Ming-Han; Poirey, Remy; Delecluse, Henri-Jacques

2018-04-06

The Epstein-Barr virus (EBV) genome encodes several hundred transcripts. We have used ribosome profiling to characterize viral translation in infected cells and map new translation initiation sites. We show here that EBV transcripts are translated with highly variable efficiency, owing to variable transcription and translation rates, variable ribosome recruitment to the leader region and coverage by monosomes versus polysomes. Some transcripts were hardly translated, others mainly carried monosomes, showed ribosome accumulation in leader regions and most likely represent non-coding RNAs. A similar process was visible for a subset of lytic genes including the key transactivators BZLF1 and BRLF1 in cells infected with weakly replicating EBV strains. This suggests that ribosome trapping, particularly in the leader region, represents a new checkpoint for the repression of lytic replication. We could identify 25 upstream open reading frames (uORFs) located upstream of coding transcripts that displayed 5' leader ribosome trapping, six of which were located in the leader region shared by many latent transcripts. These uORFs repressed viral translation and are likely to play an important role in the regulation of EBV translation.

Elements in the murine c-mos messenger RNA 5'-untranslated region repress translation of downstream coding sequences.

PubMed

Steel, L F; Telly, D L; Leonard, J; Rice, B A; Monks, B; Sawicki, J A

1996-10-01

Murine c-mos transcripts isolated from testes have 5'-untranslated regions (5'UTRs) of approximately 300 nucleotides with a series of four overlapping open reading frames (ORFs) upstream of the AUG codon that initiates the Mos ORF. Ovarian c-mos transcripts have shorter 5'UTRs (70-80 nucleotides) and contain only 1-2 of the upstream ORFs (uORFs). To test whether these 5'UTRs affect translational efficiency, we have constructed plasmids for the expression of chimeric transcripts with a mos-derived 5'UTR fused to the Escherichia coli beta-galactosidase coding region. Translational efficiency has been evaluated by measuring beta-galactosidase activity NIH3T3 cells transiently transfected with these plasmids and with plasmids where various mutations have been introduced into the 5'UTR. We show that the 5'UTR characteristic of testis-specific c-mos mRNA strongly represses translation relative to the translation of transcripts that contain a 5'UTR derived from beta-globin mRNA, and this is mainly due to the four uORFs. Each of the four upstream AUG triplets can be recognized as a start site for translation, and no single uAUG dominates the repressive effect. The uORFs repress translation by a mechanism that is not affected by the amino acid sequence in the COOH-terminal region of the uORF-encoded peptides. The very short uORF (AUGUGA) present in ovary-specific transcripts does not repress translation. Staining of testis sections from transgenic mice carrying chimeric beta-galactosidase transgene constructs, which contain a mos 5'UTR with or without the uATGs, suggests that the uORFs can dramatically change the pattern of expression in spermatogenic cells.

Untranslated regions of diverse plant viral RNAs vary greatly in translation enhancement efficiency

PubMed Central

2012-01-01

Background Whole plants or plant cell cultures can serve as low cost bioreactors to produce massive amounts of a specific protein for pharmacological or industrial use. To maximize protein expression, translation of mRNA must be optimized. Many plant viral RNAs harbor extremely efficient translation enhancers. However, few of these different translation elements have been compared side-by-side. Thus, it is unclear which are the most efficient translation enhancers. Here, we compare the effects of untranslated regions (UTRs) containing translation elements from six plant viruses on translation in wheat germ extract and in monocotyledenous and dicotyledenous plant cells. Results The highest expressing uncapped mRNAs contained viral UTRs harboring Barley yellow dwarf virus (BYDV)-like cap-independent translation elements (BTEs). The BYDV BTE conferred the most efficient translation of a luciferase reporter in wheat germ extract and oat protoplasts, while uncapped mRNA containing the BTE from Tobacco necrosis virus-D translated most efficiently in tobacco cells. Capped mRNA containing the Tobacco mosaic virus omega sequence was the most efficient mRNA in tobacco cells. UTRs from Satellite tobacco necrosis virus, Tomato bushy stunt virus, and Crucifer-infecting tobamovirus (crTMV) did not stimulate translation efficiently. mRNA with the crTMV 5′ UTR was unstable in tobacco protoplasts. Conclusions BTEs confer the highest levels of translation of uncapped mRNAs in vitro and in vivo, while the capped omega sequence is most efficient in tobacco cells. These results provide a basis for understanding mechanisms of translation enhancement, and for maximizing protein synthesis in cell-free systems, transgenic plants, or in viral expression vectors. PMID:22559081

Gene activity during germination of spores of the fern, Onoclea sensibilis. Cell-free translation analysis of mRNA of spores and the effect of alpha-amanitin on spore germination

NASA Technical Reports Server (NTRS)

Raghavan, V.

1992-01-01

Poly(A)-RNA fractions of dormant, dark-imbibed (non-germinating) and photoinduced (germinating) spores of Onoclea sensibilis were poor templates in the rabbit reticulocyte lysate protein synthesizing system, but the translational efficiency of poly(A)+RNA was considerably higher than that of unfractionated RNA. Poly(A)+RNA isolated from photoinduced spores had a consistently higher translational efficiency than poly(A)+RNA from dark-imbibed spores. Analysis of the translation products by one-dimensional polyacrylamide gel electrophoresis showed no qualitative differences in the mRNA populations of dormant, dark-imbibed, and photoinduced spores. However, poly(A)+RNA from dark-imbibed spores appeared to encode in vitro fewer detectable polypeptides at a reduced intensity than photoinduced spores. A DNA clone encoding the large subunit of maize ribulose bisphosphate carboxylase hybridized at strong to moderate intensity to RNA isolated from dark-imbibed spores, indicating the absence of mRNA degradation. Although alpha-amanitin did not inhibit the germination of spores, the drug prevented the elongation of the rhizoid and protonemal initial with a concomitant effect on the synthesis of poly(A)+RNA. These results are consistent with the view that some form of translational control involving stored mRNA operates during dark-imbibition and photoinduced germination of spores.

Lin-28 binds IGF-2 mRNA and participates in skeletal myogenesis by increasing translation efficiency.

PubMed

Polesskaya, Anna; Cuvellier, Sylvain; Naguibneva, Irina; Duquet, Arnaud; Moss, Eric G; Harel-Bellan, Annick

2007-05-01

Lin-28 is a highly conserved, RNA-binding, microRNA-regulated protein that is involved in regulation of developmental timing in Caenorhabditis elegans. In mammals, Lin-28 is stage-specifically expressed in embryonic muscle, neurons, and epithelia, as well as in embryonic carcinoma cells, but is suppressed in most adult tissues, with the notable exception of skeletal and cardiac muscle. The specific function and mechanism of action of Lin-28 are not well understood. Here we used loss-of-function and gain-of-function assays in cultured myoblasts to show that expression of Lin-28 is essential for skeletal muscle differentiation in mice. In order to elucidate the specific function of Lin-28, we used a combination of biochemical and functional assays, which revealed that, in differentiating myoblasts, Lin-28 binds to the polysomes and increases the efficiency of protein synthesis. An important target of Lin-28 is IGF-2, a crucial growth and differentiation factor for muscle tissue. Interaction of Lin-28 with translation initiation complexes in skeletal myoblasts and in the embryonic carcinoma cell line P19 was confirmed by localization of Lin-28 to the stress granules, temporary structures that contain stalled mRNA-protein translation complexes. Our results unravel novel mechanisms of translational regulation in skeletal muscle and suggest that Lin-28 performs the role of "translational enhancer" in embryonic and adult cells and tissues.

The control of lambda DNA terminase synthesis.

PubMed Central

Murialdo, H; Davidson, A; Chow, S; Gold, M

1987-01-01

Nu1 and A, the genes coding for bacteriophage lambda DNA terminase, rank among the most poorly translated genes expressed in E. coli. To understand the reason for this low level of translation the genes were cloned into plasmids and their expression measured. In addition, the wild type DNA sequences immediately preceding the genes were reduced and modified. It was found that the elements that control translation are contained in the 100 base pairs upstream from the initiation codon. Interchanging these upstream sequences with those of an efficiently translated gene dramatically increased the translation of terminase subunits. It seems unlikely that the rare codons present in the genes, and any feature of their mRNA secondary structure play a role in the control of their translation. The elimination of cos from plasmids containing Nu1 and A also resulted in an increase in terminase production. This result suggests a role for cos in the control of late gene expression. The terminase subunit overproducer strains are potentially very useful for the design of improved DNA packaging and cosmid mapping techniques. Images PMID:3029667

Shwachman–Bodian–Diamond syndrome (SBDS) protein deficiency impairs translation re-initiation from C/EBPα and C/EBPβ mRNAs

PubMed Central

In, Kyungmin; Zaini, Mohamad A.; Müller, Christine; Warren, Alan J.; von Lindern, Marieke; Calkhoven, Cornelis F.

2016-01-01

Mutations in the Shwachman–Bodian–Diamond Syndrome (SBDS) gene cause Shwachman–Diamond Syndrome (SDS), a rare congenital disease characterized by bone marrow failure with neutropenia, exocrine pancreatic dysfunction and skeletal abnormalities. The SBDS protein is important for ribosome maturation and therefore SDS belongs to the ribosomopathies. It is unknown, however, if loss of SBDS functionality affects the translation of specific mRNAs and whether this could play a role in the development of the clinical features of SDS. Here, we report that translation of the C/EBPα and -β mRNAs, that are indispensible regulators of granulocytic differentiation, is altered by SBDS mutations or knockdown. We show that SBDS function is specifically required for efficient translation re-initiation into the protein isoforms C/EBPα-p30 and C/EBPβ-LIP, which is controlled by a single cis-regulatory upstream open reading frame (uORF) in the 5′ untranslated regions (5′ UTRs) of both mRNAs. Furthermore, we show that as a consequence of the C/EBPα and -β deregulation the expression of MYC is decreased with associated reduction in proliferation, suggesting that failure of progenitor proliferation contributes to the haematological phenotype of SDS. Therefore, our study provides the first indication that disturbance of specific translation by loss of SBDS function may contribute to the development of the SDS phenotype. PMID:26762974

Research-IQ: Development and Evaluation of an Ontology-anchored Integrative Query Tool

PubMed Central

Borlawsky, Tara B.; Lele, Omkar; Payne, Philip R. O.

2011-01-01

Investigators in the translational research and systems medicine domains require highly usable, efficient and integrative tools and methods that allow for the navigation of and reasoning over emerging large-scale data sets. Such resources must cover a spectrum of granularity from bio-molecules to population phenotypes. Given such information needs, we report upon the initial design and evaluation of an ontology-anchored integrative query tool, Research-IQ, which employs a combination of conceptual knowledge engineering and information retrieval techniques to enable the intuitive and rapid construction of queries, in terms of semi-structured textual propositions, that can subsequently be applied to integrative data sets. Our initial results, based upon both quantitative and qualitative evaluations of the efficacy and usability of Research-IQ, demonstrate its potential to increase clinical and translational research throughput. PMID:21821150

How Changes in Anti-SD Sequences Would Affect SD Sequences in Escherichia coli and Bacillus subtilis.

PubMed

Abolbaghaei, Akram; Silke, Jordan R; Xia, Xuhua

2017-05-05

The 3' end of the small ribosomal RNAs (ssu rRNA) in bacteria is directly involved in the selection and binding of mRNA transcripts during translation initiation via well-documented interactions between a Shine-Dalgarno (SD) sequence located upstream of the initiation codon and an anti-SD (aSD) sequence at the 3' end of the ssu rRNA. Consequently, the 3' end of ssu rRNA (3'TAIL) is strongly conserved among bacterial species because a change in the region may impact the translation of many protein-coding genes. Escherichia coli and Bacillus subtilis differ in their 3' ends of ssu rRNA, being GAUC ACCUCCUUA 3' in E. coli and GAUC ACCUCCUU UCU3' or GAUC ACCUCCUU UCUA3' in B. subtilis Such differences in 3'TAIL lead to species-specific SDs (designated SD Ec for E. coli and SD Bs for B. subtilis ) that can form strong and well-positioned SD/aSD pairing in one species but not in the other. Selection mediated by the species-specific 3'TAIL is expected to favor SD Bs against SD Ec in B. subtilis , but favor SD Ec against SD Bs in E. coli Among well-positioned SDs, SD Ec is used more in E. coli than in B. subtilis , and SD Bs more in B. subtilis than in E. coli Highly expressed genes and genes of high translation efficiency tend to have longer SDs than lowly expressed genes and genes with low translation efficiency in both species, but more so in B. subtilis than in E. coli Both species overuse SDs matching the bolded part of the 3'TAIL shown above. The 3'TAIL difference contributes to the host specificity of phages. Copyright © 2017 Abolbaghaei et al.

The 5′-tail of antisense RNAII of pMV158 plays a critical role in binding to the target mRNA and in translation inhibition of repB

PubMed Central

López-Aguilar, Celeste; Romero-López, Cristina; Espinosa, Manuel; Berzal-Herranz, Alfredo; del Solar, Gloria

2015-01-01

Rolling-circle replication of streptococcal plasmid pMV158 is controlled by the concerted action of two trans-acting elements, namely transcriptional repressor CopG and antisense RNAII, which inhibit expression of the repB gene encoding the replication initiator protein. The pMV158-encoded antisense RNAII exerts its activity of replication control by inhibiting translation of the essential repB gene. RNAII is the smallest and simplest among the characterized antisense RNAs involved in control of plasmid replication. Structure analysis of RNAII revealed that it folds into an 8-bp-long stem containing a 1-nt bulge and closed by a 6-nt apical loop. This hairpin is flanked by a 17-nt-long single-stranded 5′-tail and an 8-nt-long 3′-terminal U-rich stretch. Here, the 3′ and 5′ regions of the 5′-tail of RNAII are shown to play a critical role in the binding to the target mRNA and in the inhibition of repB translation, respectively. In contrast, the apical loop of the single hairpin of RNAII plays a rather secondary role and the upper stem region hardly contributes to the binding or inhibition processes. The entire 5′-tail is required for efficient inhibition of repB translation, though only the 8-nt-long region adjacent to the hairpin seems to be essential for rapid binding to the mRNA. These results show that a “kissing” interaction involving base-pairing between complementary hairpin loops in RNAII and mRNA is not critical for efficient RNA/RNA binding or repB translation inhibition. A singular binding mechanism is envisaged whereby initial pairing between complementary single-stranded regions in the antisense and sense RNAs progresses upwards into the corresponding hairpin stems to form the intermolecular duplex. PMID:26175752

Differential translation efficiency of orthologous genes is involved in phenotypic divergence of yeast species.

PubMed

Man, Orna; Pilpel, Yitzhak

2007-03-01

A major challenge in comparative genomics is to understand how phenotypic differences between species are encoded in their genomes. Phenotypic divergence may result from differential transcription of orthologous genes, yet less is known about the involvement of differential translation regulation in species phenotypic divergence. In order to assess translation effects on divergence, we analyzed approximately 2,800 orthologous genes in nine yeast genomes. For each gene in each species, we predicted translation efficiency, using a measure of the adaptation of its codons to the organism's tRNA pool. Mining this data set, we found hundreds of genes and gene modules with correlated patterns of translational efficiency across the species. One signal encompassed entire modules that are either needed for oxidative respiration or fermentation and are efficiently translated in aerobic or anaerobic species, respectively. In addition, the efficiency of translation of the mRNA splicing machinery strongly correlates with the number of introns in the various genomes. Altogether, we found extensive selection on synonymous codon usage that modulates translation according to gene function and organism phenotype. We conclude that, like factors such as transcription regulation, translation efficiency affects and is affected by the process of species divergence.

Capped mRNAs with reduced secondary structure can function in extracts from poliovirus-infected cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sonenberg, N.; Guertin, D.; Lee, K.A.W.

1982-12-01

Extracts form poliovirus-infected HeLa cells were used to study ribosome binding of native and denatured reovirus mRNAs and translation of capped mRNAs with different degrees of secondary structure. Here, the authors demonstrate that ribosomes in extracts from poliovirus-infected cells could form initiation complexes with denatured reovirus mRNA, in contrast to their inability to bind native reovirus mRNA. Furthermore, the capped alfalfa mosiac virus 4 RNA, which is most probable devoid of stable secondary structure at its 5' end, could be translated at much higher efficiency than could other capped mRNAs in extracts from poliovirus-infected cells.

The Effect of an Alternate Start Codon on Heterologous Expression of a PhoA Fusion Protein in Mycoplasma gallisepticum

PubMed Central

Panicker, Indu S.; Browning, Glenn F.; Markham, Philip F.

2015-01-01

While the genomes of many Mycoplasma species have been sequenced, there are no collated data on translational start codon usage, and the effects of alternate start codons on gene expression have not been studied. Analysis of the annotated genomes found that ATG was the most prevalent translational start codon among Mycoplasma spp. However in Mycoplasma gallisepticum a GTG start codon is commonly used in the vlhA multigene family, which encodes a highly abundant, phase variable lipoprotein adhesin. Therefore, the effect of this alternate start codon on expression of a reporter PhoA lipoprotein was examined in M. gallisepticum. Mutation of the start codon from ATG to GTG resulted in a 2.5 fold reduction in the level of transcription of the phoA reporter, but the level of PhoA activity in the transformants containing phoA with a GTG start codon was only 63% of that of the transformants with a phoA with an ATG start codon, suggesting that GTG was a more efficient translational initiation codon. The effect of swapping the translational start codon in phoA reporter gene expression was less in M. gallisepticum than has been seen previously in Escherichia coli or Bacillus subtilis, suggesting the process of translational initiation in mycoplasmas may have some significant differences from those used in other bacteria. This is the first study of translational start codon usage in mycoplasmas and the impact of the use of an alternate start codon on expression in these bacteria. PMID:26010086

A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS).

PubMed

Unwin, Richard D; Griffiths, John R; Whetton, Anthony D

2009-01-01

The application of a targeted mass spectrometric workflow to the sensitive identification of post-translational modifications is described. This protocol employs multiple reaction monitoring (MRM) to search for all putative peptides specifically modified in a target protein. Positive MRMs trigger an MS/MS experiment to confirm the nature and site of the modification. This approach, termed MIDAS (MRM-initiated detection and sequencing), is more sensitive than approaches using neutral loss scanning or precursor ion scanning methodologies, due to a more efficient use of duty cycle along with a decreased background signal associated with MRM. We describe the use of MIDAS for the identification of phosphorylation, with a typical experiment taking just a couple of hours from obtaining a peptide sample. With minor modifications, the MIDAS method can be applied to other protein modifications or unmodified peptides can be used as a MIDAS target.

Passive damping concepts for tubular beams with partial rotational and translational end restraints

NASA Technical Reports Server (NTRS)

Razzaq, Zia; Muyundo, David K.

1991-01-01

The main objectives of the study are: (1) identification of potential passive damping concepts for slender tubular structural members with rotational and translational end springs under natural and forced-free vibrations; (2) evaluation of damping efficiencies of the various damping concepts; and (3) evaluation of the suitability of a theoretical finite difference analysis by comparison to the experimental results for the case of natural vibrations. Only member flexural an translation motion is considered. The natural vibration study is conducted on the seven damping concepts and for only one specific initial deflection. The most suitable of the seven dampers is further investigated under forced-free vibrations. In addition only one set of end springs is used for all of the experiments. The results show that passive damping provides a possible approach to structural vibration reduction.

The Quantitative Evaluation of the Clinical and Translational Science Awards (CTSA) Program Based on Science Mapping and Scientometric Analysis

PubMed Central

Zhang, Yin; Wang, Lei

2013-01-01

Abstract The Clinical and Translational Science Awards (CTSA) program is one of the most important initiatives in translational medical funding. The quantitative evaluation of the efficiency and performance of the CTSA program has a significant referential meaning for the decision making of global translational medical funding. Using science mapping and scientometric analytic tools, this study quantitatively analyzed the scientific articles funded by the CTSA program. The results of the study showed that the quantitative productivities of the CTSA program had a stable increase since 2008. In addition, the emerging trends of the research funded by the CTSA program covered clinical and basic medical research fields. The academic benefits from the CTSA program were assisting its members to build a robust academic home for the Clinical and Translational Science and to attract other financial support. This study provided a quantitative evaluation of the CTSA program based on science mapping and scientometric analysis. Further research is required to compare and optimize other quantitative methods and to integrate various research results. PMID:24330689

The quantitative evaluation of the Clinical and Translational Science Awards (CTSA) program based on science mapping and scientometric analysis.

PubMed

Zhang, Yin; Wang, Lei; Diao, Tianxi

2013-12-01

The Clinical and Translational Science Awards (CTSA) program is one of the most important initiatives in translational medical funding. The quantitative evaluation of the efficiency and performance of the CTSA program has a significant referential meaning for the decision making of global translational medical funding. Using science mapping and scientometric analytic tools, this study quantitatively analyzed the scientific articles funded by the CTSA program. The results of the study showed that the quantitative productivities of the CTSA program had a stable increase since 2008. In addition, the emerging trends of the research funded by the CTSA program covered clinical and basic medical research fields. The academic benefits from the CTSA program were assisting its members to build a robust academic home for the Clinical and Translational Science and to attract other financial support. This study provided a quantitative evaluation of the CTSA program based on science mapping and scientometric analysis. Further research is required to compare and optimize other quantitative methods and to integrate various research results. © 2013 Wiley Periodicals, Inc.

The unique Leishmania EIF4E4 N-terminus is a target for multiple phosphorylation events and participates in critical interactions required for translation initiation.

PubMed

de Melo Neto, Osvaldo P; da Costa Lima, Tamara D C; Xavier, Camila C; Nascimento, Larissa M; Romão, Tatiany P; Assis, Ludmila A; Pereira, Mariana M C; Reis, Christian R S; Papadopoulou, Barbara

2015-01-01

The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa.

The unique Leishmania EIF4E4 N-terminus is a target for multiple phosphorylation events and participates in critical interactions required for translation initiation

PubMed Central

de Melo Neto, Osvaldo P; da Costa Lima, Tamara D C; Xavier, Camila C; Nascimento, Larissa M; Romão, Tatiany P; Assis, Ludmila A; Pereira, Mariana M C; Reis, Christian R S; Papadopoulou, Barbara

2015-01-01

The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa. PMID:26338184

The helicase Ded1p controls use of near-cognate translation initiation codons in 5' UTRs.

PubMed

Guenther, Ulf-Peter; Weinberg, David E; Zubradt, Meghan M; Tedeschi, Frank A; Stawicki, Brittany N; Zagore, Leah L; Brar, Gloria A; Licatalosi, Donny D; Bartel, David P; Weissman, Jonathan S; Jankowsky, Eckhard

2018-06-27

The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation 1 . Mutations in DDX3 are linked to tumorigenesis 2-4 and intellectual disability 5 , and the enzyme is targeted by a range of viruses 6 . How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5' untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5' untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5' untranslated regions.

In Silico Estimation of Translation Efficiency in Human Cell Lines: Potential Evidence for Widespread Translational Control

PubMed Central

Stevens, Stewart G.; Brown, Chris M

2013-01-01

Recently large scale transcriptome and proteome datasets for human cells have become available. A striking finding from these studies is that the level of an mRNA typically predicts no more than 40% of the abundance of protein. This correlation represents the overall figure for all genes. We present here a bioinformatic analysis of translation efficiency – the rate at which mRNA is translated into protein. We have analysed those human datasets that include genome wide mRNA and protein levels determined in the same study. The analysis comprises five distinct human cell lines that together provide comparable data for 8,170 genes. For each gene we have used levels of mRNA and protein combined with protein stability data from the HeLa cell line to estimate translation efficiency. This was possible for 3,990 genes in one or more cell lines and 1,807 genes in all five cell lines. Interestingly, our analysis and modelling shows that for many genes this estimated translation efficiency has considerable consistency between cell lines. Some deviations from this consistency likely result from the regulation of protein degradation. Others are likely due to known translational control mechanisms. These findings suggest it will be possible to build improved models for the interpretation of mRNA expression data. The results we present here provide a view of translation efficiency for many genes. We provide an online resource allowing the exploration of translation efficiency in genes of interest within different cell lines (http://bioanalysis.otago.ac.nz/TranslationEfficiency). PMID:23460887

Role of the open reading frames of Rous sarcoma virus leader RNA in translation and genome packaging.

PubMed Central

Donzé, O; Spahr, P F

1992-01-01

The Rous sarcoma virus (RSV) RNA leader sequence carries three open reading frames (uORFs) upstream of the AUG initiator of the gag gene. We studied, in vivo, the role of these uORFs by changing two or three nucleotides of the three AUGs or by deleting the first uORF. Our results show that (i) unlike most previously characterized uORFs, which decrease translation, the first uORF (AUG1) of RSV acts as an enhancer of translation, since absence of the first AUG decreased translation; AUG3 also modulates translation, probably by interfering with scanning ribosomes as described for other upstream ORFs, and mutation of AUG2 had no effect on translation. (ii) Mutation of each of the upstream AUGs lowered the infectivity of progeny virions. (iii) Unexpectedly, mutation of AUG1 and/or AUG3 dramatically reduced RNA packaging by 50-to 100-fold, unlike mutation of AUG2 which did not alter RNA packaging efficiency. Additional mutants in the vicinity of uORF1 and uORF3 were constructed in order to elucidate the mechanism by which uORFs affect RNA packaging: a translation model requiring uORFs 1 and 3, and involving ribosome pausing at AUG 3 is discussed. Images PMID:1327749

Evidence of translation efficiency adaptation of the coding regions of the bacteriophage lambda.

PubMed

Goz, Eli; Mioduser, Oriah; Diament, Alon; Tuller, Tamir

2017-08-01

Deciphering the way gene expression regulatory aspects are encoded in viral genomes is a challenging mission with ramifications related to all biomedical disciplines. Here, we aimed to understand how the evolution shapes the bacteriophage lambda genes by performing a high resolution analysis of ribosomal profiling data and gene expression related synonymous/silent information encoded in bacteriophage coding regions.We demonstrated evidence of selection for distinct compositions of synonymous codons in early and late viral genes related to the adaptation of translation efficiency to different bacteriophage developmental stages. Specifically, we showed that evolution of viral coding regions is driven, among others, by selection for codons with higher decoding rates; during the initial/progressive stages of infection the decoding rates in early/late genes were found to be superior to those in late/early genes, respectively. Moreover, we argued that selection for translation efficiency could be partially explained by adaptation to Escherichia coli tRNA pool and the fact that it can change during the bacteriophage life cycle.An analysis of additional aspects related to the expression of viral genes, such as mRNA folding and more complex/longer regulatory signals in the coding regions, is also reported. The reported conclusions are likely to be relevant also to additional viruses. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Efficient engineering of chromosomal ribosome binding site libraries in mismatch repair proficient Escherichia coli.

PubMed

Oesterle, Sabine; Gerngross, Daniel; Schmitt, Steven; Roberts, Tania Michelle; Panke, Sven

2017-09-26

Multiplexed gene expression optimization via modulation of gene translation efficiency through ribosome binding site (RBS) engineering is a valuable approach for optimizing artificial properties in bacteria, ranging from genetic circuits to production pathways. Established algorithms design smart RBS-libraries based on a single partially-degenerate sequence that efficiently samples the entire space of translation initiation rates. However, the sequence space that is accessible when integrating the library by CRISPR/Cas9-based genome editing is severely restricted by DNA mismatch repair (MMR) systems. MMR efficiency depends on the type and length of the mismatch and thus effectively removes potential library members from the pool. Rather than working in MMR-deficient strains, which accumulate off-target mutations, or depending on temporary MMR inactivation, which requires additional steps, we eliminate this limitation by developing a pre-selection rule of genome-library-optimized-sequences (GLOS) that enables introducing large functional diversity into MMR-proficient strains with sequences that are no longer subject to MMR-processing. We implement several GLOS-libraries in Escherichia coli and show that GLOS-libraries indeed retain diversity during genome editing and that such libraries can be used in complex genome editing operations such as concomitant deletions. We argue that this approach allows for stable and efficient fine tuning of chromosomal functions with minimal effort.

Application of sorting and next generation sequencing to study 5΄-UTR influence on translation efficiency in Escherichia coli

PubMed Central

Evfratov, Sergey A.; Osterman, Ilya A.; Komarova, Ekaterina S.; Pogorelskaya, Alexandra M.; Rubtsova, Maria P.; Zatsepin, Timofei S.; Semashko, Tatiana A.; Kostryukova, Elena S.; Mironov, Andrey A.; Burnaev, Evgeny; Krymova, Ekaterina; Gelfand, Mikhail S.; Govorun, Vadim M.; Bogdanov, Alexey A.; Dontsova, Olga A.

2017-01-01

Abstract Yield of protein per translated mRNA may vary by four orders of magnitude. Many studies analyzed the influence of mRNA features on the translation yield. However, a detailed understanding of how mRNA sequence determines its propensity to be translated is still missing. Here, we constructed a set of reporter plasmid libraries encoding CER fluorescent protein preceded by randomized 5΄ untranslated regions (5΄-UTR) and Red fluorescent protein (RFP) used as an internal control. Each library was transformed into Escherchia coli cells, separated by efficiency of CER mRNA translation by a cell sorter and subjected to next generation sequencing. We tested efficiency of translation of the CER gene preceded by each of 48 natural 5΄-UTR sequences and introduced random and designed mutations into natural and artificially selected 5΄-UTRs. Several distinct properties could be ascribed to a group of 5΄-UTRs most efficient in translation. In addition to known ones, several previously unrecognized features that contribute to the translation enhancement were found, such as low proportion of cytidine residues, multiple SD sequences and AG repeats. The latter could be identified as translation enhancer, albeit less efficient than SD sequence in several natural 5΄-UTRs. PMID:27899632

[Molecular mechanisms of protein biosynthesis initiation--biochemical and biomedical implications of a new model of translation enhanced by the RNA hypoxia response element (rHRE)].

PubMed

Master, Adam; Nauman, Alicja

2014-01-01

Translation initiation is a key rate-limiting step in cellular protein synthesis. A cap-dependent initiation is the most effective mechanism of the translation. However, some physiological (mitosis) and pathological (oxidative stress) processes may switch the classic mechanism to an alternative one that is regulated by an mRNA element such as IRES, uORF, IRE, CPE, DICE, AURE or CITE. A recently discovered mechanism of RNA hypoxia response element (rHRE)-dependent translation initiation, may change the view of oxygen-regulated translation and give a new insight into unexplained biochemical processes. Hypoxia is one of the better-known factors that may trigger an alternative mechanism of the translation initiation. Temporal events of oxygen deficiency within tissues and organs may activate processes such as angiogenesis, myogenesis, regeneration, wound healing, and may promote an adaptive response in cardiovascular and neurodegenerative diseases. On the other hand, growth of solid tumors may be accompanied by cyclic hypoxia, allowing for synthesis of proteins required for further progression of cancer cells. This paper provides a review of current knowledge on translational control in the context of alternative models of translation initiation.

A search for structurally similar cellular internal ribosome entry sites

PubMed Central

Baird, Stephen D.; Lewis, Stephen M.; Turcotte, Marcel; Holcik, Martin

2007-01-01

Internal ribosome entry sites (IRES) allow ribosomes to be recruited to mRNA in a cap-independent manner. Some viruses that impair cap-dependent translation initiation utilize IRES to ensure that the viral RNA will efficiently compete for the translation machinery. IRES are also employed for the translation of a subset of cellular messages during conditions that inhibit cap-dependent translation initiation. IRES from viruses like Hepatitis C and Classical Swine Fever virus share a similar structure/function without sharing primary sequence similarity. Of the cellular IRES structures derived so far, none were shown to share an overall structural similarity. Therefore, we undertook a genome-wide search of human 5′UTRs (untranslated regions) with an empirically derived structure of the IRES from the key inhibitor of apoptosis, X-linked inhibitor of apoptosis protein (XIAP), to identify novel IRES that share structure/function similarity. Three of the top matches identified by this search that exhibit IRES activity are the 5′UTRs of Aquaporin 4, ELG1 and NF-kappaB repressing factor (NRF). The structures of AQP4 and ELG1 IRES have limited similarity to the XIAP IRES; however, they share trans-acting factors that bind the XIAP IRES. We therefore propose that cellular IRES are not defined by overall structure, as viral IRES, but are instead dependent upon short motifs and trans-acting factors for their function. PMID:17591613

Mean of the typical decoding rates: a new translation efficiency index based on the analysis of ribosome profiling data.

PubMed

Dana, Alexandra; Tuller, Tamir

2014-12-01

Gene translation modeling and prediction is a fundamental problem that has numerous biomedical implementations. In this work we present a novel, user-friendly tool/index for calculating the mean of the typical decoding rates that enables predicting translation elongation efficiency of protein coding genes for different tissue types, developmental stages, and experimental conditions. The suggested translation efficiency index is based on the analysis of the organism's ribosome profiling data. This index could be used for example to predict changes in translation elongation efficiency of lowly expressed genes that usually have relatively low and/or biased ribosomal densities and protein levels measurements, or can be used for example for predicting translation efficiency of new genetically engineered genes. We demonstrate the usability of this index via the analysis of six organisms in different tissues and developmental stages. Distributable cross platform application and guideline are available for download at: http://www.cs.tau.ac.il/~tamirtul/MTDR/MTDR_Install.html. Copyright © 2015 Dana and Tuller.

Porcine reproductive and respiratory syndrome virus infection induces both eIF2α-phosphorylation-dependent and -independent host translation shutoff.

PubMed

Li, Yang; Fang, Liurong; Zhou, Yanrong; Tao, Ran; Wang, Dang; Xiao, Shaobo

2018-06-13

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has caused tremendous economic losses in the global swine industry since it was discovered in the late 1980s. Inducing host translation shutoff is a strategy used by many viruses to optimize their replication and spread. Here, we demonstrate that PRRSV infection causes host translation suppression, which is strongly dependent on viral replication. By screening PRRSV-encoded nonstructural proteins (nsps), we found that nsp2 participates in the induction of host translation shutoff and that its transmembrane (TM) domain is required for this process. Nsp2-induced translation suppression is independent of protein degradation pathways and the phosphorylation of eukaryotic initiation factor 2α (eIF2α). However, the overexpression of nsp2 or its TM domain significantly attenuated the mammalian target of rapamycin (mTOR) signaling pathway, an alternative pathway for modulating host gene expression. PRRSV infection also attenuated the mTOR signaling pathway, and PRRSV-induced host translation shutoff could be partly reversed when the attenuated mTOR phosphorylation was reactivated by an activator of the mTOR pathway. PRRSV infection still negatively regulated the host translation when the effects of eIF2α phosphorylation were completely reversed. Taken together, our results demonstrate that PRRSV infection induces host translation shutoff and that nsp2 is associated with this process. Both eIF2α phosphorylation and the attenuation of the mTOR signaling pathway contribute to PRRSV-induced host translation arrest. IMPORTANCE Viruses are obligate parasites, and the production of progeny viruses relies strictly on the host translation machinery. Therefore, the efficient modulation of host mRNA translation benefits viral replication, spread, and evolution. In this study, we provide evidence that porcine reproductive and respiratory syndrome virus (PRRSV) infection induces host translation shutoff and that the viral nonstructural protein nsp2 is associated with this process. Many viruses induce host translation shutoff by phosphorylating eukaryotic initiation factor 2α (eIF2α). However, PRRSV nsp2 does not induce eIF2α phosphorylation but attenuates the mTOR signaling pathway, another pathway regulating the host cell translational machinery. We also found that PRRSV-induced host translation shutoff was partly reversed by dephosphorylating eIF2α or reactivating the mTOR pathway, indicating that PRRSV infection induces both eIF2α-phosphorylation-dependent and -independent host translation shutoff. Copyright © 2018 American Society for Microbiology.

Hda Monomerization by ADP Binding Promotes Replicase Clamp-mediated DnaA-ATP Hydrolysis*S⃞

PubMed Central

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-01-01

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only ∼100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain. PMID:18977760

Hda monomerization by ADP binding promotes replicase clamp-mediated DnaA-ATP hydrolysis.

PubMed

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-12-26

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only approximately 100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.

Demonstration of GTG as an endogenous initiation codon for a human mRNA transcript revealed by molecular cloning of the serpin endopin 2B.

PubMed

Hwang, Shin-Rong; Garza, Christina Z; Wegrzyn, Jill; Hook, Vivian Y H

2004-08-16

This study demonstrates utilization of the novel GTG initiation codon for translation of a human mRNA transcript that encodes the serpin endopin 2B, a protease inhibitor. Molecular cloning revealed the nucleotide sequence of the human endopin 2B cDNA. Its deduced primary sequence shows high homology to bovine endopin 2A that possesses cross-class protease inhibition of elastase and papain. Notably, the human endopin 2B cDNA sequence revealed GTG as the predicted translation initiation codon; the predicted translation product of 46 kDa endopin 2B was produced by in vitro translation of 35S-endopin 2B with mammalian (rabbit) protein translation components. Importantly, bioinformatic studies demonstrated the presence of the entire human endopin 2B cDNA sequence with GTG as initiation codon within the human genome on chromosome 14. Further evidence for GTG as a functional initiation codon was illustrated by GTG-mediated in vitro translation of the heterologous protein EGFP, and by GTG-mediated expression of EGFP in mammalian PC12 cells. Mutagenesis of GTG to GTC resulted in the absence of EGFP expression in PC12 cells, indicating the function of GTG as an initiation codon. In addition, it was apparent that the GTG initiation codon produces lower levels of translated protein compared to ATG as initiation codon. Significantly, GTG-mediated translation of endopin 2B demonstrates a functional human gene product not previously predicted from initial analyses of the human genome. Further analyses based on GTG as an alternative initiation codon may predict new candidate genes of the human genome.

Two-step membrane binding by the bacterial SRP receptor enable efficient and accurate Co-translational protein targeting.

PubMed

Hwang Fu, Yu-Hsien; Huang, William Y C; Shen, Kuang; Groves, Jay T; Miller, Thomas; Shan, Shu-Ou

2017-07-28

The signal recognition particle (SRP) delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum, or the bacterial plasma membrane. The precise mechanism by which the bacterial SRP receptor, FtsY, interacts with and is regulated at the target membrane remain unclear. Here, quantitative analysis of FtsY-lipid interactions at single-molecule resolution revealed a two-step mechanism in which FtsY initially contacts membrane via a Dynamic mode, followed by an SRP-induced conformational transition to a Stable mode that activates FtsY for downstream steps. Importantly, mutational analyses revealed extensive auto-inhibitory mechanisms that prevent free FtsY from engaging membrane in the Stable mode; an engineered FtsY pre-organized into the Stable mode led to indiscriminate targeting in vitro and disrupted FtsY function in vivo. Our results show that the two-step lipid-binding mechanism uncouples the membrane association of FtsY from its conformational activation, thus optimizing the balance between the efficiency and fidelity of co-translational protein targeting.

A Novel Latin Hypercube Algorithm via Translational Propagation

PubMed Central

Pan, Guang; Ye, Pengcheng

2014-01-01

Metamodels have been widely used in engineering design to facilitate analysis and optimization of complex systems that involve computationally expensive simulation programs. The accuracy of metamodels is directly related to the experimental designs used. Optimal Latin hypercube designs are frequently used and have been shown to have good space-filling and projective properties. However, the high cost in constructing them limits their use. In this paper, a methodology for creating novel Latin hypercube designs via translational propagation and successive local enumeration algorithm (TPSLE) is developed without using formal optimization. TPSLE algorithm is based on the inspiration that a near optimal Latin Hypercube design can be constructed by a simple initial block with a few points generated by algorithm SLE as a building block. In fact, TPSLE algorithm offers a balanced trade-off between the efficiency and sampling performance. The proposed algorithm is compared to two existing algorithms and is found to be much more efficient in terms of the computation time and has acceptable space-filling and projective properties. PMID:25276844

Ribosome Profiling Reveals a Cell-Type-Specific Translational Landscape in Brain Tumors

PubMed Central

Gonzalez, Christian; Sims, Jennifer S.; Hornstein, Nicholas; Mela, Angeliki; Garcia, Franklin; Lei, Liang; Gass, David A.; Amendolara, Benjamin; Bruce, Jeffrey N.

2014-01-01

Glioma growth is driven by signaling that ultimately regulates protein synthesis. Gliomas are also complex at the cellular level and involve multiple cell types, including transformed and reactive cells in the brain tumor microenvironment. The distinct functions of the various cell types likely lead to different requirements and regulatory paradigms for protein synthesis. Proneural gliomas can arise from transformation of glial progenitors that are driven to proliferate via mitogenic signaling that affects translation. To investigate translational regulation in this system, we developed a RiboTag glioma mouse model that enables cell-type-specific, genome-wide ribosome profiling of tumor tissue. Infecting glial progenitors with Cre-recombinant retrovirus simultaneously activates expression of tagged ribosomes and delivers a tumor-initiating mutation. Remarkably, we find that although genes specific to transformed cells are highly translated, their translation efficiencies are low compared with normal brain. Ribosome positioning reveals sequence-dependent regulation of ribosomal activity in 5′-leaders upstream of annotated start codons, leading to differential translation in glioma compared with normal brain. Additionally, although transformed cells express a proneural signature, untransformed tumor-associated cells, including reactive astrocytes and microglia, express a mesenchymal signature. Finally, we observe the same phenomena in human disease by combining ribosome profiling of human proneural tumor and non-neoplastic brain tissue with computational deconvolution to assess cell-type-specific translational regulation. PMID:25122893

Norovirus translation requires an interaction between the C Terminus of the genome-linked viral protein VPg and eukaryotic translation initiation factor 4G.

PubMed

Chung, Liliane; Bailey, Dalan; Leen, Eoin N; Emmott, Edward P; Chaudhry, Yasmin; Roberts, Lisa O; Curry, Stephen; Locker, Nicolas; Goodfellow, Ian G

2014-08-01

Viruses have evolved a variety of mechanisms to usurp the host cell translation machinery to enable translation of the viral genome in the presence of high levels of cellular mRNAs. Noroviruses, a major cause of gastroenteritis in man, have evolved a mechanism that relies on the interaction of translation initiation factors with the virus-encoded VPg protein covalently linked to the 5' end of the viral RNA. To further characterize this novel mechanism of translation initiation, we have used proteomics to identify the components of the norovirus translation initiation factor complex. This approach revealed that VPg binds directly to the eIF4F complex, with a high affinity interaction occurring between VPg and eIF4G. Mutational analyses indicated that the C-terminal region of VPg is important for the VPg-eIF4G interaction; viruses with mutations that alter or disrupt this interaction are debilitated or non-viable. Our results shed new light on the unusual mechanisms of protein-directed translation initiation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cap analogs modified with 1,2-dithiodiphosphate moiety protect mRNA from decapping and enhance its translational potential

PubMed Central

Strenkowska, Malwina; Grzela, Renata; Majewski, Maciej; Wnek, Katarzyna; Kowalska, Joanna; Lukaszewicz, Maciej; Zuberek, Joanna; Darzynkiewicz, Edward; Kuhn, Andreas N.; Sahin, Ugur; Jemielity, Jacek

2016-01-01

Along with a growing interest in mRNA-based gene therapies, efforts are increasingly focused on reaching the full translational potential of mRNA, as a major obstacle for in vivo applications is sufficient expression of exogenously delivered mRNA. One method to overcome this limitation is chemically modifying the 7-methylguanosine cap at the 5′ end of mRNA (m7Gppp-RNA). We report a novel class of cap analogs designed as reagents for mRNA modification. The analogs carry a 1,2-dithiodiphosphate moiety at various positions along a tri- or tetraphosphate bridge, and thus are termed 2S analogs. These 2S analogs have high affinities for translation initiation factor 4E, and some exhibit remarkable resistance against the SpDcp1/2 decapping complex when introduced into RNA. mRNAs capped with 2S analogs combining these two features exhibit high translation efficiency in cultured human immature dendritic cells. These properties demonstrate that 2S analogs are potentially beneficial for mRNA-based therapies such as anti-cancer immunization. PMID:27903882

Eukaryotic translation initiation factor 4E availability controls the switch between cap-dependent and internal ribosomal entry site-mediated translation.

PubMed

Svitkin, Yuri V; Herdy, Barbara; Costa-Mattioli, Mauro; Gingras, Anne-Claude; Raught, Brian; Sonenberg, Nahum

2005-12-01

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5' end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.

Duplex unwinding and ATPase activities of the DEAD-box helicase eIF4A are coupled by eIF4G and eIF4B

PubMed Central

Özeş, Ali R.; Feoktistova, Kateryna; Avanzino, Brian C.; Fraser, Christopher S.

2011-01-01

Eukaryotic initiation factor 4A (eIF4A) is a DEAD-box helicase that stimulates translation initiation by unwinding mRNA secondary structure. The accessory proteins, eIF4G, eIF4B, and eIF4H enhance the duplex unwinding activity of eIF4A, but the extent to which they modulate eIF4A activity is poorly understood. Here, we use real time fluorescence assays to determine the kinetic parameters of duplex unwinding and ATP hydrolysis by these initiation factors. To ensure efficient duplex unwinding, eIF4B and eIF4G cooperatively activate the duplex unwinding activity of eIF4A. Our data reveal that eIF4H is much less efficient at stimulating eIF4A unwinding activity than eIF4B, implying that eIF4H is not able to completely substitute for eIF4B in duplex unwinding. By monitoring unwinding and ATPase assays using identical conditions, we demonstrate that eIF4B couples the ATP hydrolysis cycle of eIF4A with strand separation, thereby minimizing non-productive unwinding events. Using duplex substrates with altered GC contents, but with similar predicted thermal stabilities, we further show that the rate of formation of productive unwinding complexes is strongly influenced by the local stability per base pair in addition to the stability of the entire duplex. This finding explains how a change in the GC content of a hairpin while maintaining overall predicted thermal stability is able to influence translation initiation. PMID:21840318

Clinical and Translational Science Awards: can they increase the efficiency and speed of clinical and translational research?

PubMed

Heller, Caren; de Melo-Martín, Inmaculada

2009-04-01

Most agree that the recent decades-long boom in biomedical research discoveries has not had a sufficient effect on the public's health. To overcome some of the barriers to speeding clinical and translational (C/T) research, the National Institutes of Health has established the Institutional Clinical and Translational Science Award (CTSA). To explore whether the CTSA proposal addresses major C/T barriers and whether funded institutions offer adequate solutions, the authors reviewed the obstacles to C/T research described in the literature and examined the completeness of the solutions offered by the 12 initial CTSA awardees. Through an analysis of the literature, the authors categorized C/T barriers into three categories (research workforce, research operations, and organizational silos). They then analyzed each CTSA proposal regarding the types of programs offered to address these barriers. They found that, in general, institutions developed detailed programs to address research workforce and research operations barriers but had limited to no solutions for organizational silos. The authors suggest that differences in how barriers are addressed are consistent with the degree of control that CTSA centers have over these obstacles and solutions. They argue that although CTSA centers might have an important role in successfully addressing some of the barriers to C/T research, CTSA centers might ultimately have difficulties achieving their purported goal of facilitating and increasing the efficiency and speed of C/T research because of a lack of control over solutions to some important obstacles facing such research.

Temperature sensing in Yersinia pestis: translation of the LcrF activator protein is thermally regulated.

PubMed Central

Hoe, N P; Goguen, J D

1993-01-01

The lcrF gene of Yersinia pestis encodes a transcription activator responsible for inducing expression of several virulence-related proteins in response to temperature. The mechanism of this thermoregulation was investigated. An lcrF clone was found to produce much lower levels of LcrF protein at 26 than at 37 degrees C in Y. pestis, although it was transcribed at similar levels at both temperatures. High-level T7 polymerase-directed transcription of the lcrF gene in Escherichia coli also resulted in temperature-dependent production of the LcrF protein. Pulse-chase experiments showed that the LcrF protein was stable at 26 and 37 degrees C, suggesting that translation rate or message degradation is thermally controlled. The lcrF mRNA appears to be highly unstable and could not be reliably detected in Y. pestis. Insertion of the lcrF gene into plasmid pET4a, which produces high levels of plasmid-length RNA, aided detection of lcrF-specific message in E. coli. Comparison of the amount of LcrF protein produced per unit of message at 26 and 37 degrees C indicated that the efficiency of translation of lcrF message increased with temperature. mRNA secondary structure predictions suggest that the lcrF Shine-Dalgarno sequence is sequestered in a stem-loop. A model in which decreased stability of this stem-loop with increasing temperature leads to increased efficiency of translation initiation of lcrF message is presented. Images PMID:7504666

Improved initial guess with semi-subpixel level accuracy in digital image correlation by feature-based method

NASA Astrophysics Data System (ADS)

Zhang, Yunlu; Yan, Lei; Liou, Frank

2018-05-01

The quality initial guess of deformation parameters in digital image correlation (DIC) has a serious impact on convergence, robustness, and efficiency of the following subpixel level searching stage. In this work, an improved feature-based initial guess (FB-IG) scheme is presented to provide initial guess for points of interest (POIs) inside a large region. Oriented FAST and Rotated BRIEF (ORB) features are semi-uniformly extracted from the region of interest (ROI) and matched to provide initial deformation information. False matched pairs are eliminated by the novel feature guided Gaussian mixture model (FG-GMM) point set registration algorithm, and nonuniform deformation parameters of the versatile reproducing kernel Hilbert space (RKHS) function are calculated simultaneously. Validations on simulated images and real-world mini tensile test verify that this scheme can robustly and accurately compute initial guesses with semi-subpixel level accuracy in cases with small or large translation, deformation, or rotation.

Fabricating binary optics: An overview of binary optics process technology

NASA Technical Reports Server (NTRS)

Stern, Margaret B.

1993-01-01

A review of binary optics processing technology is presented. Pattern replication techniques have been optimized to generate high-quality efficient microoptics in visible and infrared materials. High resolution optical photolithography and precision alignment is used to fabricate maximally efficient fused silica diffractive microlenses at lambda = 633 nm. The degradation in optical efficiency of four-phase-level fused silica microlenses resulting from an intentional 0.35 micron translational error has been systematically measured as a function of lens speed (F/2 - F/60). Novel processes necessary for high sag refractive IR microoptics arrays, including deep anisotropic Si-etching, planarization of deep topography and multilayer resist techniques, are described. Initial results are presented for monolithic integration of photonic and microoptic systems.

Eukaryotic Translation Initiation Factor 4E Availability Controls the Switch between Cap-Dependent and Internal Ribosomal Entry Site-Mediated Translation†

PubMed Central

Svitkin, Yuri V.; Herdy, Barbara; Costa-Mattioli, Mauro; Gingras, Anne-Claude; Raught, Brian; Sonenberg, Nahum

2005-01-01

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5′ end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection. PMID:16287867

Binary translation using peephole translation rules

DOEpatents

Bansal, Sorav; Aiken, Alex

2010-05-04

An efficient binary translator uses peephole translation rules to directly translate executable code from one instruction set to another. In a preferred embodiment, the translation rules are generated using superoptimization techniques that enable the translator to automatically learn translation rules for translating code from the source to target instruction set architecture.

Collisions of excited Na atoms with H/sub 2/ molecules. I. Ab initio potential energy surfaces and qualitative discussion of the quenching process

DOE Office of Scientific and Technical Information (OSTI.GOV)

Botschwina, P.; Meyer, W.; Hertel, I.V.

Potential energy surfaces have been calculated for the four lowest electronic states of Na (3 /sup 2/S, 3 /sup 2/P)+H/sub 2/(/sup 1/..sigma../sup +//sub g/) by means of the RHF--SCF and PNO--CEPA methods. For the so-called quenching process of Na (3 /sup 2/P) by H/sub 2/ at low initial translational energies (E--VRT energy transfer) the energetically most favorable path occurs in C/sub 2v/ symmetry, since: at intermediate Na--H/sub 2/ separation: the A /sup 2/B/sub 2/ potential energy surface is attractive. From the CEPA calculations, the crossing point of minimal energy between the X /sup 2/A/sub 1/ and A /sup 2/B/sub 2/more » surfaces is obtained at R/sub c/ = 3.57 a.u. and r/sub c/ = 2.17 a.u. with an energy difference to the asymptotic limit (R = infinity, r = r/sub e/) of -0.06 eV. It is thus classically accessible without any initial translational energy, but at low initial translational energies (approx.0.1 eV) quenching will be efficient only for arrangements of collision partners close to C/sub 2v/ symmetry. There is little indication of an avoiding crossing with an ionic intermediate correlating asymptotically with Na/sup +/ and H/sub 2//sup -/ as was assumed in previous discussions of the quenching process. The dependence of the total quenching cross sections on the initial translational energy is discussed by means of the ''absorbing sphere'' model, taking the initial zero-point vibrational energy of the hydrogen molecule into account. New experimental data of the product channel distribution in H/sub 2/ for center-of-mass forward scattering are presented. The final vibrational states v' = 3, 2, 1, and 0 of H/sub 2/ are populated to about 26%, 61%, 13%, and 0%, respectively. The observed distributions in H/sub 2/ (and D/sub 2/) may be rationalized by simple dynamic considerations on the basis of the calculated surfaces.« less

Engineering bacterial translation initiation - Do we have all the tools we need?

PubMed

Vigar, Justin R J; Wieden, Hans-Joachim

2017-11-01

Reliable tools that allow precise and predictable control over gene expression are critical for the success of nearly all bioengineering applications. Translation initiation is the most regulated phase during protein biosynthesis, and is therefore a promising target for exerting control over gene expression. At the translational level, the copy number of a protein can be fine-tuned by altering the interaction between the translation initiation region of an mRNA and the ribosome. These interactions can be controlled by modulating the mRNA structure using numerous approaches, including small molecule ligands, RNAs, or RNA-binding proteins. A variety of naturally occurring regulatory elements have been repurposed, facilitating advances in synthetic gene regulation strategies. The pursuit of a comprehensive understanding of mechanisms governing translation initiation provides the framework for future engineering efforts. Here we outline state-of-the-art strategies used to predictably control translation initiation in bacteria. We also discuss current limitations in the field and future goals. Due to its function as the rate-determining step, initiation is the ideal point to exert effective translation regulation. Several engineering tools are currently available to rationally design the initiation characteristics of synthetic mRNAs. However, improvements are required to increase the predictability, effectiveness, and portability of these tools. Predictable and reliable control over translation initiation will allow greater predictability when designing, constructing, and testing genetic circuits. The ability to build more complex circuits predictably will advance synthetic biology and contribute to our fundamental understanding of the underlying principles of these processes. "This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

Influence of Translation Initiation on Organellar Protein Targeting in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sally A. Mackenzie

2011-04-18

A primary focus of the Mackenzie laboratory is the elucidation of processes and machinery for mitochondrial genome maintenance and transmission in higher plants. We have found that numerous organellar DNA maintenance components in plants appear to be dual targeted to mitochondria and plastids. Of particular interest was the observation that some twin (tandemly arrayed) dual targeting presequences appeared to utilize non-AUG alternative translation initiation, allowing for multiple translation starts at a single gene. Two aspects of this phenomenon were of particular interest: (1) Alternative translation initiation might provide a mechanism to regulate protein targeting temporally and spatially, a possibility thatmore » had not been demonstrated previously, and (2) alternative translation initiation might occur in genes involved in nuclear-controlled mitochondrial genome recombination, thought to be exclusively mitochondrial in their function. During the course of this research, we pursued three aims, with an emphasis on two specific genes of interest: POLgamma2, an organellar DNA polymerase, and MSH1, a MutS homolog thought to participate in mitochondrial, but not plastid, genome recombination surveillance. Our aims were to (1) Identify additional genes within Arabidopsis and other genomes that employ non-AUG alternative translation initiation, (2) Locate sequences upstream to the annotated AUG that confer alternative non-AUG translation initiation activity, and (3) Identify cis and trans factors that influence start site selection in genes with non-AUG starts. Toward these ends, we have shown that non-AUG initiation occurs in a number of genes, likely influencing targeting behavior of the protein. We have also shown that start site selection is strongly influenced by Kozak consensus sequence environment, indicating that alternative translation initiation in plants occurs by relaxation of ribosome scanning.« less

Tissue specific expression of the retinoic acid receptor-beta 2: regulation by short open reading frames in the 5'-noncoding region

PubMed Central

1994-01-01

The 40-S subunit of eukaryotic ribosomes binds to the capped 5'-end of mRNA and scans for the first AUG in a favorable sequence context to initiate translation. Most eukaryotic mRNAs therefore have a short 5'- untranslated region (5'-UTR) and no AUGs upstream of the translational start site; features that seem to assure efficient translation. However, approximately 5-10% of all eukaryotic mRNAs, particularly those encoding for regulatory proteins, have complex leader sequences that seem to compromise translational initiation. The retinoic-acid- receptor-beta 2 (RAR beta 2) mRNA is such a transcript with a long (461 nucleotides) 5'-UTR that contains five, partially overlapping, upstream open reading frames (uORFs) that precede the major ORF. We have begun to investigate the function of this complex 5'-UTR in transgenic mice, by introducing mutations in the start/stop codons of the uORFs in RAR beta 2-lacZ reporter constructs. When we compared the expression patterns of mutant and wild-type constructs we found that these mutations affected expression of the downstream RAR beta 2-ORF, resulting in an altered regulation of RAR beta 2-lacZ expression in heart and brain. Other tissues were unaffected. RNA analysis of adult tissues demonstrated that the uORFs act at the level of translation; adult brains and hearts of transgenic mice carrying a construct with either the wild-type or a mutant UTR, had the same levels of mRNA, but only the mutant produced protein. Our study outlines an unexpected role for uORFs: control of tissue-specific and developmentally regulated gene expression. PMID:7962071

Supporting knowledge translation through collaborative translational research initiatives: ‘Bridging’ versus ‘blurring’ boundary-spanning approaches in the UK CLAHRC initiative

PubMed Central

Evans, Sarah; Scarbrough, Harry

2014-01-01

Recent policy initiatives in the UK and internationally have sought to promote knowledge translation between the ‘producers’ and ‘users’ of research. Within this paper we explore how boundary-spanning interventions used within such initiatives can support knowledge translation between diverse groups. Using qualitative data from a 3-year research study conducted from January 2010 to December 2012 of two case-sites drawn from the CLAHRC initiative in the UK, we distinguish two different approaches to supporting knowledge translation; a ‘bridging’ approach that involves designated roles, discrete events and activities to span the boundaries between communities, and a ‘blurring’ approach that de-emphasises the boundaries between groups, enabling a more continuous process of knowledge translation as part of day-to-day work-practices. In this paper, we identify and differentiate these boundary-spanning approaches and describe how they emerged from the context defined by the wider CLAHRC networks. This highlights the need to develop a more contextualised analysis of the boundary-spanning that underpins knowledge translation processes, relating this to the distinctive features of a particular case. PMID:24561773

Engineering an efficient and tight D-amino acid-inducible gene expression system in Rhodosporidium/Rhodotorula species.

PubMed

Liu, Yanbin; Koh, Chong Mei John; Ngoh, Si Te; Ji, Lianghui

2015-10-26

Rhodosporidium and Rhodotorula are two genera of oleaginous red yeast with great potential for industrial biotechnology. To date, there is no effective method for inducible expression of proteins and RNAs in these hosts. We have developed a luciferase gene reporter assay based on a new codon-optimized LUC2 reporter gene (RtLUC2), which is flanked with CAR2 homology arms and can be integrated into the CAR2 locus in the nuclear genome at >90 % efficiency. We characterized the upstream DNA sequence of a D-amino acid oxidase gene (DAO1) from R. toruloides ATCC 10657 by nested deletions. By comparing the upstream DNA sequences of several putative DAO1 homologs of Basidiomycetous fungi, we identified a conserved DNA motif with a consensus sequence of AGGXXGXAGX11GAXGAXGG within a 0.2 kb region from the mRNA translation initiation site. Deletion of this motif led to strong mRNA transcription under non-inducing conditions. Interestingly, DAO1 promoter activity was enhanced about fivefold when the 108 bp intron 1 was included in the reporter construct. We identified a conserved CT-rich motif in the intron with a consensus sequence of TYTCCCYCTCCYCCCCACWYCCGA, deletion or point mutations of which drastically reduced promoter strength under both inducing and non-inducing conditions. Additionally, we created a selection marker-free DAO1-null mutant (∆dao1e) which displayed greatly improved inducible gene expression, particularly when both glucose and nitrogen were present in high levels. To avoid adding unwanted peptide to proteins to be expressed, we converted the original translation initiation codon to ATC and re-created a translation initiation codon at the start of exon 2. This promoter, named P DAO1-in1m1 , showed very similar luciferase activity to the wild-type promoter upon induction with D-alanine. The inducible system was tunable by adjusting the levels of inducers, carbon source and nitrogen source. The intron 1-containing DAO1 promoters coupled with a DAO1 null mutant makes an efficient and tight D-amino acid-inducible gene expression system in Rhodosporidium and Rhodotorula genera. The system will be a valuable tool for metabolic engineering and enzyme expression in these yeast hosts.

Structural domains within the HIV-1 mRNA and the ribosomal protein S25 influence cap-independent translation initiation.

PubMed

Carvajal, Felipe; Vallejos, Maricarmen; Walters, Beth; Contreras, Nataly; Hertz, Marla I; Olivares, Eduardo; Cáceres, Carlos J; Pino, Karla; Letelier, Alejandro; Thompson, Sunnie R; López-Lastra, Marcelo

2016-07-01

The 5' leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work, we examine the internal ribosome entry site (IRES) located in the 5' leader that drives translation initiation of the viral Gag protein under conditions that hinder cap-dependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25). Additionally, a mechanistic and mutational analysis revealed that the HIV-1 IRES is modular in nature and that once the 40S ribosomal subunit is recruited to the IRES, translation initiates without the need of ribosome scanning. These findings elucidate a mechanism of initiation by the HIV-1 IRES whereby a number of highly structured sites present within the HIV-1 5' leader leads to the recruitment of the 40S subunit directly at the site of initiation of protein synthesis. © 2016 Federation of European Biochemical Societies.

Structural domains within the HIV-1 mRNA and the ribosomal protein S25 influence cap-independent translation initiation

PubMed Central

Carvajal, Felipe; Vallejos, Maricarmen; Walters, Beth A.; Contreras, Nataly; Hertz, Marla I.; Olivares, Eduardo; Cáceres, C. Joaquín; Pino, Karla; Letelier, Alejandro; Thompson, Sunnie R.; López-Lastra, Marcelo

2016-01-01

The 5′leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work we examine the internal ribosome entry site (IRES) located in the 5′leader that drives translation initiation of the viral Gag protein under conditions that hinder cap-dependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25). Additionally, a mechanistic and mutational analysis revealed that the HIV-1 IRES is modular in nature and that once the 40S ribosomal subunit is recruited to the IRES, translation initiates without the need of ribosome scanning. These findings elucidate a mechanism of initiation by the HIV-1 IRES whereby a number of highly structured sites present within the HIV-1 5′leader leads to the recruitment of the 40S subunit directly at the site of initiation of protein synthesis. PMID:27191820

Eukaryotic Initiation Factor 4H Is under Transcriptional Control of p65/NF-κB

PubMed Central

Fiume, Giuseppe; Rossi, Annalisa; de Laurentiis, Annamaria; Falcone, Cristina; Pisano, Antonio; Vecchio, Eleonora; Pontoriero, Marilena; Scala, Iris; Scialdone, Annarita; Masci, Francesca Fasanella; Mimmi, Selena; Palmieri, Camillo; Scala, Giuseppe; Quinto, Ileana

2013-01-01

Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and spatial control of gene expression. Initiation of protein synthesis requires at least 13 translation initiation factors to assemble the 80S ribosomal initiation complex. Loss of translation control may result in cell malignant transformation. Here, we asked whether translational initiation factors could be regulated by NF-κB transcription factor, a major regulator of genes involved in cell proliferation, survival, and inflammatory response. We show that the p65 subunit of NF-κB activates the transcription of eIF4H gene, which is the regulatory subunit of eIF4A, the most relevant RNA helicase in translation initiation. The p65-dependent transcriptional activation of eIF4H increased the eIF4H protein content augmenting the rate of global protein synthesis. In this context, our results provide novel insights into protein synthesis regulation in response to NF-κB activation signalling, suggesting a transcription-translation coupled mechanism of control. PMID:23776612

Demonstration of GTG as an alternative initiation codon for the serpin endopin 2B-2.

PubMed

Hwang, Shin-Rong; Garza, Christina Z; Wegrzyn, Jill L; Hook, Vivian Y H

2005-02-18

This study demonstrates GTG as a novel, alternative initiation codon for translation of bovine endopin 2B-2, a serpin protease inhibitor. Molecular cDNA cloning revealed the endopin 2B-1 and endopin 2B-2 isoforms that are predicted to inhibit papain and elastase. Notably, GTG was demonstrated as the initiation codon for endopin 2B-2, whereas endopin 2B-1 possesses ATG as its initiation codon. GTG mediated in vitro translation of 46kDa endopin 2B-2. GTG also mediated translation of EGFP by in vitro translation and by expression in mammalian cells. Notably, mutagenesis of GTG to GTC resulted in the absence of EGFP expression in cells. GTG produced a lower level of protein expression compared to ATG. The use of GTG as an initiation codon to direct translation of endopin 2B, as well as the heterologous protein EGFP, demonstrates the role of GTG in the regulation of mRNA translation in mammalian cells. Significantly, further analyses of mammalian genomes based on GTG as an alternative initiation codon may predict new candidate gene products expressed by mammalian and human genomes.

Interfamilial recombination between viruses led to acquisition of a novel translation-enhancing RNA element that allows resistance breaking

PubMed Central

Miras, Manuel; Sempere, Raquel N.; Kraft, Jelena J.; Miller, W. Allen; Aranda, Miguel A.; Truniger, Veronica

2015-01-01

Summary Many plant viruses depend on functional RNA elements, called 3′-UTR cap-independent translation enhancers (3′-CITEs), for translation of their RNAs. In this manuscript we provide direct proof for the existing hypothesis that 3′-CITEs are modular and transferable by recombination in nature, and that this is associated with an advantage for the created virus. By characterizing a newly identified Melon necrotic spot virus (MNSV; Tombusviridae) isolate, which is able to overcome eukaryotic translation initiation factor 4E (eIF4E)-mediated resistance, we found that it contains a 55 nucleotide insertion in its 3′-UTR. We provide strong evidence that this insertion was acquired by interfamilial recombination with the 3′-UTR of an Asiatic Cucurbit aphid-borne yellows virus (CABYV; Luteoviridae). By constructing chimeric viruses, we showed that this recombined sequence is responsible for resistance breaking. Analysis of the translational efficiency of reporter constructs showed that this sequence functions as a novel 3′-CITE in both resistant and susceptible plants, being essential for translation control in resistant plants. In conclusion, we showed that a recombination event between two clearly identified viruses from different families led to the transfer of exactly the sequence corresponding to a functional RNA element, giving rise to a new isolate with the capacity to infect an otherwise non-susceptible host. PMID:24372390

Interfamilial recombination between viruses led to acquisition of a novel translation-enhancing RNA element that allows resistance breaking.

PubMed

Miras, Manuel; Sempere, Raquel N; Kraft, Jelena J; Miller, W Allen; Aranda, Miguel A; Truniger, Veronica

2014-04-01

Many plant viruses depend on functional RNA elements, called 3'-UTR cap-independent translation enhancers (3'-CITEs), for translation of their RNAs. In this manuscript we provide direct proof for the existing hypothesis that 3'-CITEs are modular and transferable by recombination in nature, and that this is associated with an advantage for the created virus. By characterizing a newly identified Melon necrotic spot virus (MNSV; Tombusviridae) isolate, which is able to overcome eukaryotic translation initiation factor 4E (eIF4E)-mediated resistance, we found that it contains a 55 nucleotide insertion in its 3'-UTR. We provide strong evidence that this insertion was acquired by interfamilial recombination with the 3'-UTR of an Asiatic Cucurbit aphid-borne yellows virus (CABYV; Luteoviridae). By constructing chimeric viruses, we showed that this recombined sequence is responsible for resistance breaking. Analysis of the translational efficiency of reporter constructs showed that this sequence functions as a novel 3'-CITE in both resistant and susceptible plants, being essential for translation control in resistant plants. In conclusion, we showed that a recombination event between two clearly identified viruses from different families led to the transfer of exactly the sequence corresponding to a functional RNA element, giving rise to a new isolate with the capacity to infect an otherwise nonsusceptible host. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

The Yeast Eukaryotic Translation Initiation Factor 2B Translation Initiation Complex Interacts with the Fatty Acid Synthesis Enzyme YBR159W and Endoplasmic Reticulum Membranes

PubMed Central

Browne, Christopher M.; Samir, Parimal; Fites, J. Scott; Villarreal, Seth A.

2013-01-01

Using affinity purifications coupled with mass spectrometry and yeast two-hybrid assays, we show the Saccharomyces cerevisiae translation initiation factor complex eukaryotic translation initiation factor 2B (eIF2B) and the very-long-chain fatty acid (VLCFA) synthesis keto-reductase enzyme YBR159W physically interact. The data show that the interaction is specifically between YBR159W and eIF2B and not between other members of the translation initiation or VLCFA pathways. A ybr159wΔ null strain has a slow-growth phenotype and a reduced translation rate but a normal GCN4 response to amino acid starvation. Although YBR159W localizes to the endoplasmic reticulum membrane, subcellular fractionation experiments show that a fraction of eIF2B cofractionates with lipid membranes in a YBR159W-independent manner. We show that a ybr159wΔ yeast strain and other strains with null mutations in the VLCFA pathway cause eIF2B to appear as numerous foci throughout the cytoplasm. PMID:23263984

Hypermethylated-capped selenoprotein mRNAs in mammals

PubMed Central

Wurth, Laurence; Gribling-Burrer, Anne-Sophie; Verheggen, Céline; Leichter, Michael; Takeuchi, Akiko; Baudrey, Stéphanie; Martin, Franck; Krol, Alain; Bertrand, Edouard; Allmang, Christine

2014-01-01

Mammalian mRNAs are generated by complex and coordinated biogenesis pathways and acquire 5′-end m7G caps that play fundamental roles in processing and translation. Here we show that several selenoprotein mRNAs are not recognized efficiently by translation initiation factor eIF4E because they bear a hypermethylated cap. This cap modification is acquired via a 5′-end maturation pathway similar to that of the small nucle(ol)ar RNAs (sn- and snoRNAs). Our findings also establish that the trimethylguanosine synthase 1 (Tgs1) interacts with selenoprotein mRNAs for cap hypermethylation and that assembly chaperones and core proteins devoted to sn- and snoRNP maturation contribute to recruiting Tgs1 to selenoprotein mRNPs. We further demonstrate that the hypermethylated-capped selenoprotein mRNAs localize to the cytoplasm, are associated with polysomes and thus translated. Moreover, we found that the activity of Tgs1, but not of eIF4E, is required for the synthesis of the GPx1 selenoprotein in vivo. PMID:25013170

The Implementation of Novel Collaborative Structures for the Identification and Resolution of Barriers to Pluripotent Stem Cell Translation

PubMed Central

Brindley, David A.; French, Anna; Suh, Jane; Roberts, MacKenna; Davies, Benjamin; Pinedo-Villanueva, Rafael; Wartolowska, Karolina; Rooke, Kelly; Kramm, Anneke; Judge, Andrew; Morrey, Mark; Chandra, Amit; Hurley, Hannah; Grover, Liam; Bingham, Ian; Siegel, Bernard; Rattley, Matt S.; Buckler, R. Lee; McKeon, David; Krumholz, Katie; Hook, Lilian; May, Michael; Rikabi, Sarah; Pigott, Rosie; Morys, Megan; Sabokbar, Afsie; Titus, Emily; Laabi, Yacine; Lemaitre, Gilles; Zahkia, Raymond; Sipp, Doug; Horne, Robert; Bravery, Christopher; Williams, David; Wall, Ivan; Snyder, Evan Y.; Karp, Jeffrey M.; Barker, Richard W.; Bure, Kim; Carr, Andrew J.; Reeve, Brock

2013-01-01

Abstract Increased global connectivity has catalyzed technological development in almost all industries, in part through the facilitation of novel collaborative structures. Notably, open innovation and crowd-sourcing—of expertise and/or funding—has tremendous potential to increase the efficiency with which biomedical ecosystems interact to deliver safe, efficacious and affordable therapies to patients. Consequently, such practices offer tremendous potential in advancing development of cellular therapies. In this vein, the CASMI Translational Stem Cell Consortium (CTSCC) was formed to unite global thought-leaders, producing academically rigorous and commercially practicable solutions to a range of challenges in pluripotent stem cell translation. Critically, the CTSCC research agenda is defined through continuous consultation with its international funding and research partners. Herein, initial findings for all research focus areas are presented to inform global product development strategies, and to stimulate continued industry interaction around biomanufacturing, strategic partnerships, standards, regulation and intellectual property and clinical adoption. PMID:24304079

Using the knowledge-to-action framework to guide the timing of dialysis initiation.

PubMed

Sood, Manish M; Manns, Braden; Nesrallah, Gihad

2014-05-01

The optimal time at which to initiate chronic dialysis remains unknown. Using a contemporary knowledge translation approach (the knowledge-to-action framework), a pan-Canadian collaboration (CANN-NET) set out to study the scope of the problem, then develop and disseminate evidence-based guidelines addressing the timing of dialysis initiation. The purpose of this review is to summarize the key findings and describe the planned Canadian knowledge translation strategy for improving knowledge and practices pertaining to the timing dialysis initiation. New research has provided considerable insights regarding the initiation of dialysis. A Canadian cohort study identified significant variation in the estimated glomerular filtration rate level at dialysis initiation, and a survey of providers identified related knowledge gaps that might be amenable to knowledge translation interventions. A recent knowledge synthesis/guideline concluded that early dialysis initiation is costly, and provides no measureable clinical benefits. A systematic knowledge translation intervention including a multifaceted approach may aid in reducing variation in practice and improving the quality of care. Utilizing the knowledge-to-action framework, we identified practice variation and key barriers to the optimal timing for dialysis initiation that may be amenable to knowledge translation strategies.

Interrelations between translation and general mRNA degradation in yeast

PubMed Central

Huch, Susanne; Nissan, Tracy

2014-01-01

Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system. How to cite this article: WIREs RNA 2014, 5:747–763. doi: 10.1002/wrna.1244 PMID:24944158

Interrelations between translation and general mRNA degradation in yeast.

PubMed

Huch, Susanne; Nissan, Tracy

2014-01-01

Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system. © 2014 The Authors. WIREs RNA published by John Wiley & Sons, Ltd.

Eukaryotic Initiation Factor 4E Binding Protien Family of Protiens: Sentinels at a Translational Control Checkpoint in Lung Tumor Defense

PubMed Central

Kim, Yong Y; Von Weymarn, Linda; Larsson, Ola; Fan, Danhua; Underwood, Jon M; Hecht, Stephen S; Polunovsky, Vitaly A; Bitterman, Peter B

2009-01-01

The usurping of translational control by sustained activation of translation initiation factors is oncogenic. Here we show that the primary negative regulators of these oncogenic initiation factors - the 4E-BP protein family - operate as guardians of a translational control checkpoint in lung tumor defense. When challenged with the tobacco carcinogen NNK, 4ebp1−/−/4ebp2−/− mice showed increased sensitivity to tumorigenesis compared to their wild type counterparts. The 4E-BP deficient state per se creates pro-oncogenic, genome-wide skewing of the molecular landscape - with translational activation of genes governing angiogenesis, growth and proliferation; and translational activation of the precise cytochrome p450 enzyme isoform (CYP2A5) that bioactivates NNK into mutagenic metabolites. Our study provides in vivo proof for a translational control checkpoint in lung tumor defense. PMID:19843855

Archaeal translation initiation revisited: the initiation factor 2 and eukaryotic initiation factor 2B alpha-beta-delta subunit families

NASA Technical Reports Server (NTRS)

Kyrpides, N. C.; Woese, C. R.

1998-01-01

As the amount of available sequence data increases, it becomes apparent that our understanding of translation initiation is far from comprehensive and that prior conclusions concerning the origin of the process are wrong. Contrary to earlier conclusions, key elements of translation initiation originated at the Universal Ancestor stage, for homologous counterparts exist in all three primary taxa. Herein, we explore the evolutionary relationships among the components of bacterial initiation factor 2 (IF-2) and eukaryotic IF-2 (eIF-2)/eIF-2B, i.e., the initiation factors involved in introducing the initiator tRNA into the translation mechanism and performing the first step in the peptide chain elongation cycle. All Archaea appear to posses a fully functional eIF-2 molecule, but they lack the associated GTP recycling function, eIF-2B (a five-subunit molecule). Yet, the Archaea do posses members of the gene family defined by the (related) eIF-2B subunits alpha, beta, and delta, although these are not specifically related to any of the three eukaryotic subunits. Additional members of this family also occur in some (but by no means all) Bacteria and even in some eukaryotes. The functional significance of the other members of this family is unclear and requires experimental resolution. Similarly, the occurrence of bacterial IF-2-like molecules in all Archaea and in some eukaryotes further complicates the picture of translation initiation. Overall, these data lend further support to the suggestion that the rudiments of translation initiation were present at the Universal Ancestor stage.

Translation initiation mediated by nuclear cap-binding protein complex.

PubMed

Ryu, Incheol; Kim, Yoon Ki

2017-04-01

In mammals, cap-dependent translation of mRNAs is initiated by two distinct mechanisms: cap-binding complex (CBC; a heterodimer of CBP80 and 20)-dependent translation (CT) and eIF4E-dependent translation (ET). Both translation initiation mechanisms share common features in driving cap- dependent translation; nevertheless, they can be distinguished from each other based on their molecular features and biological roles. CT is largely associated with mRNA surveillance such as nonsense-mediated mRNA decay (NMD), whereas ET is predominantly involved in the bulk of protein synthesis. However, several recent studies have demonstrated that CT and ET have similar roles in protein synthesis and mRNA surveillance. In a subset of mRNAs, CT preferentially drives the cap-dependent translation, as ET does, and ET is responsible for mRNA surveillance, as CT does. In this review, we summarize and compare the molecular features of CT and ET with a focus on the emerging roles of CT in translation. [BMB Reports 2017; 50(4): 186-193].

Role of messenger RNA-ribosome complex in complementary DNA display.

PubMed

Naimuddin, Mohammed; Ohtsuka, Isao; Kitamura, Koichiro; Kudou, Motonori; Kimura, Shinnosuke

2013-07-15

In vitro display technologies such as ribosome display and messenger RNA (mRNA)/complementary DNA (cDNA) display are powerful methods that can generate library diversities on the order of 10(10-14). However, in mRNA and cDNA display methods, the end use diversity is two orders of magnitude lower than initial diversity and is dependent on the downstream processes that act as limiting factors. We found that in our previous cDNA display protocol, the purification of protein fusions by the use of streptavidin matrices from cell-free translation mixtures had poor efficiency (∼10-15%) that seriously affected the diversity of the purified library. Here, we have investigated and optimized the protocols that provided remarkable purification efficiencies. The stalled ribosome in the mRNA-ribosome complex was found to impede this purification efficiency. Among the various conditions tested, destabilization of ribosomes by appropriate concentration of metal chelating agents in combination with an optimal temperature of 30°C were found to be crucial and effective for nearly complete isolation of protein fusions from the cell-free translation system. Thus, this protocol provided 8- to 10-fold increased efficiency of purification over the previous method and results in retaining the diversity of the library by approximately an order of magnitude-important for directed evolution. We also discuss the possible effects in the fabrication of protein chips. Copyright © 2013 Elsevier Inc. All rights reserved.

[Correlation of codon biases and potential secondary structures with mRNA translation efficiency in unicellular organisms].

PubMed

Vladimirov, N V; Likhoshvaĭ, V A; Matushkin, Iu G

2007-01-01

Gene expression is known to correlate with degree of codon bias in many unicellular organisms. However, such correlation is absent in some organisms. Recently we demonstrated that inverted complementary repeats within coding DNA sequence must be considered for proper estimation of translation efficiency, since they may form secondary structures that obstruct ribosome movement. We have developed a program for estimation of potential coding DNA sequence expression in defined unicellular organism using its genome sequence. The program computes elongation efficiency index. Computation is based on estimation of coding DNA sequence elongation efficiency, taking into account three key factors: codon bias, average number of inverted complementary repeats, and free energy of potential stem-loop structures formed by the repeats. The influence of these factors on translation is numerically estimated. An optimal proportion of these factors is computed for each organism individually. Quantitative translational characteristics of 384 unicellular organisms (351 bacteria, 28 archaea, 5 eukaryota) have been computed using their annotated genomes from NCBI GenBank. Five potential evolutionary strategies of translational optimization have been determined among studied organisms. A considerable difference of preferred translational strategies between Bacteria and Archaea has been revealed. Significant correlations between elongation efficiency index and gene expression levels have been shown for two organisms (S. cerevisiae and H. pylori) using available microarray data. The proposed method allows to estimate numerically the coding DNA sequence translation efficiency and to optimize nucleotide composition of heterologous genes in unicellular organisms. http://www.mgs.bionet.nsc.ru/mgs/programs/eei-calculator/.

Insights into factorless translational initiation by the tRNA-like pseudoknot domain of a viral IRES.

PubMed

Au, Hilda H T; Jan, Eric

2012-01-01

The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae family adopts an overlapping triple pseudoknot structure to directly recruit the 80S ribosome in the absence of initiation factors. The pseudoknot I (PKI) domain of the IRES mimics a tRNA-like codon:anticodon interaction in the ribosomal P site to direct translation initiation from a non-AUG initiation codon in the A site. In this study, we have performed a comprehensive mutational analysis of this region to delineate the molecular parameters that drive IRES translation. We demonstrate that IRES-mediated translation can initiate at an alternate adjacent and overlapping start site, provided that basepairing interactions within PKI remain intact. Consistent with this, IGR IRES translation tolerates increases in the variable loop region that connects the anticodon- and codon-like elements within the PKI domain, as IRES activity remains relatively robust up to a 4-nucleotide insertion in this region. Finally, elements from an authentic tRNA anticodon stem-loop can functionally supplant corresponding regions within PKI. These results verify the importance of the codon:anticodon interaction of the PKI domain and further define the specific elements within the tRNA-like domain that contribute to optimal initiator Met-tRNA(i)-independent IRES translation.

Insights into Factorless Translational Initiation by the tRNA-Like Pseudoknot Domain of a Viral IRES

PubMed Central

Au, Hilda H. T.; Jan, Eric

2012-01-01

The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae family adopts an overlapping triple pseudoknot structure to directly recruit the 80S ribosome in the absence of initiation factors. The pseudoknot I (PKI) domain of the IRES mimics a tRNA-like codon:anticodon interaction in the ribosomal P site to direct translation initiation from a non-AUG initiation codon in the A site. In this study, we have performed a comprehensive mutational analysis of this region to delineate the molecular parameters that drive IRES translation. We demonstrate that IRES-mediated translation can initiate at an alternate adjacent and overlapping start site, provided that basepairing interactions within PKI remain intact. Consistent with this, IGR IRES translation tolerates increases in the variable loop region that connects the anticodon- and codon-like elements within the PKI domain, as IRES activity remains relatively robust up to a 4-nucleotide insertion in this region. Finally, elements from an authentic tRNA anticodon stem-loop can functionally supplant corresponding regions within PKI. These results verify the importance of the codon:anticodon interaction of the PKI domain and further define the specific elements within the tRNA-like domain that contribute to optimal initiator Met-tRNAi-independent IRES translation. PMID:23236506

Antitumor activity and mechanism of action of the cyclopenta[b]benzofuran, silvestrol.

PubMed

Cencic, Regina; Carrier, Marilyn; Galicia-Vázquez, Gabriela; Bordeleau, Marie-Eve; Sukarieh, Rami; Bourdeau, Annie; Brem, Brigitte; Teodoro, Jose G; Greger, Harald; Tremblay, Michel L; Porco, John A; Pelletier, Jerry

2009-01-01

Flavaglines are a family of natural products from the genus Aglaia that exhibit anti-cancer activity in vitro and in vivo and inhibit translation initiation. They have been shown to modulate the activity of eIF4A, the DEAD-box RNA helicase subunit of the eukaryotic initiation factor (eIF) 4F complex, a complex that stimulates ribosome recruitment during translation initiation. One flavagline, silvestrol, is capable of modulating chemosensitivity in a mechanism-based mouse model. Among a number of flavagline family members tested herein, we find that silvestrol is the more potent translation inhibitor among these. We find that silvestrol impairs the ribosome recruitment step of translation initiation by affecting the composition of the eukaryotic initiation factor (eIF) 4F complex. We show that silvestrol exhibits significant anticancer activity in human breast and prostate cancer xenograft models, and that this is associated with increased apoptosis, decreased proliferation, and inhibition of angiogenesis. We demonstrate that targeting translation by silvestrol results in preferential inhibition of weakly initiating mRNAs. Our results indicate that silvestrol is a potent anti-cancer compound in vivo that exerts its activity by affecting survival pathways as well as angiogenesis. We propose that silvestrol mediates its effects by preferentially inhibiting translation of malignancy-related mRNAs. Silvestrol appears to be well tolerated in animals.

Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches

PubMed Central

Guedich, Sondés; Puffer-Enders, Barbara; Baltzinger, Mireille; Hoffmann, Guillaume; Da Veiga, Cyrielle; Jossinet, Fabrice; Thore, Stéphane; Bec, Guillaume; Ennifar, Eric; Burnouf, Dominique; Dumas, Philippe

2016-01-01

ABSTRACT Riboswitches are non-coding elements upstream or downstream of mRNAs that, upon binding of a specific ligand, regulate transcription and/or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitches regulating translation of thiM operon and transcription and translation of thiC operon in E. coli, and that of THIC in the plant A. thaliana. For all, we ascertained an induced-fit mechanism involving initial binding of the TPP followed by a conformational change leading to a higher-affinity complex. The experimental values obtained for all kinetic and thermodynamic parameters of TPP binding imply that the regulation by A. thaliana riboswitch is governed by mass-action law, whereas it is of kinetic nature for the two bacterial riboswitches. Kinetic regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentration range of TPP. We verified the existence of these pauses and the model prediction on their duration. Our analysis also led to quantitative estimates of the respective efficiency of kinetic and thermodynamic regulations, which shows that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation and confirms empirical observations that were obtained by numerical simulations. PMID:26932506

Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches.

PubMed

Guedich, Sondés; Puffer-Enders, Barbara; Baltzinger, Mireille; Hoffmann, Guillaume; Da Veiga, Cyrielle; Jossinet, Fabrice; Thore, Stéphane; Bec, Guillaume; Ennifar, Eric; Burnouf, Dominique; Dumas, Philippe

2016-01-01

Riboswitches are non-coding elements upstream or downstream of mRNAs that, upon binding of a specific ligand, regulate transcription and/or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitches regulating translation of thiM operon and transcription and translation of thiC operon in E. coli, and that of THIC in the plant A. thaliana. For all, we ascertained an induced-fit mechanism involving initial binding of the TPP followed by a conformational change leading to a higher-affinity complex. The experimental values obtained for all kinetic and thermodynamic parameters of TPP binding imply that the regulation by A. thaliana riboswitch is governed by mass-action law, whereas it is of kinetic nature for the two bacterial riboswitches. Kinetic regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentration range of TPP. We verified the existence of these pauses and the model prediction on their duration. Our analysis also led to quantitative estimates of the respective efficiency of kinetic and thermodynamic regulations, which shows that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation and confirms empirical observations that were obtained by numerical simulations.

PreTIS: A Tool to Predict Non-canonical 5’ UTR Translational Initiation Sites in Human and Mouse

PubMed Central

Reuter, Kerstin; Helms, Volkhard

2016-01-01

Translation of mRNA sequences into proteins typically starts at an AUG triplet. In rare cases, translation may also start at alternative non–AUG codons located in the annotated 5’ UTR which leads to an increased regulatory complexity. Since ribosome profiling detects translational start sites at the nucleotide level, the properties of these start sites can then be used for the statistical evaluation of functional open reading frames. We developed a linear regression approach to predict in–frame and out–of–frame translational start sites within the 5’ UTR from mRNA sequence information together with their translation initiation confidence. Predicted start codons comprise AUG as well as near–cognate codons. The underlying datasets are based on published translational start sites for human HEK293 and mouse embryonic stem cells that were derived by the original authors from ribosome profiling data. The average prediction accuracy of true vs. false start sites for HEK293 cells was 80%. When applied to mouse mRNA sequences, the same model predicted translation initiation sites observed in mouse ES cells with an accuracy of 76%. Moreover, we illustrate the effect of in silico mutations in the flanking sequence context of a start site on the predicted initiation confidence. Our new webservice PreTIS visualizes alternative start sites and their respective ORFs and predicts their ability to initiate translation. Solely, the mRNA sequence is required as input. PreTIS is accessible at http://service.bioinformatik.uni-saarland.de/pretis. PMID:27768687

Consequences of germline variation disrupting the constitutional translational initiation codon start sites of MLH1 and BRCA2: use of potential alternative start sites and implications for predicting variant pathogenicity

PubMed Central

Parsons, Michael T.; Whiley, Phillip J.; Beesley, Jonathan; Drost, Mark; de Wind, Niels; Thompson, Bryony A.; Marquart, Louise; Hopper, John L.; Jenkins, Mark A.; Brown, Melissa A.; Tucker, Kathy; Warwick, Linda; Buchanan, Daniel D.; Spurdle, Amanda B.

2014-01-01

Variants that disrupt the translation initiation sequences in cancer predisposition genes are generally assumed to be deleterious. However few studies have validated these assumptions with functional and clinical data. Two cancer syndrome gene variants likely to affect native translation initiation were identified by clinical genetic testing: MLH1:c.1A>G p.(Met1?) and BRCA2:c.67+3A>G. In vitro GFP-reporter assays were conducted to assess the consequences of translation initiation disruption on alternative downstream initiation codon usage. Analysis of MLH1:c.1A>G p.(Met1?) showed that translation was mostly initiated at an in-frame position 103 nucleotides downstream, but also at two ATG sequences downstream. The protein product encoded by the in-frame transcript initiating from position c.103 showed loss of in vitro mismatch repair activity comparable to known pathogenic mutations. BRCA2:c.67+3A>G was shown by mRNA analysis to result in an aberrantly spliced transcript deleting exon 2 and the consensus ATG site. In the absence of exon 2, translation initiated mostly at an out-of-frame ATG 323 nucleotides downstream, and to a lesser extent at an in-frame ATG 370 nucleotides downstream. Initiation from any of the downstream alternative sites tested in both genes would lead to loss of protein function, but further clinical data is required to confirm if these variants are associated with a high cancer risk. Importantly, our results highlight the need for caution in interpreting the functional and clinical consequences of variation that leads to disruption of the initiation codon, since translation may not necessarily occur from the first downstream alternative start site, or from a single alternative start site. PMID:24302565

47 CFR 74.1283 - Station identification.

Code of Federal Regulations, 2010 CFR

2010-10-01

..., AUXILIARY, SPECIAL BROADCAST AND OTHER PROGRAM DISTRIBUTIONAL SERVICES FM Broadcast Translator Stations and... translator station will consist of the initial letter K or W followed by the channel number assigned to the translator and two letters. The use of the initial letter will generally conform to the pattern used in the...

Upf1 senses 3′UTR length to potentiate mRNA decay

PubMed Central

Hogg, J. Robert; Goff, Stephen P.

2010-01-01

Summary The selective degradation of mRNAs by the nonsense-mediated decay pathway is a quality control process with important consequences for human disease. From initial studies using RNA hairpin-tagged mRNAs for purification of messenger ribonucleoproteins assembled on transcripts with HIV-1 3′ untranslated region (3′UTR) sequences, we uncover a two-step mechanism for Upf1-dependent degradation of mRNAs with long 3′UTRs. We demonstrate that Upf1 associates with mRNAs in a 3′UTR length-dependent manner and is highly enriched on transcripts containing 3′UTRs known to elicit NMD. Surprisingly, Upf1 recruitment and subsequent RNA decay can be antagonized by retroviral RNA elements that promote translational readthrough. By modulating the efficiency of translation termination, recognition of long 3′UTRs by Upf1 is uncoupled from the initiation of decay. We propose a model for 3′UTR length surveillance in which equilibrium binding of Upf1 to mRNAs precedes a kinetically distinct commitment to RNA decay. PMID:21029861

Co-adaption of tRNA gene copy number and amino acid usage influences translation rates in three life domains

PubMed Central

Du, Meng-Ze; Wei, Wen; Qin, Lei; Liu, Shuo; Zhang, An-Ying; Zhang, Yong; Zhou, Hong

2017-01-01

Abstract Although more and more entangled participants of translation process were realized, how they cooperate and co-determine the final translation efficiency still lacks details. Here, we reasoned that the basic translation components, tRNAs and amino acids should be consistent to maximize the efficiency and minimize the cost. We firstly revealed that 310 out of 410 investigated genomes of three domains had significant co-adaptions between the tRNA gene copy numbers and amino acid compositions, indicating that maximum efficiency constitutes ubiquitous selection pressure on protein translation. Furthermore, fast-growing and larger bacteria are found to have significantly better co-adaption and confirmed the effect of this pressure. Within organism, highly expressed proteins and those connected to acute responses have higher co-adaption intensity. Thus, the better co-adaption probably speeds up the growing of cells through accelerating the translation of special proteins. Experimentally, manipulating the tRNA gene copy number to optimize co-adaption between enhanced green fluorescent protein (EGFP) and tRNA gene set of Escherichia coli indeed lifted the translation rate (speed). Finally, as a newly confirmed translation rate regulating mechanism, the co-adaption reflecting translation rate not only deepens our understanding on translation process but also provides an easy and practicable method to improve protein translation rates and productivity. PMID:28992099

Modulation of translation-initiation in CHO-K1 cells by rapamycin-induced heterodimerization of engineered eIF4G fusion proteins.

PubMed

Schlatter, Stefan; Senn, Claudia; Fussenegger, Martin

2003-07-20

Translation-initiation is a predominant checkpoint in mammalian cells which controls protein synthesis and fine-tunes the flow of information from gene to protein. In eukaryotes, translation-initiation is typically initiated at a 7-methyl-guanylic acid cap posttranscriptionally linked to the 5' end of mRNAs. Alternative cap-independent translation-initiation involves 5' untranslated regions (UTR) known as internal ribosome entry sites, which adopt a particular secondary structure. Translation-initiating ribosome assembly at cap or IRES elements is mediated by a multiprotein complex of which the initiation factor 4F (eIF4F) consisting of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G is a major constituent. eIF4G is a key target of picornaviral protease 2A, which cleaves this initiation factor into eIF4G(Delta) and (Delta)eIF4G to redirect the cellular translation machinery exclusively to its own IRES-containing transcripts. We have designed a novel translation control system (TCS) for conditional as well as adjustable translation of cap- and IRES-dependent transgene mRNAs in mammalian cells. eIF4G(Delta) and (Delta)eIF4G were fused C- and N-terminally to the FK506-binding protein (FKBP) and the FKBP-rapamycin-binding domain (FRB) of the human FKBP-rapamycin-associated protein (FRAP), respectively. Rapamycin-induced heterodimerization of eIF4G(Delta)-FKBP and FRB-(Delta)eIF4G fusion proteins reconstituted a functional chimeric elongation factor 4G in a dose-dependent manner. Rigorous quantitative expression analysis of cap- and IRES-dependent SEAP- (human placental secreted alkaline phosphatase) and luc- (Photinus pyralis luciferase) encoding reporter constructs confirmed adjustable translation control and revealed increased production of desired proteins in response to dimerization-induced heterologous eIF4G in Chinese hamster ovary (CHO-K1) cells. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 210-225, 2003.

Machine-Aided Translation: From Terminology Banks to Interactive Translation Systems.

ERIC Educational Resources Information Center

Greenfield, Concetta C.; Serain, Daniel

The rapid growth of the need for technical translations in recent years has led specialists to utilize computer technology to improve the efficiency and quality of translation. The two approaches considered were automatic translation and terminology banks. Since the results of fully automatic translation were considered unsatisfactory by various…

Silibinin inhibits translation initiation: implications for anticancer therapy.

PubMed

Lin, Chen-Ju; Sukarieh, Rami; Pelletier, Jerry

2009-06-01

Silibinin is a nontoxic flavonoid reported to have anticancer properties. In this study, we show that silibinin exhibits antiproliferative activity on MCF-7 breast cancer cells. Exposure to silibinin leads to a concentration-dependent decrease in global protein synthesis associated with reduced levels of eukaryotic initiation factor 4F complex. Moreover, polysome profile analysis of silibinin-treated cells shows a decrease in polysome content and translation of cyclin D1 mRNA. Silibinin exerts its effects on translation initiation by inhibiting the mammalian target of rapamycin signaling pathway by acting upstream of TSC2. Our results show that silibinin blocks mammalian target of rapamycin signaling with a concomitant reduction in translation initiation, thus providing a possible molecular mechanism of how silibinin can inhibit growth of transformed cells.

A Herpesvirus Ribosome-Associated, RNA-Binding Protein Confers a Growth Advantage upon Mutants Deficient in a GADD34-Related Function†

PubMed Central

Mulvey, Matthew; Poppers, Jeremy; Ladd, Alison; Mohr, Ian

1999-01-01

The herpes simplex virus type 1 γ34.5 gene product and the cellular GADD34 protein both contain similar domains that can regulate the activity of eukaryotic initiation factor 2 (eIF2), a critical translation initiation factor. Viral mutants that lack the GADD34-related function grow poorly on a variety of malignant human cells, as activation of the cellular PKR kinase leads to the accumulation of inactive, phosphorylated eIF2 at late times postinfection. Termination of translation prior to the completion of the viral reproductive cycle leads to impaired growth. Extragenic suppressors that regain the ability to synthesize proteins efficiently in the absence of the viral GADD34-related function have been isolated. These suppressor alleles are dominant in trans and affect the steady-state accumulation of several viral mRNA species. We demonstrate that deregulated expression of Us11, a virus-encoded RNA-binding, ribosome-associated protein is necessary and sufficient to confer a growth advantage upon viral mutants that lack a GADD34-related function. Ectopic expression of Us11 reduces the accumulation of the activated cellular PKR kinase and allows for sustained protein synthesis. Thus, an RNA-binding, ribosome-associated protein (Us11) and a GADD34-related protein (γ34.5) both function in a signal pathway that regulates translation by modulating eIF2 phosphorylation. PMID:10074192

4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression

PubMed Central

Shives, Katherine D.; Massey, Aaron R.; May, Nicholas A.; Morrison, Thomas E.; Beckham, J. David

2016-01-01

West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome. PMID:27763553

An efficient blocking M2L translation for low-frequency fast multipole method in three dimensions

NASA Astrophysics Data System (ADS)

Takahashi, Toru; Shimba, Yuta; Isakari, Hiroshi; Matsumoto, Toshiro

2016-05-01

We propose an efficient scheme to perform the multipole-to-local (M2L) translation in the three-dimensional low-frequency fast multipole method (LFFMM). Our strategy is to combine a group of matrix-vector products associated with M2L translation into a matrix-matrix product in order to diminish the memory traffic. For this purpose, we first developed a grouping method (termed as internal blocking) based on the congruent transformations (rotational and reflectional symmetries) of M2L-translators for each target box in the FMM hierarchy (adaptive octree). Next, we considered another method of grouping (termed as external blocking) that was able to handle M2L translations for multiple target boxes collectively by using the translational invariance of the M2L translation. By combining these internal and external blockings, the M2L translation can be performed efficiently whilst preservingthe numerical accuracy exactly. We assessed the proposed blocking scheme numerically and applied it to the boundary integral equation method to solve electromagnetic scattering problems for perfectly electrical conductor. From the numerical results, it was found that the proposed M2L scheme achieved a few times speedup compared to the non-blocking scheme.

Improved transcription and translation with L-leucine stimulation of mTORC1 in Roberts syndrome.

PubMed

Xu, Baoshan; Gogol, Madelaine; Gaudenz, Karin; Gerton, Jennifer L

2016-01-05

Roberts syndrome (RBS) is a human developmental disorder caused by mutations in the cohesin acetyltransferase ESCO2. We previously reported that mTORC1 signaling was depressed and overall translation was reduced in RBS cells and zebrafish models for RBS. Treatment of RBS cells and zebrafish RBS models with L-leucine partially rescued mTOR function and protein synthesis, correlating with increased cell division and improved development. In this study, we use RBS cells to model mTORC1 repression and analyze transcription and translation with ribosome profiling to determine gene-level effects of L-leucine. L-leucine treatment partially rescued translational efficiency of ribosomal subunits, translation initiation factors, snoRNA production, and mitochondrial function in RBS cells, consistent with these processes being mTORC1 controlled. In contrast, other genes are differentially expressed independent of L-leucine treatment, including imprinted genes such as H19 and GTL2, miRNAs regulated by GTL2, HOX genes, and genes in nucleolar associated domains. Our study distinguishes between gene expression changes in RBS cells that are TOR dependent and those that are independent. Some of the TOR independent gene expression changes likely reflect the architectural role of cohesin in chromatin looping and gene expression. This study reveals the dramatic rescue effects of L-leucine stimulation of mTORC1 in RBS cells and supports that normal gene expression and translation requires ESCO2 function.

TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h

PubMed Central

Schepetilnikov, Mikhail; Dimitrova, Maria; Mancera-Martínez, Eder; Geldreich, Angèle; Keller, Mario; Ryabova, Lyubov A

2013-01-01

Mammalian target-of-rapamycin (mTOR) triggers S6 kinase (S6K) activation to phosphorylate targets linked to translation in response to energy, nutrients, and hormones. Pathways of TOR activation in plants remain unknown. Here, we uncover the role of the phytohormone auxin in TOR signalling activation and reinitiation after upstream open reading frame (uORF) translation, which in plants is dependent on translation initiation factor eIF3h. We show that auxin triggers TOR activation followed by S6K1 phosphorylation at T449 and efficient loading of uORF-mRNAs onto polysomes in a manner sensitive to the TOR inhibitor Torin-1. Torin-1 mediates recruitment of inactive S6K1 to polysomes, while auxin triggers S6K1 dissociation and recruitment of activated TOR instead. A putative target of TOR/S6K1—eIF3h—is phosphorylated and detected in polysomes in response to auxin. In TOR-deficient plants, polysomes were prebound by inactive S6K1, and loading of uORF-mRNAs and eIF3h was impaired. Transient expression of eIF3h-S178D in plant protoplasts specifically upregulates uORF-mRNA translation. We propose that TOR functions in polysomes to maintain the active S6K1 (and thus eIF3h) phosphorylation status that is critical for translation reinitiation. PMID:23524850

Expression of Human Hemojuvelin (HJV) Is Tightly Regulated by Two Upstream Open Reading Frames in HJV mRNA That Respond to Iron Overload in Hepatic Cells

PubMed Central

Onofre, Cláudia; Tomé, Filipa; Barbosa, Cristina; Silva, Ana Luísa

2015-01-01

The gene encoding human hemojuvelin (HJV) is one of the genes that, when mutated, can cause juvenile hemochromatosis, an early-onset inherited disorder associated with iron overload. The 5′ untranslated region of the human HJV mRNA has two upstream open reading frames (uORFs), with 28 and 19 codons formed by two upstream AUGs (uAUGs) sharing the same in-frame stop codon. Here we show that these uORFs decrease the translational efficiency of the downstream main ORF in HeLa and HepG2 cells. Indeed, ribosomal access to the main AUG is conditioned by the strong uAUG context, which results in the first uORF being translated most frequently. The reach of the main ORF is then achieved by ribosomes that resume scanning after uORF translation. Furthermore, the amino acid sequences of the uORF-encoded peptides also reinforce the translational repression of the main ORF. Interestingly, when iron levels increase, translational repression is relieved specifically in hepatic cells. The upregulation of protein levels occurs along with phosphorylation of the eukaryotic initiation factor 2α. Nevertheless, our results support a model in which the increasing recognition of the main AUG is mediated by a tissue-specific factor that promotes uORF bypass. These results support a tight HJV translational regulation involved in iron homeostasis. PMID:25666510

Antitumor Activity and Mechanism of Action of the Cyclopenta[b]benzofuran, Silvestrol

PubMed Central

Cencic, Regina; Carrier, Marilyn; Galicia-Vázquez, Gabriela; Bordeleau, Marie-Eve; Sukarieh, Rami; Bourdeau, Annie; Brem, Brigitte; Teodoro, Jose G.; Greger, Harald; Tremblay, Michel L.; Porco, John A.; Pelletier, Jerry

2009-01-01

Background Flavaglines are a family of natural products from the genus Aglaia that exhibit anti-cancer activity in vitro and in vivo and inhibit translation initiation. They have been shown to modulate the activity of eIF4A, the DEAD-box RNA helicase subunit of the eukaryotic initiation factor (eIF) 4F complex, a complex that stimulates ribosome recruitment during translation initiation. One flavagline, silvestrol, is capable of modulating chemosensitivity in a mechanism-based mouse model. Methodology/Principal Findings Among a number of flavagline family members tested herein, we find that silvestrol is the more potent translation inhibitor among these. We find that silvestrol impairs the ribosome recruitment step of translation initiation by affecting the composition of the eukaryotic initiation factor (eIF) 4F complex. We show that silvestrol exhibits significant anticancer activity in human breast and prostate cancer xenograft models, and that this is associated with increased apoptosis, decreased proliferation, and inhibition of angiogenesis. We demonstrate that targeting translation by silvestrol results in preferential inhibition of weakly initiating mRNAs. Conclusions/Significance Our results indicate that silvestrol is a potent anti-cancer compound in vivo that exerts its activity by affecting survival pathways as well as angiogenesis. We propose that silvestrol mediates its effects by preferentially inhibiting translation of malignancy-related mRNAs. Silvestrol appears to be well tolerated in animals. PMID:19401772

Web application for automatic prediction of gene translation elongation efficiency.

PubMed

Sokolov, Vladimir; Zuraev, Bulat; Lashin, Sergei; Matushkin, Yury

2015-09-03

Expression efficiency is one of the major characteristics describing genes in various modern investigations. Expression efficiency of genes is regulated at various stages: transcription, translation, posttranslational protein modification and others. In this study, a special EloE (Elongation Efficiency) web application is described. The EloE sorts the organism's genes in a descend order on their theoretical rate of the elongation stage of translation based on the analysis of their nucleotide sequences. Obtained theoretical data have a significant correlation with available experimental data of gene expression in various organisms. In addition, the program identifies preferential codons in organism's genes and defines distribution of potential secondary structures energy in 5´ and 3´ regions of mRNA. The EloE can be useful in preliminary estimation of translation elongation efficiency for genes for which experimental data are not available yet. Some results can be used, for instance, in other programs modeling artificial genetic structures in genetically engineered experiments.

Co-adaption of tRNA gene copy number and amino acid usage influences translation rates in three life domains.

PubMed

Du, Meng-Ze; Wei, Wen; Qin, Lei; Liu, Shuo; Zhang, An-Ying; Zhang, Yong; Zhou, Hong; Guo, Feng-Biao

2017-12-01

Although more and more entangled participants of translation process were realized, how they cooperate and co-determine the final translation efficiency still lacks details. Here, we reasoned that the basic translation components, tRNAs and amino acids should be consistent to maximize the efficiency and minimize the cost. We firstly revealed that 310 out of 410 investigated genomes of three domains had significant co-adaptions between the tRNA gene copy numbers and amino acid compositions, indicating that maximum efficiency constitutes ubiquitous selection pressure on protein translation. Furthermore, fast-growing and larger bacteria are found to have significantly better co-adaption and confirmed the effect of this pressure. Within organism, highly expressed proteins and those connected to acute responses have higher co-adaption intensity. Thus, the better co-adaption probably speeds up the growing of cells through accelerating the translation of special proteins. Experimentally, manipulating the tRNA gene copy number to optimize co-adaption between enhanced green fluorescent protein (EGFP) and tRNA gene set of Escherichia coli indeed lifted the translation rate (speed). Finally, as a newly confirmed translation rate regulating mechanism, the co-adaption reflecting translation rate not only deepens our understanding on translation process but also provides an easy and practicable method to improve protein translation rates and productivity. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Sepsis and mechnaical ventilation restrain translation initiation in skeletal muscle by inducing AMPK-associated TSC[2] restriction of mTOR signaling in pigs

USDA-ARS?s Scientific Manuscript database

In skeletal muscle, AMP-activated protein kinase (AMPK) acts as a cellular energy sensor of AMP: ATP and modulates translation by repressing mammalian target of rapamycin (mTOR) activation. Endotoxin (LPS)-induced sepsis reduces muscle protein synthesis by blunting translation initiation. We hypothe...

The 5′ Untranslated Region of the Human T-Cell Lymphotropic Virus Type 1 mRNA Enables Cap-Independent Translation Initiation

PubMed Central

Olivares, Eduardo; Landry, Dori M.; Cáceres, C. Joaquín; Pino, Karla; Rossi, Federico; Navarrete, Camilo; Huidobro-Toro, Juan Pablo; Thompson, Sunnie R.

2014-01-01

ABSTRACT The human T-cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis. The mRNA of some complex retroviruses, including the human and simian immunodeficiency viruses (HIV and SIV), can initiate translation using a canonical cap-dependent mechanism or through an internal ribosome entry site (IRES). In this study, we present strong evidence showing that like HIV-1 and SIV, the 5′-untranslated region (5′UTR) of the HTLV-1 full-length mRNA harbors an IRES. Cap-independent translational activity was evaluated and demonstrated using dual luciferase bicistronic mRNAs in rabbit reticulocyte lysate, in mammalian cell culture, and in Xenopus laevis oocytes. Characterization of the HTLV-1 IRES shows that its activity is dependent on the ribosomal protein S25 (RPS25) and that its function is highly sensitive to the drug edeine. Together, these findings suggest that the 5′UTR of the HTLV-1 full-length mRNA enables internal recruitment of the eukaryotic translation initiation complex. However, the recognition of the initiation codon requires ribosome scanning. These results suggest that, after internal recruitment by the HTLV-1 IRES, a scanning step takes place for the 40S ribosomal subunit to be positioned at the translation initiation codon. IMPORTANCE The mechanism by which retroviral mRNAs recruit the 40S ribosomal subunit internally is not understood. This study provides new insights into the mechanism of translation initiation used by the human T-cell lymphotropic virus type 1 (HTLV-1). The results show that the HTLV-1 mRNA can initiate translation via a noncanonical mechanism mediated by an internal ribosome entry site (IRES). This study also provides evidence showing the involvement of cellular proteins in HTLV-1 IRES-mediated translation initiation. Together, the data presented in this report significantly contribute to the understanding of HTLV-1 gene expression. PMID:24623421

The XPO1 inhibitor Selinexor inhibits translation and enhances the radiosensitivity of glioblastoma cells grown in vitro and in vivo.

PubMed

Wahba, Amy; Rath, Barbara H; O'Neill, John W; Camphausen, Kevin; Tofilon, Philip J

2018-06-04

Analysis of the radiation-induced translatome of glioblastoma stem-like cells (GSCs) identified an interacting network in which XPO1 serves as a major hub protein. To determine whether this nuclear export protein provides a target for radiosensitization, we defined the effects of the clinically relevant XPO1 inhibitor Selinexor on the radiosensitivity of glioblastoma cells. As determined by clonogenic survival analysis, Selinexor enhanced the radiosensitivity of GSCs but not normal fibroblast cell lines. Based on γH2AX foci and neutral comet analyses, Selinexor inhibited the repair of radiation-induced DNA double strand breaks in GSCs suggesting that the Selinexor-induced radiosensitization is mediated by an inhibition of DNA repair. Consistent with a role for XPO1 in the nuclear to cytoplasm export of rRNA, Selinexor reduced 5S and 18S rRNA nuclear export in GSCs, which was accompanied by a decrease in gene translation efficiency, as determined from polysome profiles, as well as in protein synthesis. In contrast, rRNA nuclear export and protein synthesis were not reduced in normal cells treated with Selinexor. Orthotopic xenografts initiated from a GSC line were then used to define the in vivo response to Selinexor and radiation. Treatment of mice bearing orthotopic xenografts with Selinexor decreased tumor translational efficiency as determined from polysome profiles. Although Selinexor treatment alone had no effect on the survival of mice with brain tumors, it significantly enhanced the radiation-induced prolongation of survival. These results indicate that Selinexor enhances the radiosensitivity of glioblastoma cells and suggest that this effect involves a global inhibition of gene translation. Copyright ©2018, American Association for Cancer Research.

Accumulating Evidence and Research Organization (AERO) model: a new tool for representing, analyzing, and planning a translational research program.

PubMed

Hey, Spencer Phillips; Heilig, Charles M; Weijer, Charles

2013-05-30

Maximizing efficiency in drug development is important for drug developers, policymakers, and human subjects. Limited funds and the ethical imperative of risk minimization demand that researchers maximize the knowledge gained per patient-subject enrolled. Yet, despite a common perception that the current system of drug development is beset by inefficiencies, there remain few approaches for systematically representing, analyzing, and communicating the efficiency and coordination of the research enterprise. In this paper, we present the first steps toward developing such an approach: a graph-theoretic tool for representing the Accumulating Evidence and Research Organization (AERO) across a translational trajectory. This initial version of the AERO model focuses on elucidating two dimensions of robustness: (1) the consistency of results among studies with an identical or similar outcome metric; and (2) the concordance of results among studies with qualitatively different outcome metrics. The visual structure of the model is a directed acyclic graph, designed to capture these two dimensions of robustness and their relationship to three basic questions that underlie the planning of a translational research program: What is the accumulating state of total evidence? What has been the translational trajectory? What studies should be done next? We demonstrate the utility of the AERO model with an application to a case study involving the antibacterial agent, moxifloxacin, for the treatment of drug-susceptible tuberculosis. We then consider some possible elaborations for the AERO model and propose a number of ways in which the tool could be used to enhance the planning, reporting, and analysis of clinical trials. The AERO model provides an immediate visual representation of the number of studies done at any stage of research, depicting both the robustness of evidence and the relationship of each study to the larger translational trajectory. In so doing, it makes some of the invisible or inchoate properties of the research system explicit - helping to elucidate judgments about the accumulating state of evidence and supporting decision-making for future research.

Rolling Circle Translation of Circular RNA in Living Human Cells.

PubMed

Abe, Naoko; Matsumoto, Ken; Nishihara, Mizuki; Nakano, Yukiko; Shibata, Aya; Maruyama, Hideto; Shuto, Satoshi; Matsuda, Akira; Yoshida, Minoru; Ito, Yoshihiro; Abe, Hiroshi

2015-11-10

We recently reported that circular RNA is efficiently translated by a rolling circle amplification (RCA) mechanism in a cell-free Escherichia coli translation system. Recent studies have shown that circular RNAs composed of exonic sequences are abundant in human cells. However, whether these circular RNAs can be translated into proteins within cells remains unclear. In this study, we prepared circular RNAs with an infinite open reading frame and tested their translation in eukaryotic systems. Circular RNAs were translated into long proteins in rabbit reticulocyte lysate in the absence of any particular element for internal ribosome entry, a poly-A tail, or a cap structure. The translation systems in eukaryote can accept much simpler RNA as a template for protein synthesis by cyclisation. Here, we demonstrated that the circular RNA is efficiently translated in living human cells to produce abundant protein product by RCA mechanism. These findings suggest that translation of exonic circular RNAs present in human cells is more probable than previously thought.

Rolling Circle Translation of Circular RNA in Living Human Cells

PubMed Central

Abe, Naoko; Matsumoto, Ken; Nishihara, Mizuki; Nakano, Yukiko; Shibata, Aya; Maruyama, Hideto; Shuto, Satoshi; Matsuda, Akira; Yoshida, Minoru; Ito, Yoshihiro; Abe, Hiroshi

2015-01-01

We recently reported that circular RNA is efficiently translated by a rolling circle amplification (RCA) mechanism in a cell-free Escherichia coli translation system. Recent studies have shown that circular RNAs composed of exonic sequences are abundant in human cells. However, whether these circular RNAs can be translated into proteins within cells remains unclear. In this study, we prepared circular RNAs with an infinite open reading frame and tested their translation in eukaryotic systems. Circular RNAs were translated into long proteins in rabbit reticulocyte lysate in the absence of any particular element for internal ribosome entry, a poly-A tail, or a cap structure. The translation systems in eukaryote can accept much simpler RNA as a template for protein synthesis by cyclisation. Here, we demonstrated that the circular RNA is efficiently translated in living human cells to produce abundant protein product by RCA mechanism. These findings suggest that translation of exonic circular RNAs present in human cells is more probable than previously thought. PMID:26553571

Computational Modeling and Analysis of Insulin Induced Eukaryotic Translation Initiation

PubMed Central

Lequieu, Joshua; Chakrabarti, Anirikh; Nayak, Satyaprakash; Varner, Jeffrey D.

2011-01-01

Insulin, the primary hormone regulating the level of glucose in the bloodstream, modulates a variety of cellular and enzymatic processes in normal and diseased cells. Insulin signals are processed by a complex network of biochemical interactions which ultimately induce gene expression programs or other processes such as translation initiation. Surprisingly, despite the wealth of literature on insulin signaling, the relative importance of the components linking insulin with translation initiation remains unclear. We addressed this question by developing and interrogating a family of mathematical models of insulin induced translation initiation. The insulin network was modeled using mass-action kinetics within an ordinary differential equation (ODE) framework. A family of model parameters was estimated, starting from an initial best fit parameter set, using 24 experimental data sets taken from literature. The residual between model simulations and each of the experimental constraints were simultaneously minimized using multiobjective optimization. Interrogation of the model population, using sensitivity and robustness analysis, identified an insulin-dependent switch that controlled translation initiation. Our analysis suggested that without insulin, a balance between the pro-initiation activity of the GTP-binding protein Rheb and anti-initiation activity of PTEN controlled basal initiation. On the other hand, in the presence of insulin a combination of PI3K and Rheb activity controlled inducible initiation, where PI3K was only critical in the presence of insulin. Other well known regulatory mechanisms governing insulin action, for example IRS-1 negative feedback, modulated the relative importance of PI3K and Rheb but did not fundamentally change the signal flow. PMID:22102801

Translation and cultural adaptation for Brazil of the Developing Nurses' Thinking model1

PubMed Central

Jensen, Rodrigo; da Cruz, Diná de Almeida Lopes Monteiro; Tesoro, Mary Gay; Lopes, Maria Helena Baena de Moraes

2014-01-01

Objectives to translate and culturally adapt to Brazilian Portuguese the Developing Nurses' Thinking model, used as a strategy for teaching clinical reasoning. Method the translation and cultural adaptation were undertaken through initial translation, synthesis of the translations, back-translation, evaluation by a committee of specialists and a pre-test with 33 undergraduate nursing students. Results the stages of initial translation, synthesis of the translations and back-translation were undertaken satisfactorily, small adjustments being needed. In the evaluation of the translated version by the committee of specialists, all the items obtained agreement over 80% in the first round of evaluation and in the pre-test with the students, so the model was shown to be fit for purpose. Conclusion the use of the model as a complementary strategy in the teaching of diagnostic reasoning is recommended, with a view to the training of nurses who are more aware regarding the diagnostic task and the importance of patient safety. PMID:26107825

Physical and Functional Interaction between the Eukaryotic Orthologs of Prokaryotic Translation Initiation Factors IF1 and IF2

PubMed Central

Choi, Sang Ki; Olsen, DeAnne S.; Roll-Mecak, Antonina; Martung, Agnes; Remo, Keith L.; Burley, Stephen K.; Hinnebusch, Alan G.; Dever, Thomas E.

2000-01-01

To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence similarity to the factors IF1 and IF2 from prokaryotes. By a combination of two-hybrid, coimmunoprecipitation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A binding site was mapped to the C-terminal region of eIF5B. This portion of eIF5B was found to be critical for growth in vivo and for translation in vitro. Overexpression of eIF1A exacerbated the slow-growth phenotype of yeast strains expressing C-terminally truncated eIF5B. These findings indicate that the physical interaction between the evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation, perhaps to direct or stabilize the binding of methionyl-tRNA to the ribosomal P site. PMID:10982835

Two-axis tracking using translation stages for a lens-to-channel waveguide solar concentrator.

PubMed

Liu, Yuxiao; Huang, Ran; Madsen, Christi K

2014-10-20

A two-axis tracking scheme designed for <250x concentration realized by a single-axis mechanical tracker and a translation stage is discussed. The translation stage is used for adjusting positions for seasonal sun movement. It has two-dimensional x-y tracking instead of horizontal movement x-only. This tracking method is compatible with planar waveguide solar concentrators. A prototype system with 50x concentration shows >75% optical efficiency throughout the year in simulation and >65% efficiency experimentally. This efficiency can be further improved by the use of anti-reflection layers and a larger waveguide refractive index.

Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

PubMed Central

Lapierre, Pascal; Mir, Mushtaq; Chase, Michael R.; Pyle, Margaret M.; Gawande, Richa; Ahmad, Rushdy; Sarracino, David A.; Ioerger, Thomas R.; Fortune, Sarah M.; Derbyshire, Keith M.; Wade, Joseph T.; Gray, Todd A.

2015-01-01

RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5’ untranslated region (UTR) and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5’ end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5’ ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5’ UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression. PMID:26536359

Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape.

PubMed

Shell, Scarlet S; Wang, Jing; Lapierre, Pascal; Mir, Mushtaq; Chase, Michael R; Pyle, Margaret M; Gawande, Richa; Ahmad, Rushdy; Sarracino, David A; Ioerger, Thomas R; Fortune, Sarah M; Derbyshire, Keith M; Wade, Joseph T; Gray, Todd A

2015-11-01

RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5' untranslated region (UTR) and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5' end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5' ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5' UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression.

RNA turnover and protein synthesis in fish cells.

PubMed

Smith, R W; Palmer, R M; Houlihan, D F

2000-03-01

Protein synthesis in fish has been previously correlated with RNA content. The present study investigates whether protein and RNA synthesis rates are similarly related. Protein and RNA synthesis rates were determined from 3H-phenylalanine and 3H-uridine incorporation, respectively, and expressed as % x day(-1) and half-lives, respectively. Three fibroblast cell lines were used: BF-2, RTP, CHSE 214, which are derived from the bluegill, rainbow trout and Chinook salmon, respectively. These cells contained similar RNA concentrations (approximately 175 microg RNA x mg(-1) cell protein). Therefore differences in protein synthesis rates, BF-2 (31.3 +/- 1.8)>RTP (25.1 +/- 1.7)>CHSE 214 (17.6 +/-1.1), were attributable to RNA translational efficiency. The most translationally efficient RNA (BF-2 cells), 1.8 mg protein synthesised x microg(-1) RNA x day(-1), corresponded to the lowest RNA half-life, 75.4 +/- 6.4 h. Translationally efficient RNA was also energetically efficient with BF-2 cells exploiting the least costly route of nucleotide supply (i.e. exogenous salvage) 3.5-6.0 times more than the least translationally efficient RNA (CHSE 214 cells). These data suggest that differential nucleotide supply, between intracellular synthesis and exogenous salvage, constitutes the area of pre-translational flexibility exploited to maintain RNA synthesis as a fixed energetic cost component of protein synthesis.

Tuning of Recombinant Protein Expression in Escherichia coli by Manipulating Transcription, Translation Initiation Rates, and Incorporation of Noncanonical Amino Acids.

PubMed

Schlesinger, Orr; Chemla, Yonatan; Heltberg, Mathias; Ozer, Eden; Marshall, Ryan; Noireaux, Vincent; Jensen, Mogens Høgh; Alfonta, Lital

2017-06-16

Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the Eschrichia coli transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression. This effect can be rescued by introducing slow translating codons into the beginning of the gene, by shortening gene length or by reducing initiation rates. On the basis of the results, we developed a biophysical model, which suggests that the density of co-transcriptional-translation plays a role in bacterial protein synthesis. These findings indicate how cells use codon bias to tune translation speed and protein synthesis.

DsrA regulatory RNA represses both hns and rbsD mRNAs through distinct mechanisms in Escherichia coli.

PubMed

Lalaouna, David; Morissette, Audrey; Carrier, Marie-Claude; Massé, Eric

2015-10-01

The 87 nucleotide long DsrA sRNA has been mostly studied for its translational activation of the transcriptional regulator RpoS. However, it also represses hns mRNA, which encodes H-NS, a major regulator that affects expression of nearly 5% of Escherichia coli genes. A speculative model previously suggested that DsrA would block hns mRNA translation by binding simultaneously to start and stop codon regions of hns mRNA (coaxial model). Here, we show that DsrA efficiently blocked translation of hns mRNA by base-pairing immediately downstream of the start codon. In addition, DsrA induced hns mRNA degradation by actively recruiting the RNA degradosome complex. Data presented here led to a model of DsrA action on hns mRNA, which supports a canonical mechanism of sRNA-induced mRNA degradation by binding to the translation initiation region. Furthermore, using MS2-affinity purification coupled with RNA sequencing technology (MAPS), we also demonstrated that DsrA targets rbsD mRNA, involved in ribose utilization. Surprisingly, DsrA base pairs far downstream of rbsD start codon and induces rapid degradation of the transcript. Thus, our study enables us to draw an extended DsrA targetome. © 2015 John Wiley & Sons Ltd.

Moco biosynthesis and the ATAC acetyltransferase engage translation initiation by inhibiting latent PKR activity.

PubMed

Suganuma, Tamaki; Swanson, Selene K; Florens, Laurence; Washburn, Michael P; Workman, Jerry L

2016-02-01

Molybdenum cofactor (Moco) biosynthesis is linked to c-Jun N-terminal kinase (JNK) signaling in Drosophila through MoaE, a molybdopterin (MPT) synthase subunit that is also a component of the Ada Two A containing (ATAC) acetyltransferase complex. Here, we show that human MPT synthase and ATAC inhibited PKR, a double-stranded RNA-dependent protein kinase, to facilitate translation initiation of iron-responsive mRNA. MPT synthase and ATAC directly interacted with PKR and suppressed latent autophosphorylation of PKR and its downstream phosphorylation of JNK and eukaryotic initiation factor 2α (eIF2α). The suppression of eIF2α phosphorylation via MPT synthase and ATAC prevented sequestration of the guanine nucleotide exchange factor eIF2B, which recycles eIF2-GDP to eIF2-GTP, resulting in the promotion of translation initiation. Indeed, translation of the iron storage protein, ferritin, was reduced in the absence of MPT synthase or ATAC subunits. Thus, MPT synthase and ATAC regulate latent PKR signaling and link transcription and translation initiation. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Ensuring continuity of the paternal genome: potential roles for spermatozoal RNA in mammalian embryogenesis.

PubMed

Miller, David

2007-01-01

The mammalian male gamete is traditionally considered to be a less complex cell than its female counterpart, primarily because all development (bar oocyte activation) is thought to be initiated by the egg. However, the spermatozoon is exquisitely specialised to deliver the paternal genome to the oocyte and, in this respect, is pared down to carry out that job as efficiently as possible. As such, it is transcriptionally silent as a consequence of the highly condensed architecture of its chromatin and yet some spermatozoal DNA may be organised into domains that resemble sites of transcriptional activity. There is also little or no cytoplasm capable of supporting translation; however, we now understand that these cells carry a cargo of translationally competent mRNAs. Moreover, a recent report has provided intriguing new evidence that, under certain conditions, spermatozoa can translate their mRNAs de novo. Indeed, delivery of an RNA cargo by the spermatozoon to the oocyte may be as fundamental a feature of this quiescent cell as delivery of the paternal genome itself. New evidence is available showing that spermatozoal RNA can potentially affect phenotypic traits in offspring. This epigenetic phenomenon may involve the transmission of extrachromosomal episomal elements. Taken together, these data suggest that the spermatozoon is much more than the sum of its constituent parts and that the continued transmission of male-benefit genes and possibly selfish elements may be one reason for the male gamete's additional and unexpected investment in delivering the paternal genome safely and efficiently.

A web application for automatic prediction of gene translation elongation efficiency.

PubMed

Sokolov, Vladimir S; Zuraev, Bulat S; Lashin, Sergei A; Matushkin, Yury G

2015-03-01

Expression efficiency is one of the major characteristics describing genes in various modern investigations. Expression efficiency of genes is regulated at various stages: transcription, translation, posttranslational protein modification and others. In this study, a special EloE (Elongation Efficiency) web application is described. The EloE sorts the organism's genes in a descend order on their theoretical rate of the elongation stage of translation based on the analysis of their nucleotide sequences. Obtained theoretical data have a significant correlation with available experimental data of gene expression in various organisms. In addition, the program identifies preferential codons in organism's genes and defines distribution of potential secondary structures energy in 5´ and 3´ regions of mRNA. The EloE can be useful in preliminary estimation of translation elongation efficiency for genes for which experimental data are not available yet. Some results can be used, for instance, in other programs modeling artificial genetic structures in genetically engineered experiments. The EloE web application is available at http://www-bionet.sscc.ru:7780/EloE.

β-Glucuronidase as a Sensitive and Versatile Reporter in Actinomycetes ▿

PubMed Central

Myronovskyi, Maksym; Welle, Elisabeth; Fedorenko, Viktor; Luzhetskyy, Andriy

2011-01-01

Here we describe a versatile and sensitive reporter system for actinomycetes that is based on gusA, which encodes the β-glucuronidase enzyme. A series of gusA-containing transcriptional and translational fusion vectors were constructed and utilized to study the regulatory cascade of the phenalinolactone biosynthetic gene cluster. Furthermore, these vectors were used to study the efficiency of translation initiation at the ATG, GTG, TTG, and CTG start codons. Surprisingly, constructs using a TTG start codon showed the best activity, whereas those using ATG or GTG were approximately one-half or one-third as active, respectively. The CTG fusion showed only 5% of the activity of the TTG fusion. A suicide vector, pKGLP2, carrying gusA in its backbone was used to visually detect merodiploid formation and resolution, making gene targeting in actinomycetes much faster and easier. Three regulatory genes, plaR1, plaR2, and plaR3, involved in phenalinolactone biosynthesis were efficiently replaced with an apramycin resistance marker using this system. Finally, we expanded the genetic code of actinomycetes by introducing the nonproteinogenic amino acid N-epsilon-cyclopentyloxycarbonyl-l-lysine with the GusA protein as a reporter. PMID:21685164

Initiation of translation in bacteria by a structured eukaryotic IRES RNA.

PubMed

Colussi, Timothy M; Costantino, David A; Zhu, Jianyu; Donohue, John Paul; Korostelev, Andrei A; Jaafar, Zane A; Plank, Terra-Dawn M; Noller, Harry F; Kieft, Jeffrey S

2015-03-05

The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical molecular signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been observed in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.

Ribosome flow model with positive feedback

PubMed Central

Margaliot, Michael; Tuller, Tamir

2013-01-01

Eukaryotic mRNAs usually form a circular structure; thus, ribosomes that terminatae translation at the 3′ end can diffuse with increased probability to the 5′ end of the transcript, initiating another cycle of translation. This phenomenon describes ribosomal flow with positive feedback—an increase in the flow of ribosomes terminating translating the open reading frame increases the ribosomal initiation rate. The aim of this paper is to model and rigorously analyse translation with feedback. We suggest a modified version of the ribosome flow model, called the ribosome flow model with input and output. In this model, the input is the initiation rate and the output is the translation rate. We analyse this model after closing the loop with a positive linear feedback. We show that the closed-loop system admits a unique globally asymptotically stable equilibrium point. From a biophysical point of view, this means that there exists a unique steady state of ribosome distributions along the mRNA, and thus a unique steady-state translation rate. The solution from any initial distribution will converge to this steady state. The steady-state distribution demonstrates a decrease in ribosome density along the coding sequence. For the case of constant elongation rates, we obtain expressions relating the model parameters to the equilibrium point. These results may perhaps be used to re-engineer the biological system in order to obtain a desired translation rate. PMID:23720534

Translational Research on Habit and Alcohol.

PubMed

McKim, Theresa H; Shnitko, Tatiana A; Robinson, Donita L; Boettiger, Charlotte A

2016-03-01

Habitual actions enable efficient daily living, but they can also contribute to pathological behaviors that resistant change, such as alcoholism. Habitual behaviors are learned actions that appear goal-directed but are in fact no longer under the control of the action's outcome. Instead, these actions are triggered by stimuli, which may be exogenous or interoceptive, discrete or contextual. A major hallmark characteristic of alcoholism is continued alcohol use despite serious negative consequences. In essence, although the outcome of alcohol seeking and drinking is dramatically devalued, these actions persist, often triggered by environmental cues associated with alcohol use. Thus, alcoholism meets the definition of an initially goal-directed behavior that converts to a habit-based process. Habit and alcohol have been well investigated in rodent models, with comparatively less research in non-human primates and people. This review focuses on translational research on habit and alcohol with an emphasis on cross-species methodology and neural circuitry.

Global brain ischemia and reperfusion.

PubMed

White, B C; Grossman, L I; O'Neil, B J; DeGracia, D J; Neumar, R W; Rafols, J A; Krause, G S

1996-05-01

Brain damage accompanying cardiac arrest and resuscitation is frequent and devastating. Neurons in the hippocampus CA1 and CA4 zones and cortical layers III and V are selectively vulnerable to death after injury by ischemia and reperfusion. Ultrastructural evidence indicates that most of the structural damage is associated with reperfusion, during which the vulnerable neurons develop disaggregation of polyribosomes, peroxidative damage to unsaturated fatty acids in the plasma membrane, and prominent alterations in the structure of the Golgi apparatus that is responsible for membrane assembly. Reperfusion is also associated with vulnerable neurons with prominent production of messenger RNAs for stress proteins and for the proteins of the activator protein-1 complex, but these vulnerable neurons fail to efficiently translate these messages into the proteins. The inhibition of protein synthesis during reperfusion involves alteration of translation initiation factors, specifically serine phosphorylation of the alpha-subunit of eukaryotic initiation factor-2 (elF-2 alpha). Growth factors--in particular, insulin--have the potential to reverse phosphorylation of elF-2 alpha, promote effective translation of the mRNA transcripts generated in response to ischemia and reperfusion, enhance neuronal defenses against radicals, and stimulate lipid synthesis and membrane repair. There is now substantial evidence that the insulin-class growth factors have neuron-sparing effects against damage by radicals and ischemia and reperfusion. This new knowledge may provide a fundamental basis for a rational approach to "cerebral resuscitation" that will allow substantial amelioration of the often dismal neurologic outcome now associated with resuscitation from cardiac arrest.

Challenges and Benefits in Designing and Implementing a Team-Based Research Mentorship Experience in Translational Research.

PubMed

Young, Belinda-Rose; Williamson, Heather J; Burton, Donna L; Massey, Oliver Thomas; Levin, Bruce Lubotsky; Baldwin, Julie A

2015-12-01

Translational research seeks to build bridges between research and practice to address public health issues efficiently and effectively. The purpose of this paper was to evaluate a newly formed Institute that provided graduate students and adolescent behavioral health community professionals with a translational research service-learning opportunity through the creation of a community-university mentoring partnership. Goals of the team-based research mentorship approach included: 1) providing students the skills for implementing translational research; 2) providing research opportunities for community agencies to enhance operations and to encourage ongoing research involvement; and 3) developing relationships between university faculty and community agency professionals for further research collaborations. The Institute used the National Institute on Drug Abuse's Mentoring Mosaic to select a diverse group of Community and Academic Mentors. The research mentorship experience of the initial cohort was evaluated based upon the Research Mentorship Conceptual Framework and Self-Assessment Tool. As a direct result of the research mentorship, outcomes for the Academic and Community Mentors and Scholars (i.e., those seeking a graduate certificate) included improved working relationships/networking and research experience. Through experiential learning, Scholars also discovered career trajectory clarity, the need for community collaboration in research, opportunities for continuing professional development, a greater understanding of public health competencies and how they align with community-based research, and skill development in best practices for translational research. The team mentoring approach is a form of pedagogy that holds promise to enhance translational research and community-based research efforts while developing public health competencies.

The translational regulator Cup controls NMJ presynaptic terminal morphology.

PubMed

Menon, Kaushiki P; Carrillo, Robert A; Zinn, Kai

2015-07-01

During oogenesis and early embryonic development in Drosophila, translation of proteins from maternally deposited mRNAs is tightly controlled. We and others have previously shown that translational regulatory proteins that function during oogenesis also have essential roles in the nervous system. Here we examine the role of Cup in neuromuscular system development. Maternal Cup controls translation of localized mRNAs encoding the Oskar and Nanos proteins and binds to the general translation initiation factor eIF4E. In this paper, we show that zygotic Cup protein is localized to presynaptic terminals at larval neuromuscular junctions (NMJs). cup mutant NMJs have strong phenotypes characterized by the presence of small clustered boutons called satellite boutons. They also exhibit an increase in the frequency of spontaneous glutamate release events (mEPSPs). Reduction of eIF4E expression synergizes with partial loss of Cup expression to produce satellite bouton phenotypes. The presence of satellite boutons is often associated with increases in retrograde bone morphogenetic protein (BMP) signaling, and we show that synaptic BMP signaling is elevated in cup mutants. cup genetically interacts with two genes, EndoA and Dap160, that encode proteins involved in endocytosis that are also neuronal modulators of the BMP pathway. Endophilin protein, encoded by the EndoA gene, is downregulated in a cup mutant. Our results are consistent with a model in which Cup and eIF4E work together to ensure efficient localization and translation of endocytosis proteins in motor neurons and control the strength of the retrograde BMP signal. Copyright © 2015 Elsevier Inc. All rights reserved.

The translational regulator Cup controls NMJ presynaptic terminal morphology

PubMed Central

Menon, Kaushiki P.; Carrillo, Robert A.; Zinn, Kai

2015-01-01

During oogenesis and early embryonic development in Drosophila, translation of proteins from maternally deposited mRNAs is tightly controlled. We and others have previously shown that translational regulatory proteins that function during oogenesis also have essential roles in the nervous system. Here we examine the role of Cup in neuromuscular system development. Maternal Cup controls translation of localized mRNAs encoding the Oskar and Nanos proteins and binds to the general translation initiation factor eIF4E. In this paper, we show that zygotic Cup protein is localized to presynaptic terminals at larval neuromuscular junctions (NMJs). cup mutant NMJs have strong phenotypes characterized by the presence of small clustered boutons called satellite boutons. They also exhibit an increase in the frequency of spontaneous glutamate release events (mEPSPs). Reduction of eIF4E expression synergizes with partial loss of Cup expression to produce satellite bouton phenotypes. The presence of satellite boutons is often associated with increases in retrograde bone morphogenetic protein (BMP) signaling, and we show that synaptic BMP signaling is elevated in cup mutants. cup genetically interacts with four genes (EndoA, WASp, Dap160, and Synj) encoding proteins involved in endocytosis that are also neuronal modulators of the BMP pathway. Endophilin protein, encoded by the EndoA gene, is downregulated in a cup mutant. Our results are consistent with a model in which Cup and eIF4E work together to ensure efficient localization and translation of endocytosis proteins in motor neurons and control the strength of the retrograde BMP signal. PMID:26102195

Rational modular design of metabolic network for efficient production of plant polyphenol pinosylvin.

PubMed

Wu, Junjun; Zhang, Xia; Zhu, Yingjie; Tan, Qinyu; He, Jiacheng; Dong, Mingsheng

2017-05-03

Efficient biosynthesis of the plant polyphenol pinosylvin, which has numerous applications in nutraceuticals and pharmaceuticals, is necessary to make biological production economically viable. To this end, an efficient Escherichia coli platform for pinosylvin production was developed via a rational modular design approach. Initially, different candidate pathway enzymes were screened to construct de novo pinosylvin pathway directly from D-glucose. A comparative analysis of pathway intermediate pools identified that this initial construct led to the intermediate cinnamic acid accumulation. The pinosylvin synthetic pathway was then divided into two new modules separated at cinnamic acid. Combinatorial optimization of transcriptional and translational levels of these two modules resulted in a 16-fold increase in pinosylvin titer. To further improve the concentration of the limiting precursor malonyl-CoA, the malonyl-CoA synthesis module based on clustered regularly interspaced short palindromic repeats interference was assembled and optimized with other two modules. The final pinosylvin titer was improved to 281 mg/L, which was the highest pinosylvin titer even directly from D-glucose without any additional precursor supplementation. The rational modular design approach described here could bolster our capabilities in synthetic biology for value-added chemical production.

Defining the bacteroides ribosomal binding site.

PubMed

Wegmann, Udo; Horn, Nikki; Carding, Simon R

2013-03-01

The human gastrointestinal tract, in particular the colon, hosts a vast number of commensal microorganisms. Representatives of the genus Bacteroides are among the most abundant bacterial species in the human colon. Bacteroidetes diverged from the common line of eubacterial descent before other eubacterial groups. As a result, they employ unique transcription initiation signals and, because of this uniqueness, they require specific genetic tools. Although some tools exist, they are not optimal for studying the roles and functions of these bacteria in the human gastrointestinal tract. Focusing on translation initiation signals in Bacteroides, we created a series of expression vectors allowing for different levels of protein expression in this genus, and we describe the use of pepI from Lactobacillus delbrueckii subsp. lactis as a novel reporter gene for Bacteroides. Furthermore, we report the identification of the 3' end of the 16S rRNA of Bacteroides ovatus and analyze in detail its ribosomal binding site, thus defining a core region necessary for efficient translation, which we have incorporated into the design of our expression vectors. Based on the sequence logo information from the 5' untranslated region of other Bacteroidales ribosomal protein genes, we conclude that our findings are relevant to all members of this order.

Splicing of goose parvovirus pre-mRNA influences cytoplasmic translation of the processed mRNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Long; Pintel, David J., E-mail: pinteld@missouri.edu

2012-04-25

Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained inmore » unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins.« less

Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction.

PubMed

Ray, Swagat; Anderson, Emma C

2016-03-03

The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eukaryotic translation initiation factor 4G (eIF4G), we investigated whether Unr has a general role in translational control. We found that Unr strongly stimulates translation in vitro, and mutation of cold shock domains 2 or 4 inhibited its translation activity. The ability of Unr and its mutants to stimulate translation correlated with its ability to bind RNA, and to interact with PABP1. We found that Unr stimulated the binding of PABP1 to mRNA, and that Unr was required for the stable interaction of PABP1 and eIF4G in cells. siRNA-mediated knockdown of Unr reduced the overall level of cellular translation in cells, as well as that of cap-dependent and IRES-dependent reporters. These data describe a novel role for Unr in regulating cellular gene expression.

5'-Phosphorothiolate Dinucleotide Cap Analogues: Reagents for Messenger RNA Modification and Potent Small-Molecular Inhibitors of Decapping Enzymes.

PubMed

Wojtczak, Blazej A; Sikorski, Pawel J; Fac-Dabrowska, Kaja; Nowicka, Anna; Warminski, Marcin; Kubacka, Dorota; Nowak, Elzbieta; Nowotny, Marcin; Kowalska, Joanna; Jemielity, Jacek

2018-05-09

The 5' cap consists of 7-methylguanosine (m 7 G) linked by a 5'-5'-triphosphate bridge to messenger RNA (mRNA) and acts as the master regulator of mRNA turnover and translation initiation in eukaryotes. Cap analogues that influence mRNA translation and turnover (either as small molecules or as part of an RNA transcript) are valuable tools for studying gene expression, which is often also of therapeutic relevance. Here, we synthesized a series of 15 dinucleotide cap (m 7 GpppG) analogues containing a 5'-phosphorothiolate (5'-PSL) moiety (i.e., an O-to-S substitution within the 5'-phosphoester) and studied their biological properties in the context of three major cap-binding proteins: translation initiation factor 4E (eIF4E) and two decapping enzymes, DcpS and Dcp2. While the 5'-PSL moiety was neutral or slightly stabilizing for cap interactions with eIF4E, it significantly influenced susceptibility to decapping. Replacing the γ-phosphoester with the 5'-PSL moiety (γ-PSL) prevented β-γ-pyrophosphate bond cleavage by DcpS and conferred strong inhibitory properties. Combining the γ-PSL moiety with α-PSL and β-phosphorothioate (PS) moiety afforded first cap-derived hDcpS inhibitor with low nanomolar potency. Susceptibility to Dcp2 and translational properties were studied after incorporation of the new analogues into mRNA transcripts by RNA polymerase. Transcripts containing the γ-PSL moiety were resistant to cleavage by Dcp2. Surprisingly, superior translational properties were observed for mRNAs containing the α-PSL moiety, which were Dcp2-susceptible. The overall protein expression measured in HeLa cells for this mRNA was comparable to mRNA capped with the translation augmenting β-PS analogue reported previously. Overall, our study highlights 5'-PSL as a synthetically accessible cap modification, which, depending on the substitution site, can either reduce susceptibility to decapping or confer superior translational properties on the mRNA. The 5'-PSL-analogues may find application as reagents for the preparation of efficiently expressed mRNA or for investigation of the role of decapping enzymes in mRNA processing or neuromuscular disorders associated with decapping.

Structural determinants of an internal ribosome entry site that direct translational reading frame selection

PubMed Central

Ren, Qian; Au, Hilda H.T.; Wang, Qing S.; Lee, Seonghoon; Jan, Eric

2014-01-01

The dicistrovirus intergenic internal ribosome entry site (IGR IRES) directly recruits the ribosome and initiates translation using a non-AUG codon. A subset of IGR IRESs initiates translation in either of two overlapping open reading frames (ORFs), resulting in expression of the 0 frame viral structural polyprotein and an overlapping +1 frame ORFx. A U–G base pair adjacent to the anticodon-like pseudoknot of the IRES directs +1 frame translation. Here, we show that the U-G base pair is not absolutely required for +1 frame translation. Extensive mutagenesis demonstrates that 0 and +1 frame translation can be uncoupled. Ribonucleic acid (RNA) structural probing analyses reveal that the mutant IRESs adopt distinct conformations. Toeprinting analysis suggests that the reading frame is selected at a step downstream of ribosome assembly. We propose a model whereby the IRES adopts conformations to occlude the 0 frame aminoacyl-tRNA thereby allowing delivery of the +1 frame aminoacyl-tRNA to the A site to initiate translation of ORFx. This study provides a new paradigm for programmed recoding mechanisms that increase the coding capacity of a viral genome. PMID:25038250

Design and Formative Evaluation of the Policy Liaison Initiative: A Long-Term Knowledge Translation Strategy to Encourage and Support the Use of Cochrane Systematic Reviews for Informing

ERIC Educational Resources Information Center

Brennan, Sue E.; Cumpston, Miranda; Misso, Marie L.; McDonald, Steve; Murphy, Matthew J.; Green, Sally E.

2016-01-01

The Policy Liaison Initiative (PLI) is a long-term knowledge translation initiative designed to support the use of Cochrane systematic reviews in health policy. A joint initiative between the Australasian Cochrane Centre and Australian Government Department of Health and Ageing, the PLI includes: 1) a community of practice for evidence-informed…

Translational repression of mouse mu opioid receptor expression via leaky scanning

PubMed Central

Song, Kyu Young; Hwang, Cheol Kyu; Kim, Chun Sung; Choi, Hack Sun; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

2007-01-01

Mu opioid receptor (MOR) expression is under temporal and spatial controls, but expression levels of the MOR gene are relatively low in vivo. In addition to transcriptional regulations, upstream AUGs (uAUGs) and open reading frames (uORFs) profoundly affect the translation of the primary ORF and thus the protein levels in several genes. The 5′-untranslated region (UTR) of mouse MOR mRNA contains three uORFs preceding the MOR main initiation codon. In MOR-fused EGFP or MOR promoter/luciferase reporter constructs, mutating each uAUG individually or in combinations increased MOR transient heterologous expression in neuroblastoma NMB and HEK293 cells significantly. Translation of such constructs increased up to 3-fold without altering the mRNA levels if either the third uAUG or both the second and third AUGs were mutated. Additionally, these uAUG-mediated translational inhibitions were independent of their peptide as confirmed by internal mutation analyses in each uORF. Translational studies indicated that protein syntheses were initiated at these uAUG initiation sites, with the third uAUG initiating the highest translation level. These results support the hypothesis that uORFs in mouse MOR mRNA act as negative regulators through a ribosome leaky scanning mechanism. Such leaky scanning resulted in the suppression of mouse MOR under normal conditions. PMID:17284463

Eukaryotic initiation factor 3 (eIF3) and 5’ mRNA leader sequences as agents of translational regulation in Arabidopsis. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

von Arnim, Albrecht G.

2015-02-04

Protein synthesis, or translation, consumes a sizable fraction of the cell’s energy budget, estimated at 5% and up to 50% in differentiated and growing cells, respectively. Plants also invest significant energy and biomass to construct and maintain the translation apparatus. Translation is regulated by a variety of external stimuli. Compared to transcriptional control, attributes of translational control include reduced sensitivity to stochastic fluctuation, a finer gauge of control, and more rapid responsiveness to environmental stimuli. Yet, our murky understanding of translational control allows few generalizations. Consequently, translational regulation is underutilized in the context of transgene regulation, although synthetic biologists aremore » now beginning to appropriate RNA-level gene regulation into their regulatory circuits. We also know little about how translational control contributes to the diversity of plant form and function. This project explored how an emerging regulatory mRNA sequence element, upstream open reading frames (uORFs), is integrated with the general translation initiation machinery to permit translational regulation on specific mRNAs.« less

Cleavage of Poly(A)-Binding Protein by Enterovirus Proteases Concurrent with Inhibition of Translation In Vitro

PubMed Central

Joachims, Michelle; Van Breugel, Pieter C.; Lloyd, Richard E.

1999-01-01

Many enteroviruses, members of the family Picornaviridae, cause a rapid and drastic inhibition of host cell protein synthesis during infection, a process referred to as host cell shutoff. Poliovirus, one of the best-studied enteroviruses, causes marked inhibition of host cell translation while preferentially allowing translation of its own genomic mRNA. An abundance of experimental evidence has accumulated to indicate that cleavage of an essential translation initiation factor, eIF4G, during infection is responsible at least in part for this shutoff. However, evidence from inhibitors of viral replication suggests that an additional event is necessary for the complete translational shutoff observed during productive infection. This report examines the effect of poliovirus infection on a recently characterized 3′ end translational stimulatory protein, poly(A)-binding protein (PABP). PABP is involved in stimulating translation initiation in lower eukaryotes by its interaction with the poly(A) tail on mRNAs and has been proposed to facilitate 5′-end–3′-end interactions in the context of the closed-loop translational model. Here, we show that PABP is specifically degraded during poliovirus infection and that it is cleaved in vitro by both poliovirus 2A and 3C proteases and coxsackievirus B3 2A protease. Further, PABP cleavage by 2A protease is accompanied by concurrent loss of translational activity in an in vitro-translation assay. Similar loss of translational activity also occurs simultaneously with partial 3C protease-mediated cleavage of PABP in translation assays. Further, PABP is not degraded during infections in the presence of guanidine-HCl, which blocks the complete development of host translation shutoff. These results provide preliminary evidence that cleavage of PABP may contribute to inhibition of host translation in infected HeLa cells, and they are consistent with the hypothesis that PABP plays a role in facilitating translation initiation in higher eukaryotes. PMID:9847378

Sapovirus Translation Requires an Interaction between VPg and the Cap Binding Protein eIF4E

PubMed Central

Hosmillo, Myra; Chaudhry, Yasmin; Kim, Deok-Song

2014-01-01

ABSTRACT Sapoviruses of the Caliciviridae family of small RNA viruses are emerging pathogens that cause gastroenteritis in humans and animals. Molecular studies on human sapovirus have been hampered due to the lack of a cell culture system. In contrast, porcine sapovirus (PSaV) can be grown in cell culture, making it a suitable model for understanding the infectious cycle of sapoviruses and the related enteric caliciviruses. Caliciviruses are known to use a novel mechanism of protein synthesis that relies on the interaction of cellular translation initiation factors with the virus genome-encoded viral protein genome (VPg) protein, which is covalently linked to the 5′ end of the viral genome. Using PSaV as a representative member of the Sapovirus genus, we characterized the role of the viral VPg protein in sapovirus translation. As observed for other caliciviruses, the PSaV genome was found to be covalently linked to VPg, and this linkage was required for the translation and the infectivity of viral RNA. The PSaV VPg protein was associated with the 4F subunit of the eukaryotic translation initiation factor (eIF4F) complex in infected cells and bound directly to the eIF4E protein. As has been previously demonstrated for feline calicivirus, a member of the Vesivirus genus, PSaV translation required eIF4E and the interaction between eIF4E and eIF4G. Overall, our study provides new insights into the novel mechanism of sapovirus translation, suggesting that sapovirus VPg can hijack the cellular translation initiation mechanism by recruiting the eIF4F complex through a direct eIF4E interaction. IMPORTANCE Sapoviruses, which are members of the Caliciviridae family, are one of the causative agents of viral gastroenteritis in humans. However, human sapovirus remains noncultivable in cell culture, hampering the ability to characterize the virus infectious cycle. Here, we show that the VPg protein from porcine sapovirus, the only cultivatable sapovirus, is essential for viral translation and functions via a direct interaction with the cellular translation initiation factor eIF4E. This work provides new insights into the novel protein-primed mechanism of calicivirus VPg-dependent translation initiation. PMID:25142584

Sapovirus translation requires an interaction between VPg and the cap binding protein eIF4E.

PubMed

Hosmillo, Myra; Chaudhry, Yasmin; Kim, Deok-Song; Goodfellow, Ian; Cho, Kyoung-Oh

2014-11-01

Sapoviruses of the Caliciviridae family of small RNA viruses are emerging pathogens that cause gastroenteritis in humans and animals. Molecular studies on human sapovirus have been hampered due to the lack of a cell culture system. In contrast, porcine sapovirus (PSaV) can be grown in cell culture, making it a suitable model for understanding the infectious cycle of sapoviruses and the related enteric caliciviruses. Caliciviruses are known to use a novel mechanism of protein synthesis that relies on the interaction of cellular translation initiation factors with the virus genome-encoded viral protein genome (VPg) protein, which is covalently linked to the 5' end of the viral genome. Using PSaV as a representative member of the Sapovirus genus, we characterized the role of the viral VPg protein in sapovirus translation. As observed for other caliciviruses, the PSaV genome was found to be covalently linked to VPg, and this linkage was required for the translation and the infectivity of viral RNA. The PSaV VPg protein was associated with the 4F subunit of the eukaryotic translation initiation factor (eIF4F) complex in infected cells and bound directly to the eIF4E protein. As has been previously demonstrated for feline calicivirus, a member of the Vesivirus genus, PSaV translation required eIF4E and the interaction between eIF4E and eIF4G. Overall, our study provides new insights into the novel mechanism of sapovirus translation, suggesting that sapovirus VPg can hijack the cellular translation initiation mechanism by recruiting the eIF4F complex through a direct eIF4E interaction. Sapoviruses, which are members of the Caliciviridae family, are one of the causative agents of viral gastroenteritis in humans. However, human sapovirus remains noncultivable in cell culture, hampering the ability to characterize the virus infectious cycle. Here, we show that the VPg protein from porcine sapovirus, the only cultivatable sapovirus, is essential for viral translation and functions via a direct interaction with the cellular translation initiation factor eIF4E. This work provides new insights into the novel protein-primed mechanism of calicivirus VPg-dependent translation initiation. Copyright © 2014 Hosmillo et al.

Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Distinct mRNAs for Translation*

PubMed Central

Timpano, Sara; Uniacke, James

2016-01-01

Translation initiation is a focal point of translational control and requires the binding of eIF4E to the 5′ cap of mRNA. Under conditions of extreme oxygen depletion (hypoxia), human cells repress eIF4E and switch to an alternative cap-dependent translation mediated by a homolog of eIF4E, eIF4E2. This homolog forms a complex with the oxygen-regulated hypoxia-inducible factor 2α and can escape translation repression. This complex mediates cap-dependent translation under cell culture conditions of 1% oxygen (to mimic tumor microenvironments), whereas eIF4E mediates cap-dependent translation at 21% oxygen (ambient air). However, emerging evidence suggests that culturing cells in ambient air, or “normoxia,” is far from physiological or “normal.” In fact, oxygen in human tissues ranges from 1–11% or “physioxia.” Here we show that two distinct modes of cap-dependent translation initiation are active during physioxia and act on separate pools of mRNAs. The oxygen-dependent activities of eIF4E and eIF4E2 are elucidated by observing their polysome association and the status of mammalian target of rapamycin complex 1 (eIF4E-dependent) or hypoxia-inducible factor 2α expression (eIF4E2-dependent). We have identified oxygen conditions where eIF4E is the dominant cap-binding protein (21% normoxia or standard cell culture conditions), where eIF4E2 is the dominant cap-binding protein (1% hypoxia or ischemic diseases and cancerous tumors), and where both cap-binding proteins act simultaneously to initiate the translation of distinct mRNAs (1–11% physioxia or during development and stem cell differentiation). These data suggest that the physioxic proteome is generated by initiating translation of mRNAs via two distinct but complementary cap-binding proteins. PMID:27002144

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation.

PubMed

Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed; Pelletier, Jerry

2006-10-01

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2alpha phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2alpha phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2alpha to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2alpha phosphorylation-dependent and -independent pathways that target translation initiation.

Inhibition of Ribosome Recruitment Induces Stress Granule Formation Independently of Eukaryotic Initiation Factor 2α Phosphorylation

PubMed Central

Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J.; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed

2006-01-01

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2α phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2α phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2α to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2α phosphorylation-dependent and -independent pathways that target translation initiation. PMID:16870703

A highly efficient, cell-free translation/translocation system prepared from Xenopus eggs.

PubMed Central

Matthews, G; Colman, A

1991-01-01

We describe the use of a Xenopus laevis egg extract for the in vitro translation and post translational modification of membrane and secretory proteins. This extract is capable of the translation and segregation into membranes of microgram per millilitre levels of protein from added mRNAs. Signal sequences of segregated proteins are efficiently cleaved and appropriate N-linked glycosylation patterns are produced. The extract also supports the quantitative assembly of murine immunoglobulin heavy and light chains into tetramers, and two events which take place beyond the endoplasmic reticulum, mannose 6 phosphorylation of murine cathepsin D and O-linked glycosylation of coronavirus E1 protein, also occur, but at reduced efficiency. The stability of the membranes allows protease protection studies and quantitative centrifugal fractionation of segregated and unsegregated proteins to be performed. Conditions for the use of stored extract have also been determined. Images PMID:1754376

Efficiency of Iranian Translation Syllabus at BA Level; Deficiency: A New Comprehensive Model

ERIC Educational Resources Information Center

Sohrabi, Sarah; Rahimi, Ramin; Arjmandi, Masoume

2015-01-01

This study aims at investigating the practicality of the current curriculum for translation studies at national level (Iranian curriculum). It is going to have a comprehensive idea of translation students and teachers (university lecturers) over the current translation syllabus at BA level in Iran. A researcher-made CEQ questionnaire (Curriculum…

Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

NASA Astrophysics Data System (ADS)

Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

2017-01-01

Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

Distribution and diversity of ribosome binding sites in prokaryotic genomes.

PubMed

Omotajo, Damilola; Tate, Travis; Cho, Hyuk; Choudhary, Madhusudan

2015-08-14

Prokaryotic translation initiation involves the proper docking, anchoring, and accommodation of mRNA to the 30S ribosomal subunit. Three initiation factors (IF1, IF2, and IF3) and some ribosomal proteins mediate the assembly and activation of the translation initiation complex. Although the interaction between Shine-Dalgarno (SD) sequence and its complementary sequence in the 16S rRNA is important in initiation, some genes lacking an SD ribosome binding site (RBS) are still well expressed. The objective of this study is to examine the pattern of distribution and diversity of RBS in fully sequenced bacterial genomes. The following three hypotheses were tested: SD motifs are prevalent in bacterial genomes; all previously identified SD motifs are uniformly distributed across prokaryotes; and genes with specific cluster of orthologous gene (COG) functions differ in their use of SD motifs. Data for 2,458 bacterial genomes, previously generated by Prodigal (PROkaryotic DYnamic programming Gene-finding ALgorithm) and currently available at the National Center for Biotechnology Information (NCBI), were analyzed. Of the total genes examined, ~77.0% use an SD RBS, while ~23.0% have no RBS. Majority of the genes with the most common SD motifs are distributed in a manner that is representative of their abundance for each COG functional category, while motifs 13 (5'-GGA-3'/5'-GAG-3'/5'-AGG-3') and 27 (5'-AGGAGG-3') appear to be predominantly used by genes for information storage and processing, and translation and ribosome biogenesis, respectively. These findings suggest that an SD sequence is not obligatory for translation initiation; instead, other signals, such as the RBS spacer, may have an overarching influence on translation of mRNAs. Subsequent analyses of the 5' secondary structure of these mRNAs may provide further insight into the translation initiation mechanism.

Negative and Translation Termination-Dependent Positive Control of FLI-1 Protein Synthesis by Conserved Overlapping 5′ Upstream Open Reading Frames in Fli-1 mRNA

PubMed Central

Sarrazin, Sandrine; Starck, Joëlle; Gonnet, Colette; Doubeikovski, Alexandre; Melet, Fabrice; Morle, François

2000-01-01

The proto-oncogene Fli-1 encodes a transcription factor of the ets family whose overexpression is associated with multiple virally induced leukemias in mouse, inhibits murine and avian erythroid cell differentiation, and induces drastic perturbations of early development in Xenopus. This study demonstrates the surprisingly sophisticated regulation of Fli-1 mRNA translation. We establish that two FLI-1 protein isoforms (of 51 and 48 kDa) detected by Western blotting in vivo are synthesized by alternative translation initiation through the use of two highly conserved in-frame initiation codons, AUG +1 and AUG +100. Furthermore, we show that the synthesis of these two FLI-1 isoforms is regulated by two short overlapping 5′ upstream open reading frames (uORF) beginning at two highly conserved upstream initiation codons, AUG −41 and GUG −37, and terminating at two highly conserved stop codons, UGA +35 and UAA +15. The mutational analysis of these two 5′ uORF revealed that each of them negatively regulates FLI-1 protein synthesis by precluding cap-dependent scanning to the 48- and 51-kDa AUG codons. Simultaneously, the translation termination of the two 5′ uORF appears to enhance 48-kDa protein synthesis, by allowing downstream reinitiation at the 48-kDa AUG codon, and 51-kDa protein synthesis, by allowing scanning ribosomes to pile up and consequently allowing upstream initiation at the 51-kDa AUG codon. To our knowledge, this is the first example of a cellular mRNA displaying overlapping 5′ uORF whose translation termination appears to be involved in the positive control of translation initiation at both downstream and upstream initiation codons. PMID:10757781

Quantifying the Efficiency of a Translator: The Effect of Syntactical and Literal Written Translations on Language Comprehension Using the Machine Translation System FALCon

ERIC Educational Resources Information Center

McCulloh, Ian A.; Morton, Jillian; Jantzi, Jennifer K.; Rodriguez, Amy M.; Graham, John

2008-01-01

This study introduces a new method of evaluating human comprehension in the context of machine translation using a language translation program known as the FALCon (Forward Area Language Converter). The participants include 48 freshmen from the United States Military Academy enrolled in the General Psychology course, PL100. Results of this study…

PRMT1-Mediated Translation Regulation Is a Crucial Vulnerability of Cancer.

PubMed

Hsu, Jessie Hao-Ru; Hubbell-Engler, Benjamin; Adelmant, Guillaume; Huang, Jialiang; Joyce, Cailin E; Vazquez, Francisca; Weir, Barbara A; Montgomery, Philip; Tsherniak, Aviad; Giacomelli, Andrew O; Perry, Jennifer A; Trowbridge, Jennifer; Fujiwara, Yuko; Cowley, Glenn S; Xie, Huafeng; Kim, Woojin; Novina, Carl D; Hahn, William C; Marto, Jarrod A; Orkin, Stuart H

2017-09-01

Through an shRNA screen, we identified the protein arginine methyltransferase Prmt1 as a vulnerable intervention point in murine p53/Rb-null osteosarcomas, the human counterpart of which lacks effective therapeutic options. Depletion of Prmt1 in p53-deficient cells impaired tumor initiation and maintenance in vitro and in vivo Mechanistic studies reveal that translation-associated pathways were enriched for Prmt1 downstream targets, implicating Prmt1 in translation control. In particular, loss of Prmt1 led to a decrease in arginine methylation of the translation initiation complex, thereby disrupting its assembly and inhibiting translation. p53/Rb-null cells were sensitive to p53-induced translation stress, and analysis of human cancer cell line data from Project Achilles further revealed that Prmt1 and translation-associated pathways converged on the same functional networks. We propose that targeted therapy against Prmt1 and its associated translation-related pathways offer a mechanistic rationale for treatment of osteosarcomas and other cancers that exhibit dependencies on translation stress response. Cancer Res; 77(17); 4613-25. ©2017 AACR . ©2017 American Association for Cancer Research.

Insulin accelerates global and mitochondrial protein synthesis rates in neonatal muscle during sepsis

USDA-ARS?s Scientific Manuscript database

In neonatal pigs, sepsis decreases protein synthesis in skeletal muscle by decreasing translation initiation. However, insulin stimulates muscle protein synthesis despite persistent repression of translation initiation signaling. To determine whether the insulin-induced increase in global rates of m...

Adaptive changes in translation initiation activities for rat pancreatic protein synthesis with feeding of a high-protein diet.

PubMed

Hashi, Masaru; Yoshizawa, Fumiaki; Onozuka, Emi; Ogata, Momoko; Hara, Hiroshi

2005-08-01

We have previously demonstrated that dietary protein induced pancreatic hypergrowth in pancreaticobiliary diverted (PBD) rats. Dietary protein and dietary amino acids stimulate protein synthesis by regulating translation initiation in the rat skeletal muscle and liver. The aim of the present study was to determine whether feeding a high-protein diet induces activation of translation initiation for protein synthesis in the rat pancreas. In PBD rats in which the bile-pancreatic juice was surgically diverted to the upper ileum for 11-13 days, pancreatic dry weight and protein content were doubled compared with those in sham rats and further increased with feeding of a high-protein diet (60% casein diet) for 2 days. These pancreatic growth parameters were maintained at high levels for the next 5 days and were much higher than those of sham rats fed a high-protein diet. In both sham and PBD rats, feeding of a high-protein diet for 2 days induced phosphorylation of eukaryotic initiation factor 4E-binding protein 1 and 70-kDa ribosomal protein S6 kinase, indicating the activation of the initiation phase of translation for pancreatic protein synthesis. However, this increased phosphorylation returned to normal levels on Day 7 in PBD but not in sham rats. We concluded that feeding a high-protein diet induced pancreatic growth with increases in the translation initiation activities for pancreatic protein synthesis within 2 days and that prolonged feeding of a high-protein diet changed the initiation activities differently in sham and PBD rats.

Cap- and initiator tRNA-dependent initiation of TYMV polyprotein synthesis by ribosomes: evaluation of the Trojan horse model for TYMV RNA translation.

PubMed

Matsuda, Daiki; Dreher, Theo W

2007-01-01

Turnip yellow mosaic virus (TYMV) RNA directs the translation of two overlapping open reading frames. Competing models have been previously published to explain ribosome access to the downstream polyprotein cistron. The Trojan horse model, based on cell-free experiments, proposes noncanonical cap-independent initiation in which the 3'-terminal tRNA-like structure (TLS) functionally replaces initiator tRNA, and the valine bound to the TLS becomes cis-incorporated into viral protein. The initiation coupling model, based on in vivo expression and ribosome toe-printing studies, proposes a variation of canonical leaky scanning. Here, we have re-examined the wheat germ extract experiments that led to the Trojan horse model, incorporating a variety of controls. We report that (1) translation in vitro from the polyprotein AUG of TYMV RNA is unchanged after removal of the 3' TLS but is stimulated by the presence of a 5'-cap; (2) the presence of free cap analog or edeine (which interferes with initiation at the ribosomal P site and its tRNA(i) (Met) involvement) inhibits translation from the polyprotein AUG; (3) the toe-prints of immediately post-initiation ribosomes on TYMV RNA are similar with and without an intact TLS; and (4) significant deacylation of valyl-TYMV RNA in wheat germ extract can complicate the detection of cis-incorporation. These results favor the initiation coupling model.

Machine Translation Project

NASA Technical Reports Server (NTRS)

Bajis, Katie

1993-01-01

The characteristics and capabilities of existing machine translation systems were examined and procurement recommendations were developed. Four systems, SYSTRAN, GLOBALINK, PC TRANSLATOR, and STYLUS, were determined to meet the NASA requirements for a machine translation system. Initially, four language pairs were selected for implementation. These are Russian-English, French-English, German-English, and Japanese-English.

47 CFR 74.15 - Station license period.

Code of Federal Regulations, 2010 CFR

2010-10-01

... it is used. (d) Initial licenses for low power TV, TV translator, and FM translator stations will... date determined in accordance with § 73.1020 of this chapter. Lower power TV and TV translator station and FM translator station licenses will ordinarily be renewed for 8 years. However, if the FCC finds...

The 5′ RNA Terminus of Spleen Necrosis Virus Stimulates Translation of Nonviral mRNA

PubMed Central

Roberts, Tiffiney M.; Boris-Lawrie, Kathleen

2000-01-01

The RU5 region at the 5′ RNA terminus of spleen necrosis virus (SNV) has been shown to facilitate expression of human immunodeficiency virus type 1 (HIV) unspliced RNA independently of the Rev-responsive element (RRE) and Rev. The SNV sequences act as a distinct posttranscriptional control element to stimulate gag RNA nuclear export and association with polyribosomes. Here we sought to determine whether RU5 functions to neutralize the cis-acting inhibitory sequences (INSs) in HIV RNA that confer RRE/Rev dependence or functions as an independent stimulatory sequence. Experiments with HIV gag reporter plasmids that contain inactivated INS-1 indicated that neutralization of INSs does not account for RU5 function. Results with luciferase reporter gene (luc) plasmids further indicated that RU5 stimulates expression of a nonretroviral RNA that lacks INSs. Northern blot and RT-PCR analyses indicated that RU5 does not increase the steady-state levels or nuclear export of the luc transcript but rather that the U5 region facilitates efficient polyribosomal association of the mRNA. RU5 does not function as an internal ribosome entry site in bicistronic reporter plasmids, and it requires the 5′-proximal position for efficient function. Our results indicate that RU5 contains stimulatory sequences that function in a 5′-proximal position to enhance initiation of translation of a nonretroviral reporter gene RNA. We speculate that RU5 evolved to overcome the translation-inhibitory effect of the highly structured encapsidation signal and other replication motifs in the 5′ untranslated region of the retroviral RNA. PMID:10933721

Natural Language Processing: A Tutorial. Revision

DTIC Science & Technology

1990-01-01

English in word-for-word language translations. An oft-repeated (although fictional) anecdote illustrates the ... English by a language translation program, became: " The vodka is strong but 3 the steak is rotten." The point made is that vast amounts of knowledge...are required for effective language translations. The initial goal for Language Translation was "fully-automatic high-quality translation" (FAHOT).

Translation initiation at an upstream CUG codon regulates the expression of Hibiscus chlorotic ringspot virus coat protein.

PubMed

Koh, Dora Chin-Yen; Wang, Xiaoxing; Wong, Sek-Man; Liu, D X

2006-12-01

Viruses depend heavily on host cells for replication and exploit the host translation machinery for its gene expression using various unorthodox translation mechanisms. According to the conventional scanning model, only the 5'-proximal gene in the viral RNA is accessible to the ribosomes whereas other genes are silent. In this study, we use a model plant RNA virus, Hibiscus chlorotic ringspot virus (HCRSV), to investigate various translation mechanisms involved in regulation of the expression of internal genes. The 3'-end 1.2kb region of HCRSV genomic and subgenomic RNAs were shown to encode four polypeptides of 38, 27, 25 and 22.5kDa. Mutagenesis studies revealed that a CUG codon ((2570)CUG) is the initiation codon for p27, the longest of the three co-C-terminal products (p27, p25 and p22.5), and translation of p25 and p22.5 was initiated at (2603)AUG and (2666)AUG, respectively. Translation initiation of the p27 expression at the (2570)CUG codon regulates the expression of p38, the viral coat protein through a leaky scanning mechanism and mutational analysis of an upstream open reading frame (ORF) demonstrated that initiation of the p27 expression at this CUG codon (instead of an AUG) may play a role in maintaining the ratio of p27 and p38. In addition, a previously identified internal ribosome entry site was shown to control the expression of p27 and p38 in the subgenomic RNA 2.

Synaptic Plasticity and Translation Initiation

ERIC Educational Resources Information Center

Klann, Eric; Antion, Marcia D.; Banko, Jessica L.; Hou, Lingfei

2004-01-01

It is widely accepted that protein synthesis, including local protein synthesis at synapses, is required for several forms of synaptic plasticity. Local protein synthesis enables synapses to control synaptic strength independent of the cell body via rapid protein production from pre-existing mRNA. Therefore, regulation of translation initiation is…

The DnaA Tale

PubMed Central

Hansen, Flemming G.; Atlung, Tove

2018-01-01

More than 50 years have passed since the presentation of the Replicon Model which states that a positively acting initiator interacts with a specific site on a circular chromosome molecule to initiate DNA replication. Since then, the origin of chromosome replication, oriC, has been determined as a specific region that carries sequences required for binding of positively acting initiator proteins, DnaA-boxes and DnaA proteins, respectively. In this review we will give a historical overview of significant findings which have led to the very detailed knowledge we now possess about the initiation process in bacteria using Escherichia coli as the model organism, but emphasizing that virtually all bacteria have DnaA proteins that interacts with DnaA boxes to initiate chromosome replication. We will discuss the dnaA gene regulation, the special features of the dnaA gene expression, promoter strength, and translation efficiency, as well as, the DnaA protein, its concentration, its binding to DnaA-boxes, and its binding of ATP or ADP. Furthermore, we will discuss the different models for regulation of initiation which have been proposed over the years, with particular emphasis on the Initiator Titration Model. PMID:29541066

The natural compound silvestrol is a potent inhibitor of Ebola virus replication.

PubMed

Biedenkopf, Nadine; Lange-Grünweller, Kerstin; Schulte, Falk W; Weißer, Aileen; Müller, Christin; Becker, Dirk; Becker, Stephan; Hartmann, Roland K; Grünweller, Arnold

2017-01-01

The DEAD-box RNA helicase eIF4A, which is part of the heterotrimeric translation initiation complex in eukaryotes, is an important novel drug target in cancer research because its helicase activity is required to unwind extended and highly structured 5'-UTRs of several proto-oncogenes. Silvestrol, a natural compound isolated from the plant Aglaia foveolata, is a highly efficient, non-toxic and specific inhibitor of eIF4A. Importantly, 5'-capped viral mRNAs often contain structured 5'-UTRs as well, which may suggest a dependence on eIF4A for their translation by the host protein synthesis machinery. In view of the recent Ebola virus (EBOV) outbreak in West Africa, the identification of potent antiviral compounds is urgently required. Since Ebola mRNAs are 5'-capped and harbor RNA secondary structures in their extended 5'-UTRs, we initiated a BSL4 study to analyze silvestrol in EBOV-infected Huh-7 cells and in primary human macrophages for its antiviral activity. We observed that silvestrol inhibits EBOV infection at low nanomolar concentrations, as inferred from large reductions of viral titers. This correlated with an almost complete disappearance of EBOV proteins, comparable in effect to the translational shutdown of expression of the proto-oncoprotein PIM1, a cellular kinase known to be affected by silvestrol. Effective silvestrol concentrations were non-toxic in the tested cell systems. Thus, silvestrol appears to be a promising first-line drug for the treatment of acute EBOV and possibly other viral infections. Copyright © 2016 Elsevier B.V. All rights reserved.

Ribosome profiling: a Hi-Def monitor for protein synthesis at the genome-wide scale

PubMed Central

Michel, Audrey M; Baranov, Pavel V

2013-01-01

Ribosome profiling or ribo-seq is a new technique that provides genome-wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of ribosome protected mRNA fragments allowing the measurement of ribosome density along all RNA molecules present in the cell. At the same time, the high resolution of this technique allows detailed analysis of ribosome density on individual RNAs. Since its invention, the ribosome profiling technique has been utilized in a range of studies in both prokaryotic and eukaryotic organisms. Several studies have adapted and refined the original ribosome profiling protocol for studying specific aspects of translation. Ribosome profiling of initiating ribosomes has been used to map sites of translation initiation. These studies revealed the surprisingly complex organization of translation initiation sites in eukaryotes. Multiple initiation sites are responsible for the generation of N-terminally extended and truncated isoforms of known proteins as well as for the translation of numerous open reading frames (ORFs), upstream of protein coding ORFs. Ribosome profiling of elongating ribosomes has been used for measuring differential gene expression at the level of translation, the identification of novel protein coding genes and ribosome pausing. It has also provided data for developing quantitative models of translation. Although only a dozen or so ribosome profiling datasets have been published so far, they have already dramatically changed our understanding of translational control and have led to new hypotheses regarding the origin of protein coding genes. © 2013 John Wiley & Sons, Ltd. PMID:23696005

Translation of vph mRNA in Streptomyces lividans and Escherichia coli after removal of the 5' untranslated leader.

PubMed

Wu, C J; Janssen, G R

1996-10-01

The Streptomyces vinaceus viomycin phosphotransferase (vph) mRNA contains an untranslated leader with a conventional Shine-Dalgarno homology. The vph leader was removed by ligation of the vph coding sequence to the transcriptional start site of a Streptomyces or an Escherichia coli promoter, such that transcription would initiate at the first position of the vph start codon. Analysis of mRNA demonstrated that transcription initiated primarily at the A of the vph AUG translational start codon in both Streptomyces lividans and E. coli; cells expressing the unleadered vph mRNA were resistant to viomycin indicating that the Shine-Dalgarno sequence, or other features contained within the leader, was not necessary for vph translation. Addition of four nucleotides (5'-AUGC-3') onto the 5' end of the unleadered vph mRNA resulted in translation initiation from the vph start codon and the AUG triplet contained within the added sequence. Translational fusions of vph sequence to a Tn5 neo reporter gene indicated that the first 16 codons of vph coding sequence were sufficient to specify the translational start site and reading frame for expression of neomycin resistance in both E. coli and S. lividans.

The let-7 microRNA interfaces extensively with the translation machinery to regulate cell differentiation

PubMed Central

Ding, Xavier C.; Slack, Frank J.; Großhans, Helge

2010-01-01

MicroRNAs (miRNAs) are noncoding RNAs that regulate numerous target genes through a posttranscriptional mechanism and thus control major developmental pathways. The phylogenetically conserved let-7 miRNA regulates cell proliferation and differentiation, thus functioning as a key regulator of developmental timing in C. elegans and a tumor suppressor gene in humans. Using a reverse genetic screen, we have identified genetic interaction partners of C. elegans let-7, including known and novel potential target genes. Initial identification of several translation initiation factors as suppressors of a let-7 mutation led us to systematically examine genetic interaction between let-7 and the translational machinery, which we found to be widespread. In the presence of wild-type let-7, depletion of the translation initiation factor eIF3 resulted in precocious cell differentiation, suggesting that developmental timing is translationally regulated, possibly by let-7. As overexpression of eIF3 in humans promotes translation of mRNAs that are also targets of let-7-mediated repression, we suggest that eIF3 may directly or indirectly oppose let-7 activity. This might provide an explanation for the opposite functions of let-7 and eIF3 in regulating tumorigenesis. PMID:18818519

The mechanism of RNA 5' capping with NAD +, NADH and desphospho-CoA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bird, Jeremy G.; Zhang, Yu; Tian, Yuan

The chemical nature of the 5' end of RNA is a key determinant of RNA stability, processing, localization and translation efficiency and has been proposed to provide a layer of ‘epitranscriptomic’ gene regulation. Recently it has been shown that some bacterial RNA species carry a 5'-end structure reminiscent of the 5' 7-methylguanylate ‘cap’ in eukaryotic RNA. In particular, RNA species containing a 5'-end nicotinamide adenine dinucleotide (NAD+) or 3'-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria. It has been proposed that NAD+, reduced NAD+ (NADH) and dpCoA caps are added to RNA after transcription initiation, inmore » a manner analogous to the addition of 7-methylguanylate caps. Here we show instead that NAD+, NADH and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated ‘ab initio capping’ may occur in all organisms.« less

Genome-Wide and Experimental Resolution of Relative Translation Elongation Speed at Individual Gene Level in Human Cells

PubMed Central

Gu, Wei; Cui, Yizhi; Zhong, Jiayong; Jin, Jingjie; He, Qing-Yu; Wang, Tong; Zhang, Gong

2016-01-01

In the process of translation, ribosomes first assemble on mRNAs (translation initiation) and then translate along the mRNA (elongation) to synthesize proteins. Elongation pausing is deemed highly relevant to co-translational folding of nascent peptides and the functionality of protein products, which positioned the evaluation of elongation speed as one of the central questions in the field of translational control. By integrating three types of RNA-seq methods, we experimentally and computationally resolved elongation speed, with our proposed elongation velocity index (EVI), a relative measure at individual gene level and under physiological condition in human cells. We successfully distinguished slow-translating genes from the background translatome. We demonstrated that low-EVI genes encoded more stable proteins. We further identified cell-specific slow-translating codons, which might serve as a causal factor of elongation deceleration. As an example for the biological relevance, we showed that the relatively slow-translating genes tended to be associated with the maintenance of malignant phenotypes per pathway analyses. In conclusion, EVI opens a new view to understand why human cells tend to avoid simultaneously speeding up translation initiation and decelerating elongation, and the possible cancer relevance of translating low-EVI genes to gain better protein quality. PMID:26926465

Non-AUG translation: a new start for protein synthesis in eukaryotes

PubMed Central

Kearse, Michael G.; Wilusz, Jeremy E.

2017-01-01

Although it was long thought that eukaryotic translation almost always initiates at an AUG start codon, recent advancements in ribosome footprint mapping have revealed that non-AUG start codons are used at an astonishing frequency. These non-AUG initiation events are not simply errors but instead are used to generate or regulate proteins with key cellular functions; for example, during development or stress. Misregulation of non-AUG initiation events contributes to multiple human diseases, including cancer and neurodegeneration, and modulation of non-AUG usage may represent a novel therapeutic strategy. It is thus becoming increasingly clear that start codon selection is regulated by many trans-acting initiation factors as well as sequence/structural elements within messenger RNAs and that non-AUG translation has a profound impact on cellular states. PMID:28982758

The Translation of Health Research in Kinesiology

ERIC Educational Resources Information Center

Ainsworth, Barbara E.

2009-01-01

The translation of health research is a process of transforming scientific discoveries arising from laboratory, clinical, or population studies into clinical or population-based applications to improve health by reducing disease incidence, morbidity, and mortality. Initiated by the National Institutes for Health Roadmap Initiative and the U.S.…

Hippuristanol - A potent steroid inhibitor of eukaryotic initiation factor 4A

PubMed Central

Cencic, Regina; Pelletier, Jerry

2016-01-01

ABSTRACT Protein synthesis and its regulatory signaling pathways play essential roles in the initiation and maintenance of the cancer phenotype. Insight obtained over the last 3 decades on the mechanisms regulating translation in normal and transformed cells have revealed that perturbed control in cancer cells may offer an Achilles' heel for the development of novel anti-neoplastic agents. Several small molecule inhibitors have been identified and characterized that target translation initiation – more specifically, the rate-limiting step where ribosomes are recruited to mRNA templates. Among these, hippuristanol, a polyhydroxysteroid from the gorgonian Isis hippuris has been found to inhibit translation initiation by blocking the activity of eukaryotic initiation factor (eIF) 4A, an essential RNA helicase involved in this process. Herein, we highlight the biological properties of this compound, its potential development as an anti-cancer agent, and its use to validate eIF4A as an anti-neoplastic target. PMID:27335721

Fail-safe mechanism of GCN4 translational control—uORF2 promotes reinitiation by analogous mechanism to uORF1 and thus secures its key role in GCN4 expression

PubMed Central

Gunišová, Stanislava; Valášek, Leoš Shivaya

2014-01-01

One of the extensively studied mechanisms of gene-specific translational regulation is reinitiation. It takes place on messenger RNAs (mRNAs) where main ORF is preceded by upstream ORF (uORF). Even though uORFs generally down-regulate main ORF expression, specific uORFs exist that allow high level of downstream ORF expression. The key is their ability to retain 40S subunits on mRNA upon termination of their translation to resume scanning for the next AUG. Here, we took advantage of the exemplary model system of reinitiation, the mRNA of yeast transcriptional activator GCN4 containing four short uORFs, and show that contrary to previous reports, not only the first but the first two of its uORFs allow efficient reinitiation. Strikingly, we demonstrate that they utilize a similar molecular mechanism relying on several cis-acting 5′ reinitiation-promoting elements, one of which they share, and the interaction with the a/TIF32 subunit of translation initiation factor eIF3. Since a similar mechanism operates also on YAP1 uORF, our findings strongly suggest that basic principles of reinitiation are conserved. Furthermore, presence of two consecutive reinitiation-permissive uORFs followed by two reinitiation-non-permissive uORFs suggests that tightness of GCN4 translational control is ensured by a fail-safe mechanism that effectively prevents or triggers GCN4 expression under nutrient replete or deplete conditions, respectively. PMID:24623812

Arginine methylation promotes translation repression activity of eIF4G-binding protein, Scd6.

PubMed

Poornima, Gopalakrishna; Shah, Shanaya; Vignesh, Venkadasubramanian; Parker, Roy; Rajyaguru, Purusharth I

2016-11-02

Regulation of translation plays a critical role in determining mRNA fate. A new role was recently reported for a subset of RGG-motif proteins in repressing translation initiation by binding eIF4G1. However the signaling mechanism(s) that leads to spatial and temporal regulation of repression activity of RGG-motif proteins remains unknown. Here we report the role of arginine methylation in regulation of repression activity of Scd6, a conserved RGG-motif protein. We demonstrate that Scd6 gets arginine methylated at its RGG-motif and Hmt1 plays an important role in its methylation. We identify specific methylated arginine residues in the Scd6 RGG-motif in vivo We provide evidence that methylation augments Scd6 repression activity. Arginine methylation defective (AMD) mutant of Scd6 rescues the growth defect caused by overexpression of Scd6, a feature of translation repressors in general. Live-cell imaging of the AMD mutant revealed that it is defective in inducing formation of stress granules. Live-cell imaging and pull-down results indicate that it fails to bind eIF4G1 efficiently. Consistent with these results, a strain lacking Hmt1 is also defective in Scd6-eIF4G1 interaction. Our results establish that arginine methylation augments Scd6 repression activity by promoting eIF4G1-binding. We propose that arginine methylation of translation repressors with RGG-motif could be a general modulator of their repression activity. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Enzymes Involved in Post-transcriptional RNA Metabolism in Gram-negative bacteria

PubMed Central

Mohanty, Bijoy K.

2018-01-01

Gene expression in Gram-negative bacteria is regulated at many levels, including transcription initiation, RNA processing, RNA/RNA interactions, mRNA decay, and translational controls involving enzymes that alter translational efficiency. In this chapter we discuss the various enzymes that control transcription, translation and RNA stability through RNA processing and degradation. RNA processing is essential to generate functional RNAs, while degradation helps control the steady-state level of each individual transcript. For example, all the pre-tRNAs are transcribed with extra nucleotides at both their 5′ and 3′ termini, which are subsequently processed to produce mature tRNAs that can be aminoacylated. Similarly, rRNAs that are transcribed as part of a 30S polycistronic transcript, are matured to individual 16S, 23S and 5S rRNAs. Decay of mRNAs plays a key role in gene regulation through controlling the steady-state level of each transcript, which is essential for maintaining appropriate protein levels. In addition, degradation of both translated and non-translated RNAs recycles nucleotides to facilitate new RNA synthesis. To carry out all these reactions Gram-negative bacteria employ a large number of endonucleases, exonucleases, RNA helicases, and poly(A) polymerase as well as proteins that regulate the catalytic activity of particular ribonucleases. Under certain stress conditions an additional group of specialized endonucleases facilitate the cell’s ability to adapt and survive. Many of the enzymes, such as RNase E, RNase III, polynucleotide phosphorylase, RNase R, and poly(A) polymerase I participate in multiple RNA processing and decay pathways. PMID:29676246

Noise analysis of genome-scale protein synthesis using a discrete computational model of translation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Racle, Julien; Hatzimanikatis, Vassily, E-mail: vassily.hatzimanikatis@epfl.ch; Swiss Institute of Bioinformatics

2015-07-28

Noise in genetic networks has been the subject of extensive experimental and computational studies. However, very few of these studies have considered noise properties using mechanistic models that account for the discrete movement of ribosomes and RNA polymerases along their corresponding templates (messenger RNA (mRNA) and DNA). The large size of these systems, which scales with the number of genes, mRNA copies, codons per mRNA, and ribosomes, is responsible for some of the challenges. Additionally, one should be able to describe the dynamics of ribosome exchange between the free ribosome pool and those bound to mRNAs, as well as howmore » mRNA species compete for ribosomes. We developed an efficient algorithm for stochastic simulations that addresses these issues and used it to study the contribution and trade-offs of noise to translation properties (rates, time delays, and rate-limiting steps). The algorithm scales linearly with the number of mRNA copies, which allowed us to study the importance of genome-scale competition between mRNAs for the same ribosomes. We determined that noise is minimized under conditions maximizing the specific synthesis rate. Moreover, sensitivity analysis of the stochastic system revealed the importance of the elongation rate in the resultant noise, whereas the translation initiation rate constant was more closely related to the average protein synthesis rate. We observed significant differences between our results and the noise properties of the most commonly used translation models. Overall, our studies demonstrate that the use of full mechanistic models is essential for the study of noise in translation and transcription.« less

A wheat embryo cell-free protein synthesis system not requiring an exogenous supply of GTP.

PubMed

Koga, Hirohisa; Misawa, Satoru; Shibui, Tatsuro

2009-01-01

Most in vitro protein synthesis systems require a supply of GTP for the formation of translation initiation complexes, with two GTP molecules per amino acid needed as an energy source for a peptide elongation reaction. In order to optimize protein synthesis reactions in a continuous-flow wheat embryo cell-free system, we have examined the influence of adding GTP and found that the system does not require any supply of GTP. We report here the preparation of a wheat embryo extract from which endogenous GTP was removed by gel filtration, and the influence of adding GTP to the system on protein synthesis reactions. Using Green Fluorescent Protein (GFP) as a reporter, higher levels of production were observed at lower concentrations of GTP, with the optimal level of production obtained with no supply of GTP. A HPLC-based analysis of the extract and the translation mixture containing only ATP as an energy source revealed that GTP was not detectable in the extract, however, 35 microM of GTP was found in the translation mixture. This result suggests that GTP could be generated from other compounds, such as GDP and GMP, using ATP. A similar experiment with a C-terminally truncated form of human protein tyrosine phosphatase 1B (hPTP1B(1-320)) gave almost the same result. The wheat embryo cell-free translation system worked most efficiently without exogenous GTP, producing 3.5 mg/mL of translation mixture over a 48-h period at 26 degrees C. 2009 American Institute of Chemical Engineers Biotechnol.

Quantifying the Efficiency of a Translator: The Effect of Syntactical and Literal Written Translations on Language Comprehension Using the Machine Translation System FALCon (Foreign Area Language Converter)

ERIC Educational Resources Information Center

McCulloh, Ian A.; Morton, Jillian; Jantzi, Jennifer K.; Rodriguez, Amy M.; Graham, John

2008-01-01

The purpose of this study is to introduce a new method of evaluating human comprehension in the context of machine translation using a language translation program known as the FALCon (Forward Area Language Converter). The FALCon works by converting documents into digital images via scanner, and then converting those images to electronic text by…

Translation attenuation via 3′ terminal codon usage in bovine csn1s2 is responsible for the difference in αs2- and β-casein profile in milk

PubMed Central

Kim, Julie J; Yu, Jaeju; Bag, Jnanankur; Bakovic, Marica; Cant, John P

2015-01-01

The rate of secretion of αs2-casein into bovine milk is approximately 25% of that of β-casein, yet mammary expression of their respective mRNA transcripts (csn1s2 and csn2) is not different. Our objective was to identify molecular mechanisms that explain the difference in translation efficiency between csn1s2 and csn2. Cell-free translational efficiency of csn2 was 5 times that of csn1s2. Transcripts of csn1s2 distributed into heavier polysomes than csn2 transcripts, indicating an attenuation of elongation and/or termination. Stimulatory and inhibitory effects of the 5′ and 3′ UTRs on translational efficiency were different with luciferase and casein sequences in the coding regions. Substituting the 5′ and 3′ UTRs from csn2 into csn1s2 did not improve csn1s2 translation, implicating the coding region itself in the translation difference. Deletion of a 28-codon fragment from the 3′ terminus of the csn1s2 coding region, which displays codons with low correlations to cell fitness, increased translation to a par with csn2. We conclude that the usage of the last 28 codons of csn1s2 is the main regulatory element that attenuates its expression and is responsible for the differential translational expression of csn1s2 and csn2. PMID:25826667

Improving Initiation and Tracking of Research Projects at an Academic Health Center: A Case Study.

PubMed

Schmidt, Susanne; Goros, Martin; Parsons, Helen M; Saygin, Can; Wan, Hung-Da; Shireman, Paula K; Gelfond, Jonathan A L

2017-09-01

Research service cores at academic health centers are important in driving translational advancements. Specifically, biostatistics and research design units provide services and training in data analytics, biostatistics, and study design. However, the increasing demand and complexity of assigning appropriate personnel to time-sensitive projects strains existing resources, potentially decreasing productivity and increasing costs. Improving processes for project initiation, assigning appropriate personnel, and tracking time-sensitive projects can eliminate bottlenecks and utilize resources more efficiently. In this case study, we describe our application of lean six sigma principles to our biostatistics unit to establish a systematic continual process improvement cycle for intake, allocation, and tracking of research design and data analysis projects. The define, measure, analyze, improve, and control methodology was used to guide the process improvement. Our goal was to assess and improve the efficiency and effectiveness of operations by objectively measuring outcomes, automating processes, and reducing bottlenecks. As a result, we developed a web-based dashboard application to capture, track, categorize, streamline, and automate project flow. Our workflow system resulted in improved transparency, efficiency, and workload allocation. Using the dashboard application, we reduced the average study intake time from 18 to 6 days, a 66.7% reduction over 12 months (January to December 2015).

International and national initiatives in biobanking.

PubMed

Ectors, N

2011-01-01

Translational research and biobanking are "in", also in Flanders and in Belgium. In Flanders the Advice report 120 from the Flemish Council for Science and innovation, entitled "Extension of translational research in Flanders" paved the way for the Center for Medical Innovation. The Center for Medical Innovation aims at promoting collaboration between Flemish Universities, university hospitals, pharma and biotech industry and the Flemish Government specifically in the domain of translational research. The Initiative # 27 of the Cancer plan from the Federal Government aims at financing a virtual interuniversity tumor bank in order to promote "cancer" translational research in a collaborative network between academic structures, general hospitals en different industrial partners (pharmacy, biotechnology, diagnostics, ...) active in research in Belgium. However, the scientific interest in the human tissues is not new, at all. This text aims at giving an overview of the development and evolutions of "biobanking" initiatives.

Home Performance XML to Real Estate Standards Organization Data Dictionary Translator

DOE Office of Scientific and Technical Information (OSTI.GOV)

2015-10-27

This translator takes fields from the HPXML and translates them into RESO’s Data Dictionary, which is used in MLS systems for real estate transactions across the country. The purpose is to get energy efficiency data into the real estate transaction.

Phage lytic enzymes: a history.

PubMed

Trudil, David

2015-02-01

There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of 'bacteria-eaters' or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well (Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specific disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay (Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes-from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

Tolerance of a Knotted Near-Infrared Fluorescent Protein to Random Circular Permutation.

PubMed

Pandey, Naresh; Kuypers, Brianna E; Nassif, Barbara; Thomas, Emily E; Alnahhas, Razan N; Segatori, Laura; Silberg, Jonathan J

2016-07-12

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFPs to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified 27 circularly permuted iRFPs that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants that initiated translation within the PAS and GAF domains were discovered. Circularly permuted iRFPs retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a quantum yield similar to that of iRFPs but exhibited increased resistance to chemical denaturation, suggesting that the observed increase in the magnitude of the signal arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step toward the creation of near-infrared biosensors with expanded chemical sensing functions for in vivo imaging.

Tolerance of a knotted near infrared fluorescent protein to random circular permutation

PubMed Central

Pandey, Naresh; Kuypers, Brianna E.; Nassif, Barbara; Thomas, Emily E.; Alnahhas, Razan N.; Segatori, Laura; Silberg, Jonathan J.

2016-01-01

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFP to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified twenty seven circularly permuted iRFP that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants were discovered that initiated translation within the PAS and GAF domains. Circularly permuted iRFP retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a similar quantum yield as iRFP, but exhibited increased resistance to chemical denaturation, suggesting that the observed signal increase arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step towards the creation of near-infrared biosensors with expanded chemical-sensing functions for in vivo imaging. PMID:27304983

Targeted mass spectrometric analysis of N-terminally truncated isoforms generated via alternative translation initiation.

PubMed

Kobayashi, Ryuji; Patenia, Rebecca; Ashizawa, Satoshi; Vykoukal, Jody

2009-07-21

Alternative translation initiation is a mechanism whereby functionally altered proteins are produced from a single mRNA. Internal initiation of translation generates N-terminally truncated protein isoforms, but such isoforms observed in immunoblot analysis are often overlooked or dismissed as degradation products. We identified an N-terminally truncated isoform of human Dok-1 with N-terminal acetylation as seen in the wild-type. This Dok-1 isoform exhibited distinct perinuclear localization whereas the wild-type protein was distributed throughout the cytoplasm. Targeted analysis of blocked N-terminal peptides provides rapid identification of protein isoforms and could be widely applied for the general evaluation of perplexing immunoblot bands.

Specific inhibition of aphthovirus infection by RNAs transcribed from both the 5' and the 3' noncoding regions.

PubMed Central

Gutiérrez, A; Martínez-Salas, E; Pintado, B; Sobrino, F

1994-01-01

RNA molecules containing the 3' terminal region of foot-and-mouth disease virus (FMDV) RNA in both antisense and sense orientations were able to inhibit viral FMDV translation and infective particle formation in BHK-21 cells following comicroinjection or cotransfection with infectious viral RNA. Antisense, but not sense, transcripts from the 5' noncoding region including the proximal element of the internal ribosome entry site and the two functional initiation AUGs were also inhibitory, both in in vitro translation and in vivo in comicroinjected or cotransfected BHK-21 cells. This effect was not observed with nonrelated RNA transcripts from lambda phage. The inhibitions found were permanent, sequence specific, and dose dependent; an inverse correlation between the length of the transcript and the extent of the antiviral effect was seen. In all cases, the extent of inhibition increased when viral RNAs and transcripts were allowed to reanneal before transfection, concomitant with a decrease in the doses required. The antiviral effect was specific for FMDV, since transcripts failed to inhibit infective particle formation by other picornavirus, such as encephalomyocarditis virus. These results indicate that the ability of RNA transcripts to inhibit viral multiplication depends on their efficient hybridization with target regions on the viral genome. Furthermore, cells transfected with the 5'1as transcript, which is complementary to the 5' noncoding region, showed a significant reduction of plaque-forming ability during the course of a natural infection. RNA 5'1as was able to inhibit FMDV RNA translation in vitro, suggesting that the inhibitions observed are mediated by a blockage of the viral translation initiation. Conversely, hybridization of short sequences of both sense and antisense transcripts from the 3' end induces distortion of predicted highly ordered structural motifs, which could be required for the synthesis of negative-stranded viral RNA, and correlates with inhibition of viral propagation. Images PMID:7933126

5’-Terminal AUGs in Escherichia coli mRNAs with Shine-Dalgarno Sequences: Identification and Analysis of Their Roles in Non-Canonical Translation Initiation

PubMed Central

Beck, Heather J.; Fleming, Ian M. C.

2016-01-01

Analysis of the Escherichia coli transcriptome identified a unique subset of messenger RNAs (mRNAs) that contain a conventional untranslated leader and Shine-Dalgarno (SD) sequence upstream of the gene’s start codon while also containing an AUG triplet at the mRNA’s 5’- terminus (5’-uAUG). Fusion of the coding sequence specified by the 5’-terminal putative AUG start codon to a lacZ reporter gene, as well as primer extension inhibition assays, reveal that the majority of the 5’-terminal upstream open reading frames (5’-uORFs) tested support some level of lacZ translation, indicating that these mRNAs can function both as leaderless and canonical SD-leadered mRNAs. Although some of the uORFs were expressed at low levels, others were expressed at levels close to that of the respective downstream genes and as high as the naturally leaderless cI mRNA of bacteriophage λ. These 5’-terminal uORFs potentially encode peptides of varying lengths, but their functions, if any, are unknown. In an effort to determine whether expression from the 5’-terminal uORFs impact expression of the immediately downstream cistron, we examined expression from the downstream coding sequence after mutations were introduced that inhibit efficient 5’-uORF translation. These mutations were found to affect expression from the downstream cistrons to varying degrees, suggesting that some 5’-uORFs may play roles in downstream regulation. Since the 5’-uAUGs found on these conventionally leadered mRNAs can function to bind ribosomes and initiate translation, this indicates that canonical mRNAs containing 5’-uAUGs should be examined for their potential to function also as leaderless mRNAs. PMID:27467758

Specific inhibition of aphthovirus infection by RNAs transcribed from both the 5' and the 3' noncoding regions.

PubMed

Gutiérrez, A; Martínez-Salas, E; Pintado, B; Sobrino, F

1994-11-01

RNA molecules containing the 3' terminal region of foot-and-mouth disease virus (FMDV) RNA in both antisense and sense orientations were able to inhibit viral FMDV translation and infective particle formation in BHK-21 cells following comicroinjection or cotransfection with infectious viral RNA. Antisense, but not sense, transcripts from the 5' noncoding region including the proximal element of the internal ribosome entry site and the two functional initiation AUGs were also inhibitory, both in in vitro translation and in vivo in comicroinjected or cotransfected BHK-21 cells. This effect was not observed with nonrelated RNA transcripts from lambda phage. The inhibitions found were permanent, sequence specific, and dose dependent; an inverse correlation between the length of the transcript and the extent of the antiviral effect was seen. In all cases, the extent of inhibition increased when viral RNAs and transcripts were allowed to reanneal before transfection, concomitant with a decrease in the doses required. The antiviral effect was specific for FMDV, since transcripts failed to inhibit infective particle formation by other picornavirus, such as encephalomyocarditis virus. These results indicate that the ability of RNA transcripts to inhibit viral multiplication depends on their efficient hybridization with target regions on the viral genome. Furthermore, cells transfected with the 5'1as transcript, which is complementary to the 5' noncoding region, showed a significant reduction of plaque-forming ability during the course of a natural infection. RNA 5'1as was able to inhibit FMDV RNA translation in vitro, suggesting that the inhibitions observed are mediated by a blockage of the viral translation initiation. Conversely, hybridization of short sequences of both sense and antisense transcripts from the 3' end induces distortion of predicted highly ordered structural motifs, which could be required for the synthesis of negative-stranded viral RNA, and correlates with inhibition of viral propagation.

Cap- and initiator tRNA-dependent initiation of TYMV polyprotein synthesis by ribosomes: Evaluation of the Trojan horse model for TYMV RNA translation

PubMed Central

Matsuda, Daiki; Dreher, Theo W.

2007-01-01

Turnip yellow mosaic virus (TYMV) RNA directs the translation of two overlapping open reading frames. Competing models have been previously published to explain ribosome access to the downstream polyprotein cistron. The Trojan horse model, based on cell-free experiments, proposes noncanonical cap-independent initiation in which the 3′-terminal tRNA-like structure (TLS) functionally replaces initiator tRNA, and the valine bound to the TLS becomes cis-incorporated into viral protein. The initiation coupling model, based on in vivo expression and ribosome toe-printing studies, proposes a variation of canonical leaky scanning. Here, we have re-examined the wheat germ extract experiments that led to the Trojan horse model, incorporating a variety of controls. We report that (1) translation in vitro from the polyprotein AUG of TYMV RNA is unchanged after removal of the 3′ TLS but is stimulated by the presence of a 5′-cap; (2) the presence of free cap analog or edeine (which interferes with initiation at the ribosomal P site and its tRNAi Met involvement) inhibits translation from the polyprotein AUG; (3) the toe-prints of immediately post-initiation ribosomes on TYMV RNA are similar with and without an intact TLS; and (4) significant deacylation of valyl-TYMV RNA in wheat germ extract can complicate the detection of cis-incorporation. These results favor the initiation coupling model. PMID:17095542

PTENα, a PTEN isoform translated through alternative initiation, regulates mitochondrial function and energy metabolism.

PubMed

Liang, Hui; He, Shiming; Yang, Jingyi; Jia, Xinying; Wang, Pan; Chen, Xi; Zhang, Zhong; Zou, Xiajuan; McNutt, Michael A; Shen, Wen Hong; Yin, Yuxin

2014-05-06

PTEN is one of the most frequently mutated genes in human cancer. It is known that PTEN has a wide range of biological functions beyond tumor suppression. Here, we report that PTENα, an N-terminally extended form of PTEN, functions in mitochondrial metabolism. Translation of PTENα is initiated from a CUG codon upstream of and in-frame with the coding region of canonical PTEN. Eukaryotic translation initiation factor 2A (eIF2A) controls PTENα translation, which requires a CUG-centered palindromic motif. We show that PTENα induces cytochrome c oxidase activity and ATP production in mitochondria. TALEN-mediated somatic deletion of PTENα impairs mitochondrial respiratory chain function. PTENα interacts with canonical PTEN to increase PINK1 protein levels and promote energy production. Our studies demonstrate the importance of eIF2A-mediated alternative translation for generation of protein diversity in eukaryotic systems and provide insights into the mechanism by which the PTEN family is involved in multiple cellular processes. Copyright © 2014 Elsevier Inc. All rights reserved.

Aim-less translation: loss of Saccharomyces cerevisiae mitochondrial translation initiation factor mIF3/Aim23 leads to unbalanced protein synthesis.

PubMed

Kuzmenko, Anton; Derbikova, Ksenia; Salvatori, Roger; Tankov, Stoyan; Atkinson, Gemma C; Tenson, Tanel; Ott, Martin; Kamenski, Piotr; Hauryliuk, Vasili

2016-01-05

The mitochondrial genome almost exclusively encodes a handful of transmembrane constituents of the oxidative phosphorylation (OXPHOS) system. Coordinated expression of these genes ensures the correct stoichiometry of the system's components. Translation initiation in mitochondria is assisted by two general initiation factors mIF2 and mIF3, orthologues of which in bacteria are indispensible for protein synthesis and viability. mIF3 was thought to be absent in Saccharomyces cerevisiae until we recently identified mitochondrial protein Aim23 as the missing orthologue. Here we show that, surprisingly, loss of mIF3/Aim23 in S. cerevisiae does not indiscriminately abrogate mitochondrial translation but rather causes an imbalance in protein production: the rate of synthesis of the Atp9 subunit of F1F0 ATP synthase (complex V) is increased, while expression of Cox1, Cox2 and Cox3 subunits of cytochrome c oxidase (complex IV) is repressed. Our results provide one more example of deviation of mitochondrial translation from its bacterial origins.

From dental science to clinical practice: Knowledge translation and evidence-based dentistry principles.

PubMed

Afrashtehfar, Kelvin I; Assery, Mansour K

2017-07-01

It has been claimed that in order to decrease the gap between what we know and what we do, research findings must be translated from knowledge to action. Such practices better enable dentists to make evidence-based decisions instead of personal ideas and judgments. To this end, this literature review aims to revisit the concepts of knowledge translation and evidence-based dentistry (EBD) and depict their role and influence within dental education. It addresses some possible strategies to facilitate knowledge translation (KT), encourage dental students to use EBD principles, and to encourage dental educators to create an environment in which students become self-directed learners. It concludes with a call to develop up-to-date and efficient online platforms that could grant dentists better access to EBD sources in order to more efficiently translate research evidence into the clinic.

Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation

USDA-ARS?s Scientific Manuscript database

In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study...

Study of RNA-A Initiation Translation of The Infectious Pancreatic Necrosis Virus.

PubMed

Rivas-Aravena, Andrea; Muñoz, Patricio; Jorquera, Patricia; Diaz, Alvaro; Reinoso, Claudia; González-Catrilelbún, Sebastián; Sandino, Ana María

2017-08-15

The infectious pancreatic necrosis virus (IPNV) is a salmonid pathogen that causes significant economic losses to the aquaculture industry. IPNV is a non-enveloped virus containing two uncapped and non-polyadenylated double strand RNA genomic segments, RNA-A and RNA-B. The viral protein Vpg is covalently attached to the 5' end of both segments. There is little knowledge about its viral cycle, particularly about the translation of the RNAs. Through experiments using mono and bicistronic reporters, in this work we show that the 120-nucleotide-long 5'-UTR of RNA-A contains an internal ribosome entry site (IRES) that functions efficiently both in vitro and in salmon cells. IRES activity is strongly dependent on temperature. Also, the IRES structure is confined to the 5'UTR and is not affected by the viral coding sequence. This is the first report of IRES activity in a fish virus and can give us tools to generate antivirals to attack the virus without affecting fish directly. Copyright © 2017. Published by Elsevier B.V.

A Novel Approach to Measuring Efficiency of Scientific Research Projects: Data Envelopment Analysis.

PubMed

Dilts, David M; Zell, Adrienne; Orwoll, Eric

2015-10-01

Measuring the efficiency of resource allocation for the conduct of scientific projects in medical research is difficult due to, among other factors, the heterogeneity of resources supplied (e.g., dollars or FTEs) and outcomes expected (e.g., grants, publications). While this is an issue in medical science, it has been approached successfully in other fields by using data envelopment analysis (DEA). DEA has a number of advantages over other techniques as it simultaneously uses multiple heterogeneous inputs and outputs to determine which projects are performing most efficiently, referred to as being at the efficiency frontier, when compared to others in the data set. This research uses DEA for the evaluation of supported translational science projects by the Oregon Clinical and Translational Research Institute (OCTRI), a NCATS Clinical & Translational Science Award (CTSA) recipient. These results suggest that the primary determinate of overall project efficiency at OCTRI is the amount of funding, with smaller amounts of funding providing more efficiency than larger funding amounts. These results, and the use of DEA, highlight both the success of using this technique in helping determine medical research efficiency and those factors to consider when distributing funds for new projects at CTSAs. © 2015 Wiley Periodicals, Inc.

From what we know to what we do: lessons learned from the translational CLAHRC initiative in England.

PubMed

Currie, Graeme; Lockett, Andy; El Enany, Nellie

2013-10-01

There exists a translation gap between academic research and clinical practice in health care systems. One policy-driven initiative to address the translation gap in England are the Collaborations for Leadership in Applied Health Research and Care (CLAHRCs), funded by the National Institute of Health Research (NIHR). These aim to bring together NHS organizations and universities to accelerate the translation of evidence-based innovation into clinical practice. Our aim was to draw out lessons for policy-makers regarding the mobilization of such initiatives. Qualitative semi-structured interviews with 174 participants across nine CLAHRCs plus in-depth case studies across four CLAHRCs. Those interviewed were staff who were central to the CLAHRCs including senior managers and directors, junior and senior academics, and health care practitioners. Social positions of the CLAHRC leaders, conceived as institutional entrepreneurs, together with the antecedent conditions for CLAHRC bids, had an impact on the vision for a CLAHRC. The process of envisioning encompassed diagnostic and prognostic framing. Within the envisioning process, the utilization of existing activities and established relationships in the CLAHRC bid influenced early mobilization. However, in some cases, it led to a translational 'lock in' towards established models regarding applied research. The CLAHRC experiment in England holds important lessons for policy-makers regarding how to address the translation gap. First, policy makers need to consider whether they set out a defined template for translational initiatives or whether variation is encouraged. We might expect a degree of learning from pilot activities within a CLAHRC that allows for greater clarity in the design of subsequent translational initiatives. Second, policy makers and practitioners need to understand the importance of both antecedent conditions and the social position of senior members of a CLAHRC (institutional entrepreneurs) leading the development of a bid. Whilst established and well-known clinical academics are likely to be trusted to lead CLAHRCs, and the presence of pre-existing organizational relationships are important for mobilization, privileging these aspects may constrain more radical change.

Translational Control of Viral Gene Expression in Eukaryotes

PubMed Central

Gale, Michael; Tan, Seng-Lai; Katze, Michael G.

2000-01-01

As obligate intracellular parasites, viruses rely exclusively on the translational machinery of the host cell for the synthesis of viral proteins. This relationship has imposed numerous challenges on both the infecting virus and the host cell. Importantly, viruses must compete with the endogenous transcripts of the host cell for the translation of viral mRNA. Eukaryotic viruses have thus evolved diverse mechanisms to ensure translational efficiency of viral mRNA above and beyond that of cellular mRNA. Mechanisms that facilitate the efficient and selective translation of viral mRNA may be inherent in the structure of the viral nucleic acid itself and can involve the recruitment and/or modification of specific host factors. These processes serve to redirect the translation apparatus to favor viral transcripts, and they often come at the expense of the host cell. Accordingly, eukaryotic cells have developed antiviral countermeasures to target the translational machinery and disrupt protein synthesis during the course of virus infection. Not to be outdone, many viruses have answered these countermeasures with their own mechanisms to disrupt cellular antiviral pathways, thereby ensuring the uncompromised translation of virion proteins. Here we review the varied and complex translational programs employed by eukaryotic viruses. We discuss how these translational strategies have been incorporated into the virus life cycle and examine how such programming contributes to the pathogenesis of the host cell. PMID:10839817

Design of an efficient music-speech discriminator.

PubMed

Tardón, Lorenzo J; Sammartino, Simone; Barbancho, Isabel

2010-01-01

In this paper, the problem of the design of a simple and efficient music-speech discriminator for large audio data sets in which advanced music playing techniques are taught and voice and music are intrinsically interleaved is addressed. In the process, a number of features used in speech-music discrimination are defined and evaluated over the available data set. Specifically, the data set contains pieces of classical music played with different and unspecified instruments (or even lyrics) and the voice of a teacher (a top music performer) or even the overlapped voice of the translator and other persons. After an initial test of the performance of the features implemented, a selection process is started, which takes into account the type of classifier selected beforehand, to achieve good discrimination performance and computational efficiency, as shown in the experiments. The discrimination application has been defined and tested on a large data set supplied by Fundacion Albeniz, containing a large variety of classical music pieces played with different instrument, which include comments and speeches of famous performers.

A new MIF4G domain-containing protein, CTIF, directs nuclear cap-binding protein CBP80/20-dependent translation

PubMed Central

Kim, Kyoung Mi; Cho, Hana; Choi, Kobong; Kim, Jaedong; Kim, Bong-Woo; Ko, Young-Gyu; Jang, Sung Key; Kim, Yoon Ki

2009-01-01

During or right after mRNA export via the nuclear pore complex (NPC) in mammalian cells, mRNAs undergo translation mediated by nuclear cap-binding proteins 80 and 20 (CBP80/20). After CBP80/20-dependent translation, CBP80/20 is replaced by cytoplasmic cap-binding protein eIF4E, which directs steady-state translation. Nonsense-mediated mRNA decay (NMD), one of the best-characterized mRNA surveillance mechanisms, has been shown to occur on CBP80/20-bound mRNAs. However, despite the tight link between CBP80/20-dependent translation and NMD, the underlying molecular mechanism and cellular factors that mediate CBP80/20-dependent translation remain obscure. Here, we identify a new MIF4G domain-containing protein, CTIF (CBP80/20-dependent translation initiation factor). CTIF interacts directly with CBP80 and is part of the CBP80/20-dependent translation initiation complex. Depletion of endogenous CTIF from an in vitro translation system selectively blocks the translation of CBP80-bound mRNAs, while addition of purified CTIF restores it. Accordingly, down-regulation of endogenous CTIF abrogates NMD. Confocal microscopy shows that CTIF is localized to the perinuclear region. Our observations demonstrate the existence of CBP80/20-dependent translation and support the idea that CBP80/20-dependent translation is mechanistically different from steady-state translation through identification of a specific cellular protein, CTIF. PMID:19648179

Trapping dynamics of xenon on Pt(111)

NASA Astrophysics Data System (ADS)

Arumainayagam, Christopher R.; Madix, Robert J.; Mcmaster, Mark C.; Suzawa, Valerie M.; Tully, John C.

1990-02-01

The dynamics of Xe trapping on Pt(111) was studied using supersonic atomic beam techniques. Initial trapping probabilities ( S0) were measured directly as a function of incident translational energy ( EinT) and angle of incidence (θ i) at a surface temperature ( Tins) 95 K. The initial trapping probability decreases smoothly with increasing ET cosθ i;, rather than ET cos 2θ i, suggesting participation of parallel momentum in the trapping process. Accordingly, the measured initial trapping probability falls off more slowly with increasing incident translational energy than predicted by one-dimensional theories. This finding is in near agreement with previous mean translational energy measurements for Xe desorbing near the Pt(111) surface normal, assuming detailed balance applies. Three-dimensional stochastic classical trajectory calculations presented herein also exhibit the importance of tangential momentum in trapping and satisfactorily reproduce the experimental initial trapping probabilities.

The Enterococcus faecalis EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT

PubMed Central

Montealegre, Maria Camila; La Rosa, Sabina Leanti; Roh, Jung Hyeob; Harvey, Barrett R.

2015-01-01

ABSTRACT The endocarditis and biofilm-associated pili (Ebp) are important in Enterococcus faecalis pathogenesis, and the pilus tip, EbpA, has been shown to play a major role in pilus biogenesis, biofilm formation, and experimental infections. Based on in silico analyses, we previously predicted that ATT is the EbpA translational start codon, not the ATG codon, 120 bp downstream of ATT, which is annotated as the translational start. ATT is rarely used to initiate protein synthesis, leading to our hypothesis that this codon participates in translational regulation of Ebp production. To investigate this possibility, site-directed mutagenesis was used to introduce consecutive stop codons in place of two lysines at positions 5 and 6 from the ATT, to replace the ATT codon in situ with ATG, and then to revert this ATG to ATT; translational fusions of ebpA to lacZ were also constructed to investigate the effect of these start codons on translation. Our results showed that the annotated ATG does not start translation of EbpA, implicating ATT as the start codon; moreover, the presence of ATT, compared to the engineered ATG, resulted in significantly decreased EbpA surface display, attenuated biofilm, and reduced adherence to fibrinogen. Corroborating these findings, the translational fusion with the native ATT as the initiation codon showed significantly decreased expression of β-galactosidase compared to the construct with ATG in place of ATT. Thus, these results demonstrate that the rare initiation codon of EbpA negatively regulates EbpA surface display and negatively affects Ebp-associated functions, including biofilm and adherence to fibrinogen. PMID:26015496

Structural variant of the intergenic internal ribosome entry site elements in dicistroviruses and computational search for their counterparts

PubMed Central

HATAKEYAMA, YOSHINORI; SHIBUYA, NORIHIRO; NISHIYAMA, TAKASHI; NAKASHIMA, NOBUHIKO

2004-01-01

The intergenic region (IGR) located upstream of the capsid protein gene in dicistroviruses contains an internal ribosome entry site (IRES). Translation initiation mediated by the IRES does not require initiator methionine tRNA. Comparison of the IGRs among dicistroviruses suggested that Taura syndrome virus (TSV) and acute bee paralysis virus have an extra side stem loop in the predicted IRES. We examined whether the side stem is responsible for translation activity mediated by the IGR using constructs with compensatory mutations. In vitro translation analysis showed that TSV has an IGR-IRES that is structurally distinct from those previously described. Because IGR-IRES elements determine the translation initiation site by virtue of their own tertiary structure formation, the discovery of this initiation mechanism suggests the possibility that eukaryotic mRNAs might have more extensive coding regions than previously predicted. To test this hypothesis, we searched full-length cDNA databases and whole genome sequences of eukaryotes using the pattern matching program, Scan For Matches, with parameters that can extract sequences containing secondary structure elements resembling those of IGR-IRES. Our search yielded several sequences, but their predicted secondary structures were suggested to be unstable in comparison to those of dicistroviruses. These results suggest that RNAs structurally similar to dicistroviruses are not common. If some eukaryotic mRNAs are translated independently of an initiator methionine tRNA, their structures are likely to be significantly distinct from those of dicistroviruses. PMID:15100433

Reactivation of stalled polyribosomes in synaptic plasticity

PubMed Central

Graber, Tyson E.; Hébert-Seropian, Sarah; Khoutorsky, Arkady; David, Alexandre; Yewdell, Jonathan W.; Lacaille, Jean-Claude; Sossin, Wayne S.

2013-01-01

Some forms of synaptic plasticity require rapid, local activation of protein synthesis. Although this is thought to reflect recruitment of mRNAs to free ribosomes, this would limit the speed and magnitude of translational activation. Here we provide compelling in situ evidence supporting an alternative model in which synaptic mRNAs are transported as stably paused polyribosomes. Remarkably, we show that metabotropic glutamate receptor activation allows the synthesis of proteins that lead to a functional long-term depression phenotype even when translation initiation has been greatly reduced. Thus, neurons evolved a unique mechanism to swiftly translate synaptic mRNAs into functional protein upon synaptic signaling using stalled polyribosomes to bypass the rate-limiting step of translation initiation. Because dysregulated plasticity is implicated in neurodevelopmental and psychiatric disorders such as fragile X syndrome, this work uncovers a unique translational target for therapies. PMID:24043809

An efficient agro-industrial complex in Almería (Spain): Towards an integrated and sustainable bioeconomy model.

PubMed

Egea, Francisco J; Torrente, Roberto G; Aguilar, Alfredo

2018-01-25

In the last ten years, bioeconomy strategies and policy-related bioeconomy initiatives have been developed all over the world. Some of them are currently in the process of translation into specific actions. In most cases, the approaches followed have been top-down policy-related initiatives, triggered by the public sector originating a dynamic which can bring together different bioeconomy stakeholders i.e. industry, academia, financial operators and farmers. This article describes a bottom-up situation with unique bioeconomy-related features that deserve specific attention. Over the last 40 years, Almería, in the south east of Spain, has developed one of the most efficient agro-industrial complexes in the world, evolving from a traditional and subsistence agriculture, to becoming the major vegetable exporter in the European Union (EU). This growth set aside issues such as sustainability, long-term perspectives on water resources or agricultural waste. However, societal concerns about a circular economy, as well as policy initiatives in the EU and in Spain on bioeconomy are changing the situation towards an integrated, efficient and sustainable bioeconomy system. Currently, the production chain demands innovations related to the use of biomass as source of bioproducts and bioenergy in order to remain competitive. Some positive aspects are the relatively small size of the agro-industrial area, making transport and communications rapid and easy, and the existence of strong and dedicated academic and financial institutions. This article outlines the current efforts and initiatives to couple the existing successful agro-industrial complex with that of a fully sustainable bioeconomy model. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a novel translation micromirror for adaptive optics

NASA Astrophysics Data System (ADS)

He, Siyuan; Ben Mrad, Ridha

2003-10-01

Conventional translation micromirrors for adaptive optics use attractive electrostatic force and therefore have two limitations: 1) the stroke is limited to less than one third of the initial gap distance between the mirror plate and the substrate. Normally the stroke is in the range of submicrometers; 2) stiction happens during operation. A novel translation micromirror, which uses a repulsive electrostatic force, is presented in this paper. This novel translation micromirror completely overcomes the limitations associated with conventional translation micromirrors and its stroke is not limited by the initial gap distance between the mirror plate and the substrate and therefore is able to achieve a much larger vertical stroke to modulate lights over a wider spectrum than that achieved by conventional translation micromirrors. The novel translation micromirror has no stiction problem and is highly compatible with mature surface micromachining technology. An analytical model is derived for the novel translation micromirror and prototypes are fabricated. The prototype of the novel translation micromirror, which is deliberately not optimized so it could be fabricated using MUMPS, achieved a vertical stroke of 1.75μm using a driving voltage of 50 volts, which is three times the stroke of conventional MUMPS translation micromirrors. It is expected that if standard surface micromachining is used instead of MUMPs, the design of the novel translation micromirror can be optimized and a much larger vertical stroke can be achieved.

Sequence and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency

PubMed Central

Sroubek, Jakub; Krishnan, Yamini; McDonald, Thomas V.

2013-01-01

Human ether-á-gogo-related gene (HERG) encodes a potassium channel that is highly susceptible to deleterious mutations resulting in susceptibility to fatal cardiac arrhythmias. Most mutations adversely affect HERG channel assembly and trafficking. Why the channel is so vulnerable to missense mutations is not well understood. Since nothing is known of how mRNA structural elements factor in channel processing, we synthesized a codon-modified HERG cDNA (HERG-CM) where the codons were synonymously changed to reduce GC content, secondary structure, and rare codon usage. HERG-CM produced typical IKr-like currents; however, channel synthesis and processing were markedly different. Translation efficiency was reduced for HERG-CM, as determined by heterologous expression, in vitro translation, and polysomal profiling. Trafficking efficiency to the cell surface was greatly enhanced, as assayed by immunofluorescence, subcellular fractionation, and surface labeling. Chimeras of HERG-NT/CM indicated that trafficking efficiency was largely dependent on 5′ sequences, while translation efficiency involved multiple areas. These results suggest that HERG translation and trafficking rates are independently governed by noncoding information in various regions of the mRNA molecule. Noncoding information embedded within the mRNA may play a role in the pathogenesis of hereditary arrhythmia syndromes and could provide an avenue for targeted therapeutics.—Sroubek, J., Krishnan, Y., McDonald, T V. Sequence- and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency. PMID:23608144

Modeling and prediction of human word search behavior in interactive machine translation

NASA Astrophysics Data System (ADS)

Ji, Duo; Yu, Bai; Ma, Bin; Ye, Na

2017-12-01

As a kind of computer aided translation method, Interactive Machine Translation technology reduced manual translation repetitive and mechanical operation through a variety of methods, so as to get the translation efficiency, and played an important role in the practical application of the translation work. In this paper, we regarded the behavior of users' frequently searching for words in the translation process as the research object, and transformed the behavior to the translation selection problem under the current translation. The paper presented a prediction model, which is a comprehensive utilization of alignment model, translation model and language model of the searching words behavior. It achieved a highly accurate prediction of searching words behavior, and reduced the switching of mouse and keyboard operations in the users' translation process.

Global Health Initiatives of the International Oncology Community.

PubMed

Al-Sukhun, Sana; de Lima Lopes, Gilberto; Gospodarowicz, Mary; Ginsburg, Ophira; Yu, Peter Paul

2017-01-01

Cancer has become one of the leading causes of morbidity and mortality in low- and middle-income countries (LMICs), where 60% of the world's total new cases are diagnosed. The challenge for effective control of cancer is multifaceted. It mandates integration of effective cancer prevention, encouraging early detection, and utilization of resource-adapted therapeutic and supportive interventions. In the resource-constrained setting, it becomes challenging to deliver each service optimally, and efficient allocation of resources is the best way to improve the outcome. This concept was translated into action through development of resource-stratified guidelines, pioneered by the Breast Health Global Initiative (BHGI), and later adopted by most oncology societies in an attempt to help physicians deliver the best possible care in a limited-resource setting. Improving outcome entails collaboration between key stakeholders, including the pharmaceutical industry, local and national health authorities, the World Health Organization (WHO), and other nonprofit, patient-oriented organizations. Therefore, we started to observe global health initiatives-led by ASCO, the Union for International Cancer Control (UICC), and the WHO-to address these challenges at the international level. This article discusses some of these initiatives.

Translational models of lung disease.

PubMed

Mercer, Paul F; Abbott-Banner, Katharine; Adcock, Ian M; Knowles, Richard G

2015-02-01

The 2nd Cross Company Respiratory Symposium (CCRS), held in Horsham, U.K. in 2012, brought together representatives from across the pharmaceutical industry with expert academics, in the common interest of improving the design and translational predictiveness of in vivo models of respiratory disease. Organized by the respiratory representatives of the European Federation of Pharmaceutical Industries and Federations (EFPIA) group of companies involved in the EU-funded project (U-BIOPRED), the aim of the symposium was to identify state-of-the-art improvements in the utility and design of models of respiratory disease, with a view to improving their translational potential and reducing wasteful animal usage. The respiratory research and development community is responding to the challenge of improving translation in several ways: greater collaboration and open sharing of data, careful selection of the species, complexity and chronicity of the models, improved practices in preclinical research, continued refinement in models of respiratory diseases and their sub-types, greater understanding of the biology underlying human respiratory diseases and their sub-types, and finally greater use of human (and especially disease-relevant) cells, tissues and explants. The present review highlights these initiatives, combining lessons from the symposium and papers published in Clinical Science arising from the symposium, with critiques of the models currently used in the settings of asthma, idiopathic pulmonary fibrosis and COPD. The ultimate hope is that this will contribute to a more rational, efficient and sustainable development of a range of new treatments for respiratory diseases that continue to cause substantial morbidity and mortality across the world.

Intracellular modifications induced by poliovirus reduce the requirement for structural motifs in the 5' noncoding region of the genome involved in internal initiation of protein synthesis.

PubMed Central

Percy, N; Belsham, G J; Brangwyn, J K; Sullivan, M; Stone, D M; Almond, J W

1992-01-01

A series of genetic deletions based partly on two RNA secondary structure models (M. A. Skinner, V. R. Racaniello, G. Dunn, J. Cooper, P. D. Minor, and J. W. Almond, J. Mol. Biol. 207:379-392, 1989; E. V. Pilipenko, V. M. Blinov, L. I. Romanova, A. N. Sinyakov, S. V. Maslova, and V. I. Agol, Virology 168:201-209, 1989) was made in the cDNA encoding the 5' noncoding region (5' NCR) of the poliovirus genome in order to study the sequences that direct the internal entry of ribosomes. The modified cDNAs were placed between two open reading frames in a single transcriptional unit and used to transfect cells in culture. Internal entry of ribosomes was detected by measuring translation from the second open reading frame in the bicistronic mRNA. When assayed alone, a large proportion of the poliovirus 5' NCR superstructure including several well-defined stem-loops was required for ribosome entry and efficient translation. However, in cells cotransfected with a complete infectious poliovirus cDNA, the requirement for the stem-loops in this large superstructure was reduced. The results suggest that virus infection modifies the cellular translational machinery, so that shortened forms of the 5' NCR are sufficient for cap-independent translation, and that the internal entry of ribosomes occurs by two distinct modes during the virus replication cycle. Images PMID:1310772

Stimulation of muscle protein synthesis by somatotropin in pigs is independent of the somatotropin-induced increase in circulating insulin.

PubMed

Wilson, Fiona A; Orellana, Renán A; Suryawan, Agus; Nguyen, Hanh V; Jeyapalan, Asumthia S; Frank, Jason; Davis, Teresa A

2008-07-01

Chronic treatment of growing pigs with porcine somatotropin (pST) promotes protein synthesis and doubles postprandial levels of insulin, a hormone that stimulates translation initiation. This study aimed to determine whether the pST-induced increase in skeletal muscle protein synthesis was mediated through an insulin-induced stimulation of translation initiation. After 7-10 days of pST (150 microg x kg(-1) x day(-1)) or control saline treatment, pancreatic glucose-amino acid clamps were performed in overnight-fasted pigs to reproduce 1) fasted (5 microU/ml), 2) fed control (25 microU/ml), and 3) fed pST-treated (50 microU/ml) insulin levels while glucose and amino acids were maintained at baseline fasting levels. Fractional protein synthesis rates and indexes of translation initiation were examined in skeletal muscle. Effectiveness of pST treatment was confirmed by reduced urea nitrogen and elevated insulin-like growth factor I levels in plasma. Skeletal muscle protein synthesis was independently increased by both insulin and pST. Insulin increased the phosphorylation of protein kinase B and the downstream effectors of the mammalian target of rapamycin, ribosomal protein S6 kinase, and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1). Furthermore, insulin reduced inactive 4E-BP1.eIF4E complex association and increased active eIF4E.eIF4G complex formation, indicating enhanced eIF4F complex assembly. However, pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of skeletal muscle protein synthesis in growing pigs is independent of the insulin-associated activation of translation initiation.

Stimulation of muscle protein synthesis by somatotropin in pigs is independent of the somatotropin-induced increase in circulating insulin

PubMed Central

Wilson, Fiona A.; Orellana, Renán A.; Suryawan, Agus; Nguyen, Hanh V.; Jeyapalan, Asumthia S.; Frank, Jason; Davis, Teresa A.

2008-01-01

Chronic treatment of growing pigs with porcine somatotropin (pST) promotes protein synthesis and doubles postprandial levels of insulin, a hormone that stimulates translation initiation. This study aimed to determine whether the pST-induced increase in skeletal muscle protein synthesis was mediated through an insulin-induced stimulation of translation initiation. After 7–10 days of pST (150 μg·kg−1·day−1) or control saline treatment, pancreatic glucose-amino acid clamps were performed in overnight-fasted pigs to reproduce 1) fasted (5 μU/ml), 2) fed control (25 μU/ml), and 3) fed pST-treated (50 μU/ml) insulin levels while glucose and amino acids were maintained at baseline fasting levels. Fractional protein synthesis rates and indexes of translation initiation were examined in skeletal muscle. Effectiveness of pST treatment was confirmed by reduced urea nitrogen and elevated insulin-like growth factor I levels in plasma. Skeletal muscle protein synthesis was independently increased by both insulin and pST. Insulin increased the phosphorylation of protein kinase B and the downstream effectors of the mammalian target of rapamycin, ribosomal protein S6 kinase, and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1). Furthermore, insulin reduced inactive 4E-BP1·eIF4E complex association and increased active eIF4E·eIF4G complex formation, indicating enhanced eIF4F complex assembly. However, pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of skeletal muscle protein synthesis in growing pigs is independent of the insulin-associated activation of translation initiation. PMID:18460595

Genomics as knowledge enterprise: Implementing an electronic research habitat at the Biopolis Experimental Therapeutics Center.

PubMed

Mitchell, Wayne; Breen, Colin; Entzeroth, Michael

2008-03-01

The Experimental Therapeutics Center (ETC) has been established at Biopolis to advance translational research by bridging the gap between discovery science and commercialization. We describe the Electronic Research Habitat at ETC, a comprehensive hardware and software infrastructure designed to effectively manage terabyte data flows and storage, increase back office efficiency, enhance the scientific work experience, and satisfy rigorous regulatory and legal requirements. Our habitat design is secure, scalable and robust, and it strives to embody the core values of the knowledge-based workplace, thus contributing to the strategic goal of building a "knowledge economy" in the context of Singapore's on-going biotechnology initiative.

Lost in Translation: Bioinformatic Analysis of Variations Affecting the Translation Initiation Codon in the Human Genome.

PubMed

Abad, Francisco; de la Morena-Barrio, María Eugenia; Fernández-Breis, Jesualdo Tomás; Corral, Javier

2018-06-01

Translation is a key biological process controlled in eukaryotes by the initiation AUG codon. Variations affecting this codon may have pathological consequences by disturbing the correct initiation of translation. Unfortunately, there is no systematic study describing these variations in the human genome. Moreover, we aimed to develop new tools for in silico prediction of the pathogenicity of gene variations affecting AUG codons, because to date, these gene defects have been wrongly classified as missense. Whole-exome analysis revealed the mean of 12 gene variations per person affecting initiation codons, mostly with high (> 0:01) minor allele frequency (MAF). Moreover, analysis of Ensembl data (December 2017) revealed 11,261 genetic variations affecting the initiation AUG codon of 7,205 genes. Most of these variations (99.5%) have low or unknown MAF, probably reflecting deleterious consequences. Only 62 variations had high MAF. Genetic variations with high MAF had closer alternative AUG downstream codons than did those with low MAF. Besides, the high-MAF group better maintained both the signal peptide and reading frame. These differentiating elements could help to determine the pathogenicity of this kind of variation. Data and scripts in Perl and R are freely available at https://github.com/fanavarro/hemodonacion. jfernand@um.es. Supplementary data are available at Bioinformatics online.

Polysome Fractionation to Analyze mRNA Distribution Profiles.

PubMed

Panda, Amaresh C; Martindale, Jennifer L; Gorospe, Myriam

2017-02-05

Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al. , 2014a and 2014b; Abdelmohsen et al. , 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ribosomes with a given mRNA. It provides valuable information about the translational status of that mRNA, depending on the number of ribosomes with which they are associated, and identifies mRNAs that are not translated (Panda et al. , 2016). mRNAs associated with many ribosomes form large polysomes that are predicted to be actively translated, while mRNAs associated with few or no ribosomes are expected to be translated poorly if at all. In sum, polysome fractionation analysis allows the direct determination of translation efficiencies at the level of the whole transcriptome as well as individual mRNAs.

Translating Alcohol Research

PubMed Central

Batman, Angela M.; Miles, Michael F.

2015-01-01

Alcohol use disorder (AUD) and its sequelae impose a major burden on the public health of the United States, and adequate long-term control of this disorder has not been achieved. Molecular and behavioral basic science research findings are providing the groundwork for understanding the mechanisms underlying AUD and have identified multiple candidate targets for ongoing clinical trials. However, the translation of basic research or clinical findings into improved therapeutic approaches for AUD must become more efficient. Translational research is a multistage process of streamlining the movement of basic biomedical research findings into clinical research and then to the clinical target populations. This process demands efficient bidirectional communication across basic, applied, and clinical science as well as with clinical practitioners. Ongoing work suggests rapid progress is being made with an evolving translational framework within the alcohol research field. This is helped by multiple interdisciplinary collaborative research structures that have been developed to advance translational work on AUD. Moreover, the integration of systems biology approaches with collaborative clinical studies may yield novel insights for future translational success. Finally, appreciation of genetic variation in pharmacological or behavioral treatment responses and optimal communication from bench to bedside and back may strengthen the success of translational research applications to AUD. PMID:26259085

Translating Alcohol Research: Opportunities and Challenges.

PubMed

Batman, Angela M; Miles, Michael F

2015-01-01

Alcohol use disorder (AUD) and its sequelae impose a major burden on the public health of the United States, and adequate long-term control of this disorder has not been achieved. Molecular and behavioral basic science research findings are providing the groundwork for understanding the mechanisms underlying AUD and have identified multiple candidate targets for ongoing clinical trials. However, the translation of basic research or clinical findings into improved therapeutic approaches for AUD must become more efficient. Translational research is a multistage process of stream-lining the movement of basic biomedical research findings into clinical research and then to the clinical target populations. This process demands efficient bidirectional communication across basic, applied, and clinical science as well as with clinical practitioners. Ongoing work suggests rapid progress is being made with an evolving translational framework within the alcohol research field. This is helped by multiple interdisciplinary collaborative research structures that have been developed to advance translational work on AUD. Moreover, the integration of systems biology approaches with collaborative clinical studies may yield novel insights for future translational success. Finally, appreciation of genetic variation in pharmacological or behavioral treatment responses and optimal communication from bench to bedside and back may strengthen the success of translational research applications to AUD.

The Role of the Clinical and Translational Science Awards Program in Improving the Quality and Efficiency of Clinical Research

PubMed Central

Rosenblum, Daniel

2011-01-01

Recognizing the need to increase the efficiency and quality of translating basic discovery into treatment and prevention strategies for patients and the public, the National Institutes of Health (NIH) announced the Clinical and Translational Science Awards (CTSAs) in 2006. Academic health centers that competed successfully for these awards agreed to work as a consortium and in cooperation with the NIH to improve the translation process by training the next generation of investigators to work in interdisciplinary teams, developing public-private partnerships in the movement of basic discovery to preclinical and clinical studies and trials, improving clinical research management, and engaging with communities to ensure their involvement in shaping research questions and in implementing research results. The CTSAs have addressed the crucial need to improve the quality and efficiency of clinical research by (1) providing training for clinical investigators and for bench researchers to facilitate their participation in the clinical and translational research environment, (2) developing more systematic approaches to clinical research management, and (3) engaging communities as active participants in the design and conduct of clinical research studies and trials and as leaders in implementing health advances that are of high importance to them. We provide an overview of the CTSA activities with attention to these three areas, which are essential to developing efficient clinical research efforts and effective implementation of research results on a national level. PMID:21896519

The Ezrin Metastatic Phenotype Is Associated with the Initiation of Protein Translation1

PubMed Central

Briggs, Joseph W; Ren, Ling; Nguyen, Rachel; Chakrabarti, Kristi; Cassavaugh, Jessica; Rahim, Said; Bulut, Gulay; Zhou, Ming; Veenstra, Timothy D; Chen, Qingrong; Wei, Jun S; Khan, Javed; Uren, Aykut; Khanna, Chand

2012-01-01

We previously associated the cytoskeleton linker protein, Ezrin, with the metastatic phenotype of pediatric sarcomas, including osteosarcoma and rhabdomyosarcoma. These studies have suggested that Ezrin contributes to the survival of cancer cells after their arrival at secondary metastatic locations. To better understand this role in metastasis, we undertook two noncandidate analyses of Ezrin function including a microarray subtraction of high-and low-Ezrin-expressing cells and a proteomic approach to identify proteins that bound the N-terminus of Ezrin in tumor lysates. Functional analyses of these data led to a novel and unifying hypothesis that Ezrin contributes to the efficiency of metastasis through regulation of protein translation. In support of this hypothesis, we found Ezrin to be part of the ribonucleoprotein complex to facilitate the expression of complex messenger RNA in cells and to bind with poly A binding protein 1 (PABP1; PABPC1). The relevance of these findings was supported by our identification of Ezrin and components of the translational machinery in pseudopodia of highly metastatic cells during the process of cell invasion. Finally, two small molecule inhibitors recently shown to inhibit the Ezrin metastatic phenotype disrupted the Ezrin/PABP1 association. Taken together, these results provide a novel mechanistic basis by which Ezrin may contribute to metastasis. PMID:22577345

Genome-Scale Analysis of Translation Elongation with a Ribosome Flow Model

PubMed Central

Meilijson, Isaac; Kupiec, Martin; Ruppin, Eytan

2011-01-01

We describe the first large scale analysis of gene translation that is based on a model that takes into account the physical and dynamical nature of this process. The Ribosomal Flow Model (RFM) predicts fundamental features of the translation process, including translation rates, protein abundance levels, ribosomal densities and the relation between all these variables, better than alternative (‘non-physical’) approaches. In addition, we show that the RFM can be used for accurate inference of various other quantities including genes' initiation rates and translation costs. These quantities could not be inferred by previous predictors. We find that increasing the number of available ribosomes (or equivalently the initiation rate) increases the genomic translation rate and the mean ribosome density only up to a certain point, beyond which both saturate. Strikingly, assuming that the translation system is tuned to work at the pre-saturation point maximizes the predictive power of the model with respect to experimental data. This result suggests that in all organisms that were analyzed (from bacteria to Human), the global initiation rate is optimized to attain the pre-saturation point. The fact that similar results were not observed for heterologous genes indicates that this feature is under selection. Remarkably, the gap between the performance of the RFM and alternative predictors is strikingly large in the case of heterologous genes, testifying to the model's promising biotechnological value in predicting the abundance of heterologous proteins before expressing them in the desired host. PMID:21909250

Application of LSP Texts in Translator Training

ERIC Educational Resources Information Center

Ilynska, Larisa; Smirnova, Tatjana; Platonova, Marina

2017-01-01

The paper presents discussion of the results of extensive empirical research into efficient methods of educating and training translators of LSP (language for special purposes) texts. The methodology is based on using popular LSP texts in the respective fields as one of the main media for translator training. The aim of the paper is to investigate…

Enabling international adoption of LOINC through translation

PubMed Central

Vreeman, Daniel J.; Chiaravalloti, Maria Teresa; Hook, John; McDonald, Clement J.

2012-01-01

Interoperable health information exchange depends on adoption of terminology standards, but international use of such standards can be challenging because of language differences between local concept names and the standard terminology. To address this important barrier, we describe the evolution of an efficient process for constructing translations of LOINC terms names, the foreign language functions in RELMA, and the current state of translations in LOINC. We also present the development of the Italian translation to illustrate how translation is enabling adoption in international contexts. We built a tool that finds the unique list of LOINC Parts that make up a given set of LOINC terms. This list enables translation of smaller pieces like the core component “hepatitis c virus” separately from all the suffixes that could appear with it, such “Ab.IgG”, “DNA”, and “RNA”. We built another tool that generates a translation of a full LOINC name from all of these atomic pieces. As of version 2.36 (June 2011), LOINC terms have been translated into 9 languages from 15 linguistic variants other than its native English. The five largest linguistic variants have all used the Part-based translation mechanism. However, even with efficient tools and processes, translation of standard terminology is a complex undertaking. Two of the prominent linguistic challenges that translators have faced include: the approach to handling acronyms and abbreviations, and the differences in linguistic syntax (e.g. word order) between languages. LOINC’s open and customizable approach has enabled many different groups to create translations that met their needs and matched their resources. Distributing the standard and its many language translations at no cost worldwide accelerates LOINC adoption globally, and is an important enabler of interoperable health information exchange PMID:22285984

Understanding Rigid Geometric Transformations: Jeff's Learning Path for Translation

ERIC Educational Resources Information Center

Yanik, Huseyin Bahadir; Flores, Alfinio

2009-01-01

This article describes the development of knowledge and understanding of translations of Jeff, a prospective elementary teacher, during a teaching experiment that also included other rigid transformations. His initial conceptions of translations and other rigid transformations were characterized as undefined motions of a single object. He…

tRNA tKUUU, tQUUG, and tEUUC wobble position modifications fine-tune protein translation by promoting ribosome A-site binding.

PubMed

Rezgui, Vanessa Anissa Nathalie; Tyagi, Kshitiz; Ranjan, Namit; Konevega, Andrey L; Mittelstaet, Joerg; Rodnina, Marina V; Peter, Matthias; Pedrioli, Patrick G A

2013-07-23

tRNA modifications are crucial to ensure translation efficiency and fidelity. In eukaryotes, the URM1 and ELP pathways increase cellular resistance to various stress conditions, such as nutrient starvation and oxidative agents, by promoting thiolation and methoxycarbonylmethylation, respectively, of the wobble uridine of cytoplasmic (tK(UUU)), (tQ(UUG)), and (tE(UUC)). Although in vitro experiments have implicated these tRNA modifications in modulating wobbling capacity and translation efficiency, their exact in vivo biological roles remain largely unexplored. Using a combination of quantitative proteomics and codon-specific translation reporters, we find that translation of a specific gene subset enriched for AAA, CAA, and GAA codons is impaired in the absence of URM1- and ELP-dependent tRNA modifications. Moreover, in vitro experiments using native tRNAs demonstrate that both modifications enhance binding of tK(UUU) to the ribosomal A-site. Taken together, our data suggest that tRNA thiolation and methoxycarbonylmethylation regulate translation of genes with specific codon content.

Role of eIF2α Kinases in Translational Control and Adaptation to Cellular Stress.

PubMed

Wek, Ronald C

2018-02-12

A central mechanism regulating translation initiation in response to environmental stress involves phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Phosphorylation of eIF2α causes inhibition of global translation, which conserves energy and facilitates reprogramming of gene expression and signaling pathways that help to restore protein homeostasis. Coincident with repression of protein synthesis, many gene transcripts involved in the stress response are not affected or are even preferentially translated in response to increased eIF2α phosphorylation by mechanisms involving upstream open reading frames (uORFs). This review highlights the mechanisms regulating eIF2α kinases, the role that uORFs play in translational control, and the impact that alteration of eIF2α phosphorylation by gene mutations or small molecule inhibitors can have on health and disease. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

The role of Myc-induced protein synthesis in cancer

PubMed Central

Ruggero, Davide

2009-01-01

Deregulation in different steps of translational control is an emerging mechanism for cancer formation. One example of an oncogene with a direct role in control of translation is the Myc transcription factor. Myc directly increases protein synthesis rates by controlling the expression of multiple components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, Pol III and rDNA. However, the contribution of Myc-dependent increases in protein synthesis towards the multi-step process leading to cancer has remained unknown. Recent evidence strongly suggests that Myc oncogenic signaling may monopolize the translational machinery to elicit cooperative effects on cell growth, cell cycle progression, and genome instability as a mechanism for cancer initiation. Moreover, new genetic tools to restore aberrant increases in protein synthesis control are now available, which should enable the dissection of important mechanisms in cancer that rely on the translational machinery. PMID:19934336

Identification and Evaluation of Medical Translator Mobile Applications Using an Adapted APPLICATIONS Scoring System.

PubMed

Khander, Amrin; Farag, Sara; Chen, Katherine T

2017-12-22

With an increasing number of patients requiring translator services, many providers are turning to mobile applications (apps) for assistance. However, there have been no published reviews of medical translator apps. To identify and evaluate medical translator mobile apps using an adapted APPLICATIONS scoring system. A list of apps was identified from the Apple iTunes and Google Play stores, using the search term, "medical translator." Apps not found on two different searches, not in an English-based platform, not used for translation, or not functional after purchase, were excluded. The remaining apps were evaluated using an adapted APPLICATIONS scoring system, which included both objective and subjective criteria. App comprehensiveness was a weighted score defined by the number of non-English languages included in each app relative to the proportion of non-English speakers in the United States. The Apple iTunes and Google Play stores. Medical translator apps identified using the search term "medical translator." Main Outcomes and Measures: Compilation of medical translator apps for provider usage. A total of 524 apps were initially found. After applying the exclusion criteria, 20 (8.2%) apps from the Google Play store and 26 (9.2%) apps from the Apple iTunes store remained for evaluation. The highest scoring apps, Canopy Medical Translator, Universal Doctor Speaker, and Vocre Translate, scored 13.5 out of 18.7 possible points. A large proportion of apps initially found did not function as medical translator apps. Using the APPLICATIONS scoring system, we have identified and evaluated medical translator apps for providers who care for non-English speaking patients.

CRISPRi-sRNA: Transcriptional-Translational Regulation of Extracellular Electron Transfer in Shewanella oneidensis.

PubMed

Cao, Yingxiu; Li, Xiaofei; Li, Feng; Song, Hao

2017-09-15

Extracellular electron transfer (EET) in Shewanella oneidensis MR-1, which is one of the most well-studied exoelectrogens, underlies many microbial electrocatalysis processes, including microbial fuel cells, microbial electrolysis cells, and microbial electrosynthesis. However, regulating the efficiency of EET remains challenging due to the lack of efficient genome regulation tools that regulate gene expression levels in S. oneidensis. Here, we systematically established a transcriptional regulation technology, i.e., clustered regularly interspaced short palindromic repeats interference (CRISPRi), in S. oneidensis MR-1 using green fluorescent protein (GFP) as a reporter. We used this CRISPRi technology to repress the expression levels of target genes, individually and in combination, in the EET pathways (e.g., the MtrCAB pathway and genes affecting the formation of electroactive biofilms in S. oneidensis), which in turn enabled the efficient regulation of EET efficiency. We then established a translational regulation technology, i.e., Hfq-dependent small regulatory RNA (sRNA), in S. oneidensis by repressing the GFP reporter and mtrA, which is a critical gene in the EET pathways in S. oneidensis. To achieve coordinated transcriptional and translational regulation at the genomic level, the CRISPRi and Hfq-dependent sRNA systems were incorporated into a single plasmid harbored in a recombinant S. oneidensis strain, which enabled an even higher efficiency of mtrA gene repression in the EET pathways than that achieved by the CRISPRi and Hfq-dependent sRNA system alone, as exhibited by the reduced electricity output. Overall, we developed a combined CRISPRi-sRNA method that enabled the synergistic transcriptional and translational regulation of target genes in S. oneidensis. This technology involving CRISPRi-sRNA transcriptional-translational regulation of gene expression at the genomic level could be applied to other microorganisms.

Neuropathological biomarker candidates in brain tumors: key issues for translational efficiency.

PubMed

Hainfellner, J A; Heinzl, H

2010-01-01

Brain tumors comprise a large spectrum of rare malignancies in children and adults that are often associated with severe neurological symptoms and fatal outcome. Neuropathological tumor typing provides both prognostic and predictive tissue information which is the basis for optimal postoperative patient management and therapy. Molecular biomarkers may extend and refine prognostic and predictive information in a brain tumor case, providing more individualized and optimized treatment options. In the recent past a few neuropathological brain tumor biomarkers have translated smoothly into clinical use whereas many candidates show protracted translation. We investigated the causes of protracted translation of candidate brain tumor biomarkers. Considering the research environment from personal, social and systemic perspectives we identified eight determinants of translational success: methodology, funding, statistics, organization, phases of research, cooperation, self-reflection, and scientific progeny. Smoothly translating biomarkers are associated with low degrees of translational complexity whereas biomarkers with protracted translation are associated with high degrees. Key issues for translational efficiency of neuropathological brain tumor biomarker research seem to be related to (i) the strict orientation to the mission of medical research, that is the improval of medical practice as primordial purpose of research, (ii) definition of research priorities according to clinical needs, and (iii) absorption of translational complexities by means of operatively beneficial standards. To this end, concrete actions should comprise adequate scientific education of young investigators, and shaping of integrative diagnostics and therapy research both on the local level and the level of influential international brain tumor research platforms.

IMPACT Is a Developmentally Regulated Protein in Neurons That Opposes the Eukaryotic Initiation Factor 2α Kinase GCN2 in the modulation of Neurite Outgrowth*

PubMed Central

Roffé, Martín; Hajj, Glaucia N. M.; Azevedo, Hátylas F.; Alves, Viviane S.; Castilho, Beatriz A.

2013-01-01

The product of the mouse Imprinted and Ancient gene, IMPACT, is preferentially expressed in neurons. We have previously shown that IMPACT overexpression inhibits the activation of the protein kinase GCN2, which signals amino acid starvation. GCN2 phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in inhibition of general protein synthesis but increased translation of specific messages, such as ATF4. GCN2 is also involved in the regulation of neuronal functions, controlling synaptic plasticity, memory, and feeding behavior. We show here that IMPACT abundance increases during differentiation of neurons and neuron-like N2a cells, whereas GCN2 displays lowered activation levels. Upon differentiation, IMPACT associates with translating ribosomes, enhances translation initiation, and down-regulates the expression of ATF4. We further show that endogenous IMPACT promotes neurite outgrowth whereas GCN2 is a strong inhibitor of spontaneous neuritogenesis. Together, these results uncover the participation of the GCN2-IMPACT module of translational regulation in a highly controlled step in the development of the nervous system. PMID:23447528

IMPACT is a developmentally regulated protein in neurons that opposes the eukaryotic initiation factor 2α kinase GCN2 in the modulation of neurite outgrowth.

PubMed

Roffé, Martín; Hajj, Glaucia N M; Azevedo, Hátylas F; Alves, Viviane S; Castilho, Beatriz A

2013-04-12

The product of the mouse Imprinted and Ancient gene, IMPACT, is preferentially expressed in neurons. We have previously shown that IMPACT overexpression inhibits the activation of the protein kinase GCN2, which signals amino acid starvation. GCN2 phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in inhibition of general protein synthesis but increased translation of specific messages, such as ATF4. GCN2 is also involved in the regulation of neuronal functions, controlling synaptic plasticity, memory, and feeding behavior. We show here that IMPACT abundance increases during differentiation of neurons and neuron-like N2a cells, whereas GCN2 displays lowered activation levels. Upon differentiation, IMPACT associates with translating ribosomes, enhances translation initiation, and down-regulates the expression of ATF4. We further show that endogenous IMPACT promotes neurite outgrowth whereas GCN2 is a strong inhibitor of spontaneous neuritogenesis. Together, these results uncover the participation of the GCN2-IMPACT module of translational regulation in a highly controlled step in the development of the nervous system.

Structure of the ribosome post-recycling complex probed by chemical cross-linking and mass spectrometry

PubMed Central

Kiosze-Becker, Kristin; Ori, Alessandro; Gerovac, Milan; Heuer, André; Nürenberg-Goloub, Elina; Rashid, Umar Jan; Becker, Thomas; Beckmann, Roland; Beck, Martin; Tampé, Robert

2016-01-01

Ribosome recycling orchestrated by the ATP binding cassette (ABC) protein ABCE1 can be considered as the final—or the first—step within the cyclic process of protein synthesis, connecting translation termination and mRNA surveillance with re-initiation. An ATP-dependent tweezer-like motion of the nucleotide-binding domains in ABCE1 transfers mechanical energy to the ribosome and tears the ribosome subunits apart. The post-recycling complex (PRC) then re-initiates mRNA translation. Here, we probed the so far unknown architecture of the 1-MDa PRC (40S/30S·ABCE1) by chemical cross-linking and mass spectrometry (XL-MS). Our study reveals ABCE1 bound to the translational factor-binding (GTPase) site with multiple cross-link contacts of the helix–loop–helix motif to the S24e ribosomal protein. Cross-linking of the FeS cluster domain to the ribosomal protein S12 substantiates an extreme lever-arm movement of the FeS cluster domain during ribosome recycling. We were thus able to reconstitute and structurally analyse a key complex in the translational cycle, resembling the link between translation initiation and ribosome recycling. PMID:27824037

T box riboswitches in Actinobacteria: Translational regulation via novel tRNA interactions

PubMed Central

Sherwood, Anna V.; Grundy, Frank J.; Henkin, Tina M.

2015-01-01

The T box riboswitch regulates many amino acid-related genes in Gram-positive bacteria. T box riboswitch-mediated gene regulation was shown previously to occur at the level of transcription attenuation via structural rearrangements in the 5′ untranslated (leader) region of the mRNA in response to binding of a specific uncharged tRNA. In this study, a novel group of isoleucyl-tRNA synthetase gene (ileS) T box leader sequences found in organisms of the phylum Actinobacteria was investigated. The Stem I domains of these RNAs lack several highly conserved elements that are essential for interaction with the tRNA ligand in other T box RNAs. Many of these RNAs were predicted to regulate gene expression at the level of translation initiation through tRNA-dependent stabilization of a helix that sequesters a sequence complementary to the Shine–Dalgarno (SD) sequence, thus freeing the SD sequence for ribosome binding and translation initiation. We demonstrated specific binding to the cognate tRNAIle and tRNAIle-dependent structural rearrangements consistent with regulation at the level of translation initiation, providing the first biochemical demonstration, to our knowledge, of translational regulation in a T box riboswitch. PMID:25583497

Protein functional features are reflected in the patterns of mRNA translation speed.

PubMed

López, Daniel; Pazos, Florencio

2015-07-09

The degeneracy of the genetic code makes it possible for the same amino acid string to be coded by different messenger RNA (mRNA) sequences. These "synonymous mRNAs" may differ largely in a number of aspects related to their overall translational efficiency, such as secondary structure content and availability of the encoded transfer RNAs (tRNAs). Consequently, they may render different yields of the translated polypeptides. These mRNA features related to translation efficiency are also playing a role locally, resulting in a non-uniform translation speed along the mRNA, which has been previously related to some protein structural features and also used to explain some dramatic effects of "silent" single-nucleotide-polymorphisms (SNPs). In this work we perform the first large scale analysis of the relationship between three experimental proxies of mRNA local translation efficiency and the local features of the corresponding encoded proteins. We found that a number of protein functional and structural features are reflected in the patterns of ribosome occupancy, secondary structure and tRNA availability along the mRNA. One or more of these proxies of translation speed have distinctive patterns around the mRNA regions coding for certain protein local features. In some cases the three patterns follow a similar trend. We also show specific examples where these patterns of translation speed point to the protein's important structural and functional features. This support the idea that the genome not only codes the protein functional features as sequences of amino acids, but also as subtle patterns of mRNA properties which, probably through local effects on the translation speed, have some consequence on the final polypeptide. These results open the possibility of predicting a protein's functional regions based on a single genomic sequence, and have implications for heterologous protein expression and fine-tuning protein function.

A set of ligation-independent in vitro translation vectors for eukaryotic protein production.

PubMed

Bardóczy, Viola; Géczi, Viktória; Sawasaki, Tatsuya; Endo, Yaeta; Mészáros, Tamás

2008-03-27

The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags. We designed four ligation independent cloning (LIC) vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein. Four newly designed in vitro translation vectors have been constructed which allow fast and parallel cloning and protein purification, thus representing useful molecular tools for high-throughput production of eukaryotic proteins.

Redox Regulation of Methionine Aminopeptidase 2 Activity*

PubMed Central

Chiu, Joyce; Wong, Jason W. H.; Hogg, Philip J.

2014-01-01

Protein translation is initiated with methionine in eukaryotes, and the majority of proteins have their N-terminal methionine removed by methionine aminopeptidases (MetAP1 and MetAP2) prior to action. Methionine removal can be important for protein function, localization, or stability. No mechanism of regulation of MetAP activity has been identified. MetAP2, but not MetAP1, contains a single Cys228-Cys448 disulfide bond that has an −RHStaple configuration and links two β-loop structures, which are hallmarks of allosteric disulfide bonds. From analysis of crystal structures and using mass spectrometry and activity assays, we found that the disulfide bond exists in oxidized and reduced states in the recombinant enzyme. The disulfide has a standard redox potential of −261 mV and is efficiently reduced by the protein reductant, thioredoxin, with a rate constant of 16,180 m−1 s−1. The MetAP2 disulfide bond also exists in oxidized and reduced states in glioblastoma tumor cells, and stressing the cells by oxygen or glucose deprivation results in more oxidized enzyme. The Cys228-Cys448 disulfide is at the rim of the active site and is only three residues distant from the catalytic His231, which suggested that cleavage of the bond would influence substrate hydrolysis. Indeed, oxidized and reduced isoforms have different catalytic efficiencies for hydrolysis of MetAP2 peptide substrates. These findings indicate that MetAP2 is post-translationally regulated by an allosteric disulfide bond, which controls substrate specificity and catalytic efficiency. PMID:24700462

A Novel Approach to Measuring Efficiency of Scientific Research Projects: Data Envelopment Analysis

PubMed Central

Zell, Adrienne; Orwoll, Eric

2015-01-01

Abstract Purpose Measuring the efficiency of resource allocation for the conduct of scientific projects in medical research is difficult due to, among other factors, the heterogeneity of resources supplied (e.g., dollars or FTEs) and outcomes expected (e.g., grants, publications). While this is an issue in medical science, it has been approached successfully in other fields by using data envelopment analysis (DEA). DEA has a number of advantages over other techniques as it simultaneously uses multiple heterogeneous inputs and outputs to determine which projects are performing most efficiently, referred to as being at the efficiency frontier, when compared to others in the data set. Method This research uses DEA for the evaluation of supported translational science projects by the Oregon Clinical and Translational Research Institute (OCTRI), a NCATS Clinical & Translational Science Award (CTSA) recipient. Results These results suggest that the primary determinate of overall project efficiency at OCTRI is the amount of funding, with smaller amounts of funding providing more efficiency than larger funding amounts. Conclusion These results, and the use of DEA, highlight both the success of using this technique in helping determine medical research efficiency and those factors to consider when distributing funds for new projects at CTSAs. PMID:26243147

A reconfigurable waveguide for energy-efficient transmission and local manipulation of information in a nanomagnetic device

NASA Astrophysics Data System (ADS)

Haldar, Arabinda; Kumar, Dheeraj; Adeyeye, Adekunle Olusola

2016-05-01

Spin-wave-based devices promise to usher in an era of low-power computing where information is carried by the precession of the electrons' spin instead of dissipative translation of their charge. This potential is, however, undermined by the need for a bias magnetic field, which must remain powered on to maintain an anisotropic device characteristic. Here, we propose a reconfigurable waveguide design that can transmit and locally manipulate spin waves without the need for any external bias field once initialized. We experimentally demonstrate the transmission of spin waves in straight as well as curved waveguides without a bias field, which has been elusive so far. Furthermore, we experimentally show a binary gating of the spin-wave signal by controlled switching of the magnetization, locally, in the waveguide. The results have potential implications in high-density integration and energy-efficient operation of nanomagnetic devices at room temperature.

Functional and genetic plasticities of the poliovirus genome: quasi-infectious RNAs modified in the 5'-untranslated region yield a variety of pseudorevertants.

PubMed Central

Gmyl, A P; Pilipenko, E V; Maslova, S V; Belov, G A; Agol, V I

1993-01-01

Poliovirus RNA species with nucleotides 564 to 571 deleted or with a secondary structure domain (positions 564 to 629) replaced by a shorter irregular oligonucleotide have been engineered previously; these RNAs have been considered quasi-infectious (yielding a single late revertant plaque) and dead, respectively (E. Pilipenko, A. Gmyl, Y. Svitkin, S. Maslova, A. Sinyakov, and V. Agol, Cell 68:119-131, 1992). By using large amounts of these RNAs for transfections, revertant clones with a great variety of genetic changes (point mutations, insertions of foreign sequences, short or extended deletions) were isolated. The pattern of these changes supported the notion that an appropriately spaced oligopyrimidine-AUG tandem is important for efficient poliovirus RNA translation. Structural features within and around this tandem modulated the initiation efficiency. The functional and genetic plasticities of the poliovirus genome are briefly discussed. Images PMID:8396686

The construction of a synthetic Escherichia coli trp promoter and its use in the expression of a synthetic interferon gene.

PubMed Central

Windass, J D; Newton, C R; De Maeyer-Guignard, J; Moore, V E; Markham, A F; Edge, M D

1982-01-01

An 82 base pair DNA fragment has been synthesised which contains the E. coli trp promoter and operator sequences and also encodes the first Shine Dalgarno sequence of the trp operon. This DNA fragment is flanked by EcoRI and ClaI/TaqI cohesive ends and is thus easy to clone, transfer between vector systems and couple to genes to drive their expression. It has been cloned into plasmid pAT153, producing a convenient trp promoter vector. We have also joined the fragment to a synthetic IFN-alpha 1 gene, using synthetic oligonucleotides to generate a completely natural, highly efficient bacterial translation initiation signal on the promoter proximal side of the IFN gene. Plasmids carrying this construction enable E. coli cells to express IFN-alpha 1 almost constitutively and with significantly higher efficiency than from a lacUV5 promoter based system. Images PMID:6184675

Soft translations and soft extensions of BCI/BCK-algebras.

PubMed

Sultana, Nazra; Rani, Nazia; Ali, Muhammad Irfan; Hussain, Azhar

2014-01-01

The concept of soft translations of soft subalgebras and soft ideals over BCI/BCK-algebras is introduced and some related properties are studied. Notions of Soft extensions of soft subalgebras and soft ideals over BCI/BCK-algebras are also initiated. Relationships between soft translations and soft extensions are explored.

On Interactive Teaching Model of Translation Course Based on Wechat

ERIC Educational Resources Information Center

Lin, Wang

2017-01-01

Constructivism is a theory related to knowledge and learning, focusing on learners' subjective initiative, based on which the interactive approach has been proved to play a crucial role in language learning. Accordingly, the interactive approach can also be applied to translation teaching since translation itself is a bilingual transformational…

Mutagenesis of the three bases preceding the start codon of the beta-galactosidase mRNA and its effect on translation in Escherichia coli.

PubMed Central

Hui, A; Hayflick, J; Dinkelspiel, K; de Boer, H A

1984-01-01

The effect on the translation efficiency of various mutations in the three bases (the -1 triplet) that precede the AUG start codon of the beta-galactosidase mRNA in Escherichia coli was studied. Of the 39 mutants examined, the level of expression varies over a 20-fold range. The most favorable combinations of bases in the -1 triplet are UAU and CUU. The expression levels in the mutants with UUC, UCA or AGG as the -1 triplet are 20-fold lower than those with UAU or CUU. In general, a U residue immediately preceding the start codon is more favorable for expression than any other base; furthermore, an A residue at the -2 position enhances the translation efficiency in most instances. In both cases, however, the degree of enhancement depends on its context, i.e. the neighboring bases. Although the rules derived from this study are complex, the results show that mutations in any of the three bases preceding the start codon can strongly affect the translational efficiency of the beta-galactosidase mRNA. PMID:6425057

Genetic code translation displays a linear trade-off between efficiency and accuracy of tRNA selection.

PubMed

Johansson, Magnus; Zhang, Jingji; Ehrenberg, Måns

2012-01-03

Rapid and accurate translation of the genetic code into protein is fundamental to life. Yet due to lack of a suitable assay, little is known about the accuracy-determining parameters and their correlation with translational speed. Here, we develop such an assay, based on Mg(2+) concentration changes, to determine maximal accuracy limits for a complete set of single-mismatch codon-anticodon interactions. We found a simple, linear trade-off between efficiency of cognate codon reading and accuracy of tRNA selection. The maximal accuracy was highest for the second codon position and lowest for the third. The results rationalize the existence of proofreading in code reading and have implications for the understanding of tRNA modifications, as well as of translation error-modulating ribosomal mutations and antibiotics. Finally, the results bridge the gap between in vivo and in vitro translation and allow us to calibrate our test tube conditions to represent the environment inside the living cell.

Training and career development in clinical and translational science: an opportunity for rehabilitation scientists.

PubMed

Kelly, Thomas H; Mattacola, Carl G

2010-11-01

The National Institutes of Health's Clinical and Translational Science Award initiative is designed to establish and promote academic centers of clinical and translational science (CTS) that are empowered to train and advance multi- and interdisciplinary investigators and research teams to apply new scientific knowledge and techniques to enhance patient care. Among the key components of a full-service center for CTS is an educational platform to support research training in CTS. Educational objectives and resources available to support the career development of the clinical and translational scientists, including clinical research education, mentored research training, and career development support, are described. The purpose of the article is to provide an overview of the CTS educational model so that rehabilitation specialists can become more aware of potential resources that are available and become more involved in the delivery and initiation of the CTS model in their own workplace. Rehabilitation clinicians and scientists are well positioned to play important leadership roles in advancing the academic mission of CTS. Rigorous academic training in rehabilitation science serves as an effective foundation for supporting the translation of basic scientific discovery into improved health care. Rehabilitation professionals are immersed in patient care, familiar with interdisciplinary health care delivery, and skilled at working with multiple health care professionals. The NIH Clinical and Translational Science Award initiative is an excellent opportunity to advance the academic development of rehabilitation scientists.

Healthcare Commercialization Programs: Improving the Efficiency of Translating Healthcare Innovations From Academia Into Practice.

PubMed

Collins, John M; Reizes, Ofer; Dempsey, Michael K

2016-01-01

Academic investigators are generating a plethora of insights and technologies that have the potential to significantly improve patient care. However, to address the imperative to improve the quality, cost and access to care with ever more constrained funding, the efficiency and the consistency with which they are translated into cost effective products and/or services need to improve. Healthcare commercialization programs (HCPs) are described and proposed as an option that institutions can add to their portfolio to improve translational research. In helping teams translate specific healthcare innovations into practice, HCPs expand the skillset of investigators and enhance an institution's innovation capacity. Lessons learned are shared from configuring and delivering HCPs, which build on the fundamentals of the National Science Foundation's Innovation Corps program, to address the unique challenges in supporting healthcare innovations and innovators.

Automated Web-Based Request Mechanism for Workflow Enhancement in an Academic Customer-Focused Biorepository.

PubMed

McDonald, Sandra A; Ryan, Benjamin J; Brink, Amy; Holtschlag, Victoria L

2012-02-01

Informatics systems, particularly those that provide capabilities for data storage, specimen tracking, retrieval, and order fulfillment, are critical to the success of biorepositories and other laboratories engaged in translational medical research. A crucial item-one easily overlooked-is an efficient way to receive and process investigator-initiated requests. A successful electronic ordering system should allow request processing in a maximally efficient manner, while also allowing streamlined tracking and mining of request data such as turnaround times and numerical categorizations (user groups, funding sources, protocols, and so on). Ideally, an electronic ordering system also facilitates the initial contact between the laboratory and customers, while still allowing for downstream communications and other steps toward scientific partnerships. We describe here the recently established Web-based ordering system for the biorepository at Washington University Medical Center, along with its benefits for workflow, tracking, and customer service. Because of the system's numerous value-added impacts, we think our experience can serve as a good model for other customer-focused biorepositories, especially those currently using manual or non-Web-based request systems. Our lessons learned also apply to the informatics developers who serve such biobanks.

Automated Web-Based Request Mechanism for Workflow Enhancement in an Academic Customer-Focused Biorepository

PubMed Central

Ryan, Benjamin J.; Brink, Amy; Holtschlag, Victoria L.

2012-01-01

Informatics systems, particularly those that provide capabilities for data storage, specimen tracking, retrieval, and order fulfillment, are critical to the success of biorepositories and other laboratories engaged in translational medical research. A crucial item—one easily overlooked—is an efficient way to receive and process investigator-initiated requests. A successful electronic ordering system should allow request processing in a maximally efficient manner, while also allowing streamlined tracking and mining of request data such as turnaround times and numerical categorizations (user groups, funding sources, protocols, and so on). Ideally, an electronic ordering system also facilitates the initial contact between the laboratory and customers, while still allowing for downstream communications and other steps toward scientific partnerships. We describe here the recently established Web-based ordering system for the biorepository at Washington University Medical Center, along with its benefits for workflow, tracking, and customer service. Because of the system's numerous value-added impacts, we think our experience can serve as a good model for other customer-focused biorepositories, especially those currently using manual or non-Web–based request systems. Our lessons learned also apply to the informatics developers who serve such biobanks. PMID:23386921

A web-based screening and accrual strategy for a cancer prevention clinical trial in healthy smokers.

PubMed

Mohebati, Arash; Knutson, Allison; Zhou, Xi Kathy; Smith, Judith J; Brown, Powel H; Dannenberg, Andrew J; Szabo, Eva

2012-09-01

Screening and recruitment of qualified subjects for clinical trials is an essential component of translational research, and it can be quite challenging if the most efficient recruitment method is not utilized. In this report, we describe a successful web-based screening and accrual method used in a randomized prospective chemoprevention clinical trial with urinary biomarker endpoints. The targeted study population was a group of at-risk healthy current smokers with no evidence of lung disease. Craigslist was used as the sole recruitment modality for this study. All interested subjects were directed to a pre-screening website, in which subject questionnaire responses were linked to the study coordinator's secure e-mail account. Of the 429 initial inquiries, 189 individuals were initially eligible based on the questionnaire response. One hundred twenty-two people were telephone-screened, of whom 98 subjects were consented, 84 were randomized and 77 subjects completed the study successfully. Utilizing this single web-based advertising strategy, accrual for the trial was completed 7 months prior to the projected date. Craigslist is a cost effective and efficient web-based resource that can be utilized in accruing subjects to some chemoprevention trials. Published by Elsevier Inc.

A web-based screening and accrual strategy for a cancer prevention clinical trial in healthy smokers

PubMed Central

Mohebati, Arash; Knutson, Allison; Zhou, Xi Kathy; Smith, Judith J.; Brown, Powel H.; Dannenberg, Andrew J.; Szabo, Eva

2012-01-01

Screening and recruitment of qualified subjects for clinical trials is an essential component of translational research, and it can be quite challenging if the most efficient recruitment method is not utilized. In this report, we describe a successful web-based screening and accrual method used in a randomized prospective chemoprevention clinical trial with urinary biomarker endpoints. The targeted study population was a group of at-risk healthy current smokers with no evidence of lung disease. Craigslist was used as the sole recruitment modality for this study. All interested subjects were directed to a pre-screening website, in which subject questionnaire responses were linked to the study coordinator's secure e-mail account. Of the 429 initial inquiries, 189 individuals were initially eligible based on the questionnaire response. One hundred twenty-two people were telephone-screened, of whom 98 subjects were consented, 84 were randomized and 77 subjects completed the study successfully. Utilizing this single web-based advertising strategy, accrual for the trial was completed 7 months prior to the projected date. Craigslist is a cost effective and efficient web-based resource that can be utilized in accruing subjects to some chemoprevention trials. PMID:22771576

Protein phosphatase PPM1G regulates protein translation and cell growth by dephosphorylating 4E binding protein 1 (4E-BP1).

PubMed

Liu, Jianyu; Stevens, Payton D; Eshleman, Nichole E; Gao, Tianyan

2013-08-09

Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.

CsrA Represses Translation of sdiA, Which Encodes the N-Acylhomoserine-l-Lactone Receptor of Escherichia coli, by Binding Exclusively within the Coding Region of sdiA mRNA ▿ †

PubMed Central

Yakhnin, Helen; Baker, Carol S.; Berezin, Igor; Evangelista, Michael A.; Rassin, Alisa; Romeo, Tony; Babitzke, Paul

2011-01-01

The RNA binding protein CsrA is the central component of a conserved global regulatory system that activates or represses gene expression posttranscriptionally. In every known example of CsrA-mediated translational control, CsrA binds to the 5′ untranslated region of target transcripts, thereby repressing translation initiation and/or altering the stability of the RNA. Furthermore, with few exceptions, repression by CsrA involves binding directly to the Shine-Dalgarno sequence and blocking ribosome binding. sdiA encodes the quorum-sensing receptor for N-acyl-l-homoserine lactone in Escherichia coli. Because sdiA indirectly stimulates transcription of csrB, which encodes a small RNA (sRNA) antagonist of CsrA, we further explored the relationship between sdiA and the Csr system. Primer extension analysis revealed four putative transcription start sites within 85 nucleotides of the sdiA initiation codon. Potential σ70-dependent promoters were identified for each of these primer extension products. In addition, two CsrA binding sites were predicted in the initially translated region of sdiA. Expression of chromosomally integrated sdiA′-′lacZ translational fusions containing the entire promoter and CsrA binding site regions indicates that CsrA represses sdiA expression. The results from gel shift and footprint studies demonstrate that tight binding of CsrA requires both of these sites. Furthermore, the results from toeprint and in vitro translation experiments indicate that CsrA represses translation of sdiA by directly competing with 30S ribosomal subunit binding. Thus, this represents the first example of CsrA preventing translation by interacting solely within the coding region of an mRNA target. PMID:21908661

Dynamic m(6)A mRNA methylation directs translational control of heat shock response.

PubMed

Zhou, Jun; Wan, Ji; Gao, Xiangwei; Zhang, Xingqian; Jaffrey, Samie R; Qian, Shu-Bing

2015-10-22

The most abundant mRNA post-transcriptional modification is N(6)-methyladenosine (m(6)A), which has broad roles in RNA biology. In mammalian cells, the asymmetric distribution of m(6)A along mRNAs results in relatively less methylation in the 5' untranslated region (5'UTR) compared to other regions. However, whether and how 5'UTR methylation is regulated is poorly understood. Despite the crucial role of the 5'UTR in translation initiation, very little is known about whether m(6)A modification influences mRNA translation. Here we show that in response to heat shock stress, certain adenosines within the 5'UTR of newly transcribed mRNAs are preferentially methylated. We find that the dynamic 5'UTR methylation is a result of stress-induced nuclear localization of YTHDF2, a well-characterized m(6)A 'reader'. Upon heat shock stress, the nuclear YTHDF2 preserves 5'UTR methylation of stress-induced transcripts by limiting the m(6)A 'eraser' FTO from demethylation. Remarkably, the increased 5'UTR methylation in the form of m(6)A promotes cap-independent translation initiation, providing a mechanism for selective mRNA translation under heat shock stress. Using Hsp70 mRNA as an example, we demonstrate that a single m(6)A modification site in the 5'UTR enables translation initiation independent of the 5' end N(7)-methylguanosine cap. The elucidation of the dynamic features of 5'UTR methylation and its critical role in cap-independent translation not only expands the breadth of physiological roles of m(6)A, but also uncovers a previously unappreciated translational control mechanism in heat shock response.

A unified view of energetic efficiency in active drag reduction, thrust generation and self-propulsion through a loss coefficient with some applications

NASA Astrophysics Data System (ADS)

Arakeri, Jaywant H.; Shukla, Ratnesh K.

2013-08-01

An analysis of the energy budget for the general case of a body translating in a stationary fluid under the action of an external force is used to define a power loss coefficient. This universal definition of power loss coefficient gives a measure of the energy lost in the wake of the translating body and, in general, is applicable to a variety of flow configurations including active drag reduction, self-propulsion and thrust generation. The utility of the power loss coefficient is demonstrated on a model bluff body flow problem concerning a two-dimensional elliptical cylinder in a uniform cross-flow. The upper and lower boundaries of the elliptic cylinder undergo continuous motion due to a prescribed reflectionally symmetric constant tangential surface velocity. It is shown that a decrease in drag resulting from an increase in the strength of tangential surface velocity leads to an initial reduction and eventual rise in the power loss coefficient. A maximum in energetic efficiency is attained for a drag reducing tangential surface velocity which minimizes the power loss coefficient. The effect of the tangential surface velocity on drag reduction and self-propulsion of both bluff and streamlined bodies is explored through a variation in the thickness ratio (ratio of the minor and major axes) of the elliptical cylinders.

Identification of cis-elements conferring high levels of gene expression in non-green plastids.

PubMed

Zhang, Jiang; Ruf, Stephanie; Hasse, Claudia; Childs, Liam; Scharff, Lars B; Bock, Ralph

2012-10-01

Although our knowledge about the mechanisms of gene expression in chloroplasts has increased substantially over the past decades, next to nothing is known about the signals and factors that govern expression of the plastid genome in non-green tissues. Here we report the development of a quantitative method suitable for determining the activity of cis-acting elements for gene expression in non-green plastids. The in vivo assay is based on stable transformation of the plastid genome and the discovery that root length upon seedling growth in the presence of the plastid translational inhibitor kanamycin is directly proportional to the expression strength of the resistance gene nptII in transgenic tobacco plastids. By testing various combinations of promoters and translation initiation signals, we have used this experimental system to identify cis-elements that are highly active in non-green plastids. Surprisingly, heterologous expression elements from maize plastids were significantly more efficient in conferring high expression levels in root plastids than homologous expression elements from tobacco. Our work has established a quantitative method for characterization of gene expression in non-green plastid types, and has led to identification of cis-elements for efficient plastid transgene expression in non-green tissues, which are valuable tools for future transplastomic studies in basic and applied research. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

A modified Delphi translation strategy and challenges of International Classification for Nursing Practice (ICNP®).

PubMed

Hou, I-Ching; Chang, Polun; Chan, Hui-Ya; Dykes, Patricia C

2013-05-01

Standardized terminology is an important infrastructure component of the electronic health record. ICNP(®) is a systemic coding system that can support the development of nursing information systems. Translation of the standardized terminology preferred terms into local terms is an important first step in the translation process. The purpose of this case report is to describe the translation strategy used and challenges faced in translating ICNP(®) Version 2 preferred terms from English to traditional Chinese. A modified Delphi strategy using forward translation and expert consensus was conducted to facilitate semantic and cultural translation and validation of the ICNP(®) and to make the process generalizable. A nursing informatics expert completed the initial forward translation. Five nursing experts with rich clinical and academic experiences joined this process and validated the initial translation. The nursing experts' consensus was then used to finalize the traditional Chinese terms. A total of 1863 preferred terms from the ICNP(®) Version 2 were translated from English into traditional Chinese. Majority agreement from two or more nursing experts was achieved for 98.3% (n=1832) of the preferred term translations. Less than 2% (n=31) of terms had no majority agreement. Translation challenges include the following: (1) changes in code structure of preferred terms from the ICNP(®) β2 version to Verson 2, (2) inability to identify resources to complete the translation that fully met ICNP recommendations for terminology translators, (3) ambiguous preferred term descriptions, and (4) ambiguous preferred term names. Most of the ICNP(®) Version 2 preferred terms were translated from English into traditional Chinese with majority consensus. For the terms without consensus, we recommend that all synonyms be included in the ICNP(®) translation. In countries like Taiwan where nursing education occurs in English, it is recommended that English terms are displayed along with the translated official language to help nurses to interpret and use the terminology correctly. Crown Copyright © 2012. Published by Elsevier Ireland Ltd. All rights reserved.

Applying a knowledge translation model to the uptake of the Baby Friendly Health Initiative in the Australian health care system.

PubMed

Atchan, Marjorie; Davis, Deborah; Foureur, Maralyn

2014-06-01

The Baby Friendly Hospital Initiative is a global, evidence-based, public health initiative. The evidence underpinning the Initiative supports practices promoting the initiation and maintenance of breastfeeding and encourages women's informed infant feeding decisions. In Australia, where the Initiative is known as the Baby Friendly Health Initiative (BFHI) the translation of evidence into practice has not been uniform, as demonstrated by a varying number of maternity facilities in each State and Territory currently accredited as 'baby friendly'. This variance has persisted regardless of BFHI implementation in Australia gaining 'in principle' support at a national and governmental level as well as inclusion in health policy in several states. There are many stakeholders that exert an influence on policy development and health care practices. Identify a theory and model to examine where and how barriers occur in the gap between evidence and practice in the uptake of the BFHI in Australia. Knowledge translation theory and the research to practice pipeline model are used to examine the identified barriers to BFHI implementation and accreditation in Australia. Australian and international studies have identified similar issues that have either enabled implementation of the BFHI or acted as a barrier. Knowledge translation theory and the research to practice pipeline model is of practical value to examine barriers. Recommendations in the form of specific targeted strategies to facilitate knowledge transfer and supportive practices into the Australian health care system and current midwifery practice are included. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

Experimental Analysis of Mimivirus Translation Initiation Factor 4a Reveals Its Importance in Viral Protein Translation during Infection of Acanthamoeba polyphaga.

PubMed

Bekliz, Meriem; Azza, Said; Seligmann, Hervé; Decloquement, Philippe; Raoult, Didier; La Scola, Bernard

2018-05-15

The Acanthamoeba polyphaga mimivirus is the first giant virus ever described, with a 1.2-Mb genome which encodes 979 proteins, including central components of the translation apparatus. One of these proteins, R458, was predicted to initiate translation, although its specific role remains unknown. We silenced the R458 gene using small interfering RNA (siRNA) and compared levels of viral fitness and protein expression in silenced versus wild-type mimivirus. Silencing decreased the growth rate, but viral particle production at the end of the viral cycle was unaffected. A comparative proteomic approach using two-dimensional difference-in-gel electrophoresis (2D-DIGE) revealed deregulation of the expression of 32 proteins in silenced mimivirus, which were defined as up- or downregulated. Besides revealing proteins with unknown functions, silencing R458 also revealed deregulation in proteins associated with viral particle structures, transcriptional machinery, oxidative pathways, modification of proteins/lipids, and DNA topology/repair. Most of these proteins belong to genes transcribed at the end of the viral cycle. Overall, our data suggest that the R458 protein regulates the expression of mimivirus proteins and, thus, that mimivirus translational proteins may not be strictly redundant in relation to those from the amoeba host. As is the case for eukaryotic initiation factor 4a (eIF4a), the R458 protein is the prototypical member of the ATP-dependent DEAD box RNA helicase mechanism. We suggest that the R458 protein is required to unwind the secondary structures at the 5' ends of mRNAs and to bind the mRNA to the ribosome, making it possible to scan for the start codon. These data are the first experimental evidence of mimivirus translation-related genes, predicted to initiate protein biosynthesis. IMPORTANCE The presence in the genome of a mimivirus of genes coding for many translational processes, with the exception of ribosome constituents, has been the subject of debate since its discovery in 2003. In this work, we focused on the R458 mimivirus gene, predicted to initiate protein biosynthesis. After silencing was performed, we observed that it has no major effect on mimivirus multiplication but that it affects protein expression and fitness. This suggests that it is effectively used by mimivirus during its developmental cycle. Until large-scale genetic manipulation of giant viruses becomes possible, the silencing strategy used here on mimivirus translation-related factors will open the way to understanding the functions of these translational genes. Copyright © 2018 American Society for Microbiology.

Technology for Large-Scale Translation of Clinical Practice Guidelines: A Pilot Study of the Performance of a Hybrid Human and Computer-Assisted Approach.

PubMed

Van de Velde, Stijn; Macken, Lieve; Vanneste, Koen; Goossens, Martine; Vanschoenbeek, Jan; Aertgeerts, Bert; Vanopstal, Klaar; Vander Stichele, Robert; Buysschaert, Joost

2015-10-09

The construction of EBMPracticeNet, a national electronic point-of-care information platform in Belgium, began in 2011 to optimize quality of care by promoting evidence-based decision making. The project involved, among other tasks, the translation of 940 EBM Guidelines of Duodecim Medical Publications from English into Dutch and French. Considering the scale of the translation process, it was decided to make use of computer-aided translation performed by certificated translators with limited expertise in medical translation. Our consortium used a hybrid approach, involving a human translator supported by a translation memory (using SDL Trados Studio), terminology recognition (using SDL MultiTerm terminology databases) from medical terminology databases, and support from online machine translation. This resulted in a validated translation memory, which is now in use for the translation of new and updated guidelines. The objective of this experiment was to evaluate the performance of the hybrid human and computer-assisted approach in comparison with translation unsupported by translation memory and terminology recognition. A comparison was also made with the translation efficiency of an expert medical translator. We conducted a pilot study in which two sets of 30 new and 30 updated guidelines were randomized to one of three groups. Comparable guidelines were translated (1) by certificated junior translators without medical specialization using the hybrid method, (2) by an experienced medical translator without this support, and (3) by the same junior translators without the support of the validated translation memory. A medical proofreader who was blinded for the translation procedure, evaluated the translated guidelines for acceptability and adequacy. Translation speed was measured by recording translation and post-editing time. The human translation edit rate was calculated as a metric to evaluate the quality of the translation. A further evaluation was made of translation acceptability and adequacy. The average number of words per guideline was 1195 and the mean total translation time was 100.2 minutes/1000 words. No meaningful differences were found in the translation speed for new guidelines. The translation of updated guidelines was 59 minutes/1000 words faster (95% CI 2-115; P=.044) in the computer-aided group. Revisions due to terminology accounted for one third of the overall revisions by the medical proofreader. Use of the hybrid human and computer-aided translation by a non-expert translator makes the translation of updates of clinical practice guidelines faster and cheaper because of the benefits of translation memory. For the translation of new guidelines, there was no apparent benefit in comparison with the efficiency of translation unsupported by translation memory (whether by an expert or non-expert translator).

Technology for Large-Scale Translation of Clinical Practice Guidelines: A Pilot Study of the Performance of a Hybrid Human and Computer-Assisted Approach

PubMed Central

2015-01-01

Background The construction of EBMPracticeNet, a national electronic point-of-care information platform in Belgium, began in 2011 to optimize quality of care by promoting evidence-based decision making. The project involved, among other tasks, the translation of 940 EBM Guidelines of Duodecim Medical Publications from English into Dutch and French. Considering the scale of the translation process, it was decided to make use of computer-aided translation performed by certificated translators with limited expertise in medical translation. Our consortium used a hybrid approach, involving a human translator supported by a translation memory (using SDL Trados Studio), terminology recognition (using SDL MultiTerm terminology databases) from medical terminology databases, and support from online machine translation. This resulted in a validated translation memory, which is now in use for the translation of new and updated guidelines. Objective The objective of this experiment was to evaluate the performance of the hybrid human and computer-assisted approach in comparison with translation unsupported by translation memory and terminology recognition. A comparison was also made with the translation efficiency of an expert medical translator. Methods We conducted a pilot study in which two sets of 30 new and 30 updated guidelines were randomized to one of three groups. Comparable guidelines were translated (1) by certificated junior translators without medical specialization using the hybrid method, (2) by an experienced medical translator without this support, and (3) by the same junior translators without the support of the validated translation memory. A medical proofreader who was blinded for the translation procedure, evaluated the translated guidelines for acceptability and adequacy. Translation speed was measured by recording translation and post-editing time. The human translation edit rate was calculated as a metric to evaluate the quality of the translation. A further evaluation was made of translation acceptability and adequacy. Results The average number of words per guideline was 1195 and the mean total translation time was 100.2 minutes/1000 words. No meaningful differences were found in the translation speed for new guidelines. The translation of updated guidelines was 59 minutes/1000 words faster (95% CI 2-115; P=.044) in the computer-aided group. Revisions due to terminology accounted for one third of the overall revisions by the medical proofreader. Conclusions Use of the hybrid human and computer-aided translation by a non-expert translator makes the translation of updates of clinical practice guidelines faster and cheaper because of the benefits of translation memory. For the translation of new guidelines, there was no apparent benefit in comparison with the efficiency of translation unsupported by translation memory (whether by an expert or non-expert translator). PMID:26453372

Translational regulation of sigma 32 synthesis: requirement for an internal control element.

PubMed Central

Kamath-Loeb, A S; Gross, C A

1991-01-01

We have investigated the sequence requirements for the translational regulation of sigma 32 by examining the behavior of a new rpoH-lacZ protein fusion containing a short N-terminal fragment of sigma 32 fused to beta-galactosidase. Although the fusion retains rpoH translational initiation signals, it lacks translational regulation, implicating coding sequences within rpoH in this regulatory process. Images PMID:2050641

A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons

PubMed Central

Tian, Tian; Salis, Howard M.

2015-01-01

Natural and engineered genetic systems require the coordinated expression of proteins. In bacteria, translational coupling provides a genetically encoded mechanism to control expression level ratios within multi-cistronic operons. We have developed a sequence-to-function biophysical model of translational coupling to predict expression level ratios in natural operons and to design synthetic operons with desired expression level ratios. To quantitatively measure ribosome re-initiation rates, we designed and characterized 22 bi-cistronic operon variants with systematically modified intergenic distances and upstream translation rates. We then derived a thermodynamic free energy model to calculate de novo initiation rates as a result of ribosome-assisted unfolding of intergenic RNA structures. The complete biophysical model has only five free parameters, but was able to accurately predict downstream translation rates for 120 synthetic bi-cistronic and tri-cistronic operons with rationally designed intergenic regions and systematically increased upstream translation rates. The biophysical model also accurately predicted the translation rates of the nine protein atp operon, compared to ribosome profiling measurements. Altogether, the biophysical model quantitatively predicts how translational coupling controls protein expression levels in synthetic and natural bacterial operons, providing a deeper understanding of an important post-transcriptional regulatory mechanism and offering the ability to rationally engineer operons with desired behaviors. PMID:26117546

The ELAV RNA-stability factor HuR binds the 5′-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation

PubMed Central

Meng, Zheng; King, Peter H.; Nabors, L. Burt; Jackson, Nateka L.; Chen, Ching-Yi; Emanuel, Peter D.; Blume, Scott W.

2005-01-01

The type I insulin-like growth factor receptor (IGF-IR) is an integral component in the control of cell proliferation, differentiation and apoptosis. The IGF-IR mRNA contains an extraordinarily long (1038 nt) 5′-untranslated region (5′-UTR), and we have characterized a diverse series of proteins interacting with this RNA sequence which may provide for intricate regulation of IGF-IR gene expression at the translational level. Here, we report the purification and identification of one of these IGF-IR 5′-UTR-binding proteins as HuR, using a novel RNA crosslinking/RNase elution strategy. Because HuR has been predominantly characterized as a 3′-UTR-binding protein, enhancing mRNA stability and generally increasing gene expression, we sought to determine whether HuR might serve a different function in the context of its binding the IGF-IR 5′-UTR. We found that HuR consistently repressed translation initiation through the IGF-IR 5′-UTR. The inhibition of translation by HuR was concentration dependent, and could be reversed in trans by addition of a fragment of the IGF-IR 5′-UTR containing the HuR binding sites as a specific competitor, or abrogated by deletion of the third RNA recognition motif of HuR. We determined that HuR repressed translation initiation through the IGF-IR 5′-UTR in cells as well, and that siRNA knockdown of HuR markedly increased IGF-IR protein levels. Interestingly, we also found that HuR potently inhibited IGF-IR translation mediated through internal ribosome entry. Kinetic assays were performed to investigate the mechanism of translation repression by HuR and the dynamic interplay between HuR and the translation apparatus. We found that HuR, occupying a cap-distal position, significantly delayed translation initiation mediated by cap-dependent scanning, but was eventually displaced from its binding site, directly or indirectly, as a consequence of ribosomal scanning. However, HuR perpetually blocked the activity of the IGF-IR IRES, apparently arresting the IRES-associated translation pre-initiation complex in an inactive state. This function of HuR as a 5′-UTR-binding protein and dual-purpose translation repressor may be critical for the precise regulation of IGF-IR expression essential to normal cellular homeostasis. PMID:15914670

Recessive Resistance to Plant Viruses: Potential Resistance Genes Beyond Translation Initiation Factors

PubMed Central

Hashimoto, Masayoshi; Neriya, Yutaro; Yamaji, Yasuyuki; Namba, Shigetou

2016-01-01

The ability of plant viruses to propagate their genomes in host cells depends on many host factors. In the absence of an agrochemical that specifically targets plant viral infection cycles, one of the most effective methods for controlling viral diseases in plants is taking advantage of the host plant’s resistance machinery. Recessive resistance is conferred by a recessive gene mutation that encodes a host factor critical for viral infection. It is a branch of the resistance machinery and, as an inherited characteristic, is very durable. Moreover, recessive resistance may be acquired by a deficiency in a negative regulator of plant defense responses, possibly due to the autoactivation of defense signaling. Eukaryotic translation initiation factor (eIF) 4E and eIF4G and their isoforms are the most widely exploited recessive resistance genes in several crop species, and they are effective against a subset of viral species. However, the establishment of efficient, recessive resistance-type antiviral control strategies against a wider range of plant viral diseases requires genetic resources other than eIF4Es. In this review, we focus on recent advances related to antiviral recessive resistance genes evaluated in model plants and several crop species. We also address the roles of next-generation sequencing and genome editing technologies in improving plant genetic resources for recessive resistance-based antiviral breeding in various crop species. PMID:27833593

NCI 1st International Workshop on the Biology, Prevention, and Treatment of Relapse After Allogeneic Hematopoietic Stem Cell Transplantation: Summary and Recommendations from the Organizing Committee

PubMed Central

Bishop, Michael R.; Alyea, Edwin P.; Cairo, Mitchell S.; Falkenburg, J.H. Frederik; June, Carl H.; Kröger, Nicolaus; Little, Richard F.; Miller, Jeffrey S.; Pavletic, Steven Z.; Porter, David L.; Riddell, Stanley R.; van Besien, Koen; Wayne, Alan S.; Weisdorf, Daniel J.; Wu, Roy S.; Giralt, Sergio

2011-01-01

The First International Workshop on The Biology, Prevention, and Treatment of Relapse After Allogeneic Hematopoietic Stem Cell Transplantation was organized and convened to identify, prioritize, and coordinate future research activities related to relapse after allogeneic hematopoietic stem cell transplantation (alloHSCT). Each of the Workshop’s six working committees have published individual reports of ongoing basic, translational and clinical research and recommended areas for future research related to the areas of relapse biology, epidemiology, prevention and treatment. This document summarizes each of the committees’ recommendations and suggests three major initiatives for a coordinated research effort to address the problem of relapse after alloHSCT. The first is the need to establish multi-center correlative and clinical trials networks for basic/translational, epidemiological, and clinical research. Second, there is a need for a network of biorepositories for the collection of samples pre- and post-alloHSCT to aid in laboratory and clinical studies. Third, there should be further refinement, implementation, and study of the proposed Workshop disease-specific response and relapse definitions and the recommendations for monitoring of minimal residual disease. These recommendations, in coordination with ongoing research initiatives and transplant organizations, provide a research framework to rapidly and efficiently address the significant problem of relapse following alloHSCT. PMID:21224011

7 CFR 1740.6 - Eligible purposes of grants.

Code of Federal Regulations, 2010 CFR

2010-01-01

..., translators, and repeaters, including all facilities required to initiate DTV broadcasting. All broadcast... transmitters or translators that may be included in an application; (b) Power upgrades of existing DTV...

Soft Translations and Soft Extensions of BCI/BCK-Algebras

PubMed Central

Sultana, Nazra; Rani, Nazia; Ali, Muhammad Irfan

2014-01-01

The concept of soft translations of soft subalgebras and soft ideals over BCI/BCK-algebras is introduced and some related properties are studied. Notions of Soft extensions of soft subalgebras and soft ideals over BCI/BCK-algebras are also initiated. Relationships between soft translations and soft extensions are explored. PMID:25298968

Translation of SNOMED CT - strategies and description of a pilot project.

PubMed

Klein, Gunnar O; Chen, Rong

2009-01-01

The translation and localization of SNOMED CT (Systematized Nomenclature of Medicine - Clinical Terms) have been initiated in a few countries. In Sweden, we conducted the first evaluation of this terminology in a project called REFTERM in which we also developed a software tool which could handle a large scale translation with a number of translators and reviewers in a web-based environment. The system makes use of existing authorized English-Swedish translations of medical terminologies such as ICD-10. The paper discusses possible strategies for a national project to translate and adapt this terminology.

Translation of dipeptide repeat proteins from the C9ORF72 expanded repeat is associated with cellular stress.

PubMed

Sonobe, Yoshifumi; Ghadge, Ghanashyam; Masaki, Katsuhisa; Sendoel, Ataman; Fuchs, Elaine; Roos, Raymond P

2018-08-01

Expansion of a hexanucleotide repeat (HRE), GGGGCC, in the C9ORF72 gene is recognized as the most common cause of familial amyotrophic lateral sclerosis (FALS), frontotemporal dementia (FTD) and ALS-FTD, as well as 5-10% of sporadic ALS. Despite the location of the HRE in the non-coding region (with respect to the main C9ORF72 gene product), dipeptide repeat proteins (DPRs) that are thought to be toxic are translated from the HRE in all three reading frames from both the sense and antisense transcript. Here, we identified a CUG that has a good Kozak consensus sequence as the translation initiation codon. Mutation of this CTG significantly suppressed polyglycine-alanine (GA) translation. GA was translated when the G 4 C 2 construct was placed as the second cistron in a bicistronic construct. CRISPR/Cas9-induced knockout of a non-canonical translation initiation factor, eIF2A, impaired GA translation. Transfection of G 4 C 2 constructs induced an integrated stress response (ISR), while triggering the ISR led to a continuation of translation of GA with a decline in conventional cap-dependent translation. These in vitro observations were confirmed in chick embryo neural cells. The findings suggest that DPRs translated from an HRE in C9ORF72 aggregate and lead to an ISR that then leads to continuing DPR production and aggregation, thereby creating a continuing pathogenic cycle. Copyright © 2018 Elsevier Inc. All rights reserved.

Amino Acid Flux from Metabolic Network Benefits Protein Translation: the Role of Resource Availability.

PubMed

Hu, Xiao-Pan; Yang, Yi; Ma, Bin-Guang

2015-06-09

Protein translation is a central step in gene expression and affected by many factors such as codon usage bias, mRNA folding energy and tRNA abundance. Despite intensive previous studies, how metabolic amino acid supply correlates with protein translation efficiency remains unknown. In this work, we estimated the amino acid flux from metabolic network for each protein in Escherichia coli and Saccharomyces cerevisiae by using Flux Balance Analysis. Integrated with the mRNA expression level, protein abundance and ribosome profiling data, we provided a detailed description of the role of amino acid supply in protein translation. Our results showed that amino acid supply positively correlates with translation efficiency and ribosome density. Moreover, with the rank-based regression model, we found that metabolic amino acid supply facilitates ribosome utilization. Based on the fact that the ribosome density change of well-amino-acid-supplied genes is smaller than poorly-amino-acid-supply genes under amino acid starvation, we reached the conclusion that amino acid supply may buffer ribosome density change against amino acid starvation and benefit maintaining a relatively stable translation environment. Our work provided new insights into the connection between metabolic amino acid supply and protein translation process by revealing a new regulation strategy that is dependent on resource availability.

Summary of NACA/NASA Variable-Sweep Research and Development Leading to the F-111 (TFX)

NASA Technical Reports Server (NTRS)

1966-01-01

On November 24, 1962, the United States ushered in a new era of aircraft development when the Department of Defense placed an initial development contract for the world's first supersonic variable-sweep aircraft - the F-111 or so-called TFX (tactical fighter-experimental). The multimission performance potential of this concept is made possible by virtue of the variable-sweep wing - a research development of the NASA and its predecessor, the NACA. With the wing swept forward into the maximum span position, the aircraft configuration is ideal for efficient subsonic flight. This provides long-range combat and ferry mission capability, short-field landing and take-off characteristics, and compatibility with naval aircraft carrier operation. With the wing swept back to about 650 of sweep, the aircraft has optimum supersonic performance to accomplish high-altitude supersonic bombing or interceptor missions. With the wing folded still further back, the aircraft provides low drag and low gust loads during supersonic flight "on the deck" (altitudes under 1000 feet). The concept of wing variable sweep, of course, is not new. Initial studies were conducted at Langley as early as 1945, and two subsonic variable-sweep prototypes (Bell X-5 and Grumman XF-IOF) were flown as early as 1951/52. These were subsonic aircraft, however, and the great advantage of variable sweep in improving supersonic flight efficiency could not be realized. Further the structures of these early aircraft were complicated by the necessity for translating the ing fore and aft to achieve satisfactory longitUdinal stability as the wing sweep was varied. Late in 1958 a research breakthrough at Langley provided the technology for designing a variable-sweep wing having satisfactory stability through a wide sweep angle range without the necessity for fore and aft translation of the wing. In this same period there evolved within the military services an urgent requirement for a versatile fighter-bomber that could fly efficiently at subsonic and supersonic speeds at high altitude and "on the deck". The application of variable sweep to this mission requirement then became obvious.

Efficient Translation of LTL Formulae into Buchi Automata

NASA Technical Reports Server (NTRS)

Giannakopoulou, Dimitra; Lerda, Flavio

2001-01-01

Model checking is a fully automated technique for checking that a system satisfies a set of required properties. With explicit-state model checkers, properties are typically defined in linear-time temporal logic (LTL), and are translated into B chi automata in order to be checked. This report presents how we have combined and improved existing techniques to obtain an efficient LTL to B chi automata translator. In particular, we optimize the core of existing tableau-based approaches to generate significantly smaller automata. Our approach has been implemented and is being released as part of the Java PathFinder software (JPF), an explicit state model checker under development at the NASA Ames Research Center.

The CTSA Consortium's Catalog of Assets for Translational and Clinical Health Research (CATCHR)

PubMed Central

Mapes, Brandy; Basford, Melissa; Zufelt, Anneliese; Wehbe, Firas; Harris, Paul; Alcorn, Michael; Allen, David; Arnim, Margaret; Autry, Susan; Briggs, Michael S.; Carnegie, Andrea; Chavis‐Keeling, Deborah; De La Pena, Carlos; Dworschak, Doris; Earnest, Julie; Grieb, Terri; Guess, Marilyn; Hafer, Nathaniel; Johnson, Tesheia; Kasper, Amanda; Kopp, Janice; Lockie, Timothy; Lombardo, Vincetta; McHale, Leslie; Minogue, Andrea; Nunnally, Beth; O'Quinn, Deanna; Peck, Kelly; Pemberton, Kieran; Perry, Cheryl; Petrie, Ginny; Pontello, Andria; Posner, Rachel; Rehman, Bushra; Roth, Deborah; Sacksteder, Paulette; Scahill, Samantha; Schieri, Lorri; Simpson, Rosemary; Skinner, Anne; Toussant, Kim; Turner, Alicia; Van der Put, Elaine; Wasser, June; Webb, Chris D.; Williams, Maija; Wiseman, Lori; Yasko, Laurel; Pulley, Jill

2014-01-01

Abstract The 61 CTSA Consortium sites are home to valuable programs and infrastructure supporting translational science and all are charged with ensuring that such investments translate quickly to improved clinical care. Catalog of Assets for Translational and Clinical Health Research (CATCHR) is the Consortium's effort to collect and make available information on programs and resources to maximize efficiency and facilitate collaborations. By capturing information on a broad range of assets supporting the entire clinical and translational research spectrum, CATCHR aims to provide the necessary infrastructure and processes to establish and maintain an open‐access, searchable database of consortium resources to support multisite clinical and translational research studies. Data are collected using rigorous, defined methods, with the resulting information made visible through an integrated, searchable Web‐based tool. Additional easy‐to‐use Web tools assist resource owners in validating and updating resource information over time. In this paper, we discuss the design and scope of the project, data collection methods, current results, and future plans for development and sustainability. With increasing pressure on research programs to avoid redundancy, CATCHR aims to make available information on programs and core facilities to maximize efficient use of resources. PMID:24456567

The Jigsaw Puzzle of mRNA Translation Initiation in Eukaryotes: A Decade of Structures Unraveling the Mechanics of the Process.

PubMed

Hashem, Yaser; Frank, Joachim

2018-03-01

Translation initiation in eukaryotes is a highly regulated and rate-limiting process. It results in the assembly and disassembly of numerous transient and intermediate complexes involving over a dozen eukaryotic initiation factors (eIFs). This process culminates in the accommodation of a start codon marking the beginning of an open reading frame at the appropriate ribosomal site. Although this process has been extensively studied by hundreds of groups for nearly half a century, it has been only recently, especially during the last decade, that we have gained deeper insight into the mechanics of the eukaryotic translation initiation process. This advance in knowledge is due in part to the contributions of structural biology, which have shed light on the molecular mechanics underlying the different functions of various eukaryotic initiation factors. In this review, we focus exclusively on the contribution of structural biology to the understanding of the eukaryotic initiation process, a long-standing jigsaw puzzle that is just starting to yield the bigger picture. Expected final online publication date for the Annual Review of Biophysics Volume 47 is May 20, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Healthcare Commercialization Programs: Improving the Efficiency of Translating Healthcare Innovations From Academia Into Practice

PubMed Central

Reizes, Ofer; Dempsey, Michael K.

2016-01-01

Academic investigators are generating a plethora of insights and technologies that have the potential to significantly improve patient care. However, to address the imperative to improve the quality, cost and access to care with ever more constrained funding, the efficiency and the consistency with which they are translated into cost effective products and/or services need to improve. Healthcare commercialization programs (HCPs) are described and proposed as an option that institutions can add to their portfolio to improve translational research. In helping teams translate specific healthcare innovations into practice, HCPs expand the skillset of investigators and enhance an institution’s innovation capacity. Lessons learned are shared from configuring and delivering HCPs, which build on the fundamentals of the National Science Foundation’s Innovation Corps program, to address the unique challenges in supporting healthcare innovations and innovators. PMID:27766188

Sox2 is translationally activated by eukaryotic initiation factor 4E in human glioma-initiating cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ge, Yuqing; Zhou, Fengbiao; Chen, Hong

2010-07-09

Sox2, a master transcription factor, contributes to the generation of induced pluripotent stem cells and plays significant roles in sustaining the self-renewal of neural stem cells and glioma-initiating cells. Understanding the functional differences of Sox2 between glioma-initiating cells and normal neural stem cells would contribute to therapeutic approach for treatment of brain tumors. Here, we first demonstrated that Sox2 could contribute to the self-renewal and proliferation of glioma-initiating cells. The following experiments showed that Sox2 was activated at translational level in a subset of human glioma-initiating cells compared with the normal neural stem cells. Further investigation revealed there was amore » positive correlation between Sox2 and eukaryotic initiation factor 4E (eIF4E) in glioma tissues. Down-regulation of eIF4E decreased Sox2 protein level without altering its mRNA level in glioma-initiating cells, indicating that Sox2 was activated by eIF4E at translational level. Furthermore, eIF4E was presumed to regulate the expression of Sox2 by its 5' untranslated region (5' UTR) sequence. Our results suggest that the eIF4E-Sox2 axis is a novel mechanism of unregulated self-renewal of glioma-initiating cells, providing a potential therapeutic target for glioma.« less

Novel coding, translation, and gene expression of a replicating covalently closed circular RNA of 220 nt.

PubMed

AbouHaidar, Mounir Georges; Venkataraman, Srividhya; Golshani, Ashkan; Liu, Bolin; Ahmad, Tauqeer

2014-10-07

The highly structured (64% GC) covalently closed circular (CCC) RNA (220 nt) of the virusoid associated with rice yellow mottle virus codes for a 16-kDa highly basic protein using novel modalities for coding, translation, and gene expression. This CCC RNA is the smallest among all known viroids and virusoids and the only one that codes proteins. Its sequence possesses an internal ribosome entry site and is directly translated through two (or three) completely overlapping ORFs (shifting to a new reading frame at the end of each round). The initiation and termination codons overlap UGAUGA (underline highlights the initiation codon AUG within the combined initiation-termination sequence). Termination codons can be ignored to obtain larger read-through proteins. This circular RNA with no noncoding sequences is a unique natural supercompact "nanogenome."

Identifying Cognitive Mechanisms Targeted for Treatment Development in Schizophrenia: An Overview of the First Meeting of the Cognitive Neuroscience Treatment Research to Improve Cognition in Schizophrenia (CNTRICS) Initiative

PubMed Central

Carter, Cameron S.; Barch, Deanna M.; Buchanan, Robert W.; Bullmore, Ed; Krystal, John H.; Cohen, Jonathan; Geyer, Mark; Green, Michael; Nuechterlein, Keith H.; Robbins, Trevor; Silverstein, Steven; Smith, Edward E.; Strauss, Milton; Wykes, Til; Heinssen, Robert

2008-01-01

This overview describes the generation and development of the ideas that led to the Cognitive Neuroscience Treatment Research to Improve Cognition in Schizophrenia (CNTRICS) initiative. It also describes the organization, process and products of the first meeting. The CNTRICS initiative involves a series of three conferences that will systematically address barriers to translating paradigms developed in the basic animal and human cognitive neuroscience fields for use in translational research aimed at developing novel treatments for cognitive impairments in schizophrenia. The articles in this special section report on the results of the first conference, which used a criterion based consensus-building process to develop a set of cognitive constructs to be targeted for translation efforts. PMID:18466880

Defect in the GTPase activating protein (GAP) function of eIF5 causes repression of GCN4 translation.

PubMed

Antony A, Charles; Alone, Pankaj V

2017-05-13

In eukaryotes, the eIF5 protein plays an important role in translation start site selection by providing the GAP (GTPase activating protein) function. However, in yeast translation initiation fidelity defective eIF5 G31R mutant causes preferential utilization of UUG as initiation codon and is termed as Suppressor of initiation codon (Sui - ) phenotype due to its hyper GTPase activity. The eIF5 G31R mutant dominantly represses GCN4 expression and confers sensitivity to 3-Amino-1,2,4-Trizole (3AT) induced starvation. The down-regulation of the GCN4 expression (Gcn - phenotype) in the eIF5 G31R mutant was not because of leaky scanning defects; rather was due to the utilization of upUUG initiation codons at the 5' regulatory region present between uORF1 and the main GCN4 ORF. Copyright © 2017 Elsevier Inc. All rights reserved.

Architecture of human translation initiation factor 3

PubMed Central

Querol-Audi, Jordi; Sun, Chaomin; Vogan, Jacob M.; Smith, Duane; Gu, Yu; Cate, Jamie; Nogales, Eva

2013-01-01

SUMMARY Eukaryotic translation initiation factor 3 (eIF3) plays a central role in protein synthesis by organizing the formation of the 43S preinitiation complex. Using genetic tag visualization by electron microscopy, we reveal the molecular organization of ten human eIF3 subunits, including an octameric core. The structure of eIF3 bears a close resemblance to that of the proteasome lid, with a conserved spatial organization of eight core subunits containing PCI and MPN domains that coordinate functional interactions in both complexes. We further show that eIF3 subunits a and c interact with initiation factors eIF1 and eIF1A, which control the stringency of start codon selection. Finally, we find that subunit j, which modulates messenger RNA interactions with the small ribosomal subunit, makes multiple independent interactions with the eIF3 octameric core. These results highlight the conserved architecture of eIF3 and how it scaffolds key factors that control translation initiation in higher eukaryotes, including humans. PMID:23623729

A New Framework for Understanding IRES-mediated Translation

PubMed Central

Komar, Anton A.; Mazumder, Barsanjit; Merrick, William C.

2012-01-01

Studies over the past 5 or so years have indicated that the traditional clustering of mechanisms for translation initiation in eukaryotes into cap-dependent and cap-independent (or IRES-mediated) is far too narrow. From individual studies of a number of mRNAs encoding proteins that are regulatory in nature (i.e. likely to be needed in small amounts such as transcription factors, protein kinases, etc.), it is now evident that mRNAs exist that blur these boundaries. This review seeks to set the basic ground rules for the analysis of different initiation pathways that are associated with these new mRNAs as well as related to the more traditional mechanisms, especially the cap-dependent translational process that is the major route of initiation of mRNAs for housekeeping proteins and thus, the bulk of protein synthesis in most cells. It will become apparent that a mixture of descriptions is likely to become the norm in the near future (i.e. m7G-assisted internal initiation). PMID:22555019

Improving laboratory efficiencies to scale-up HIV viral load testing.

PubMed

Alemnji, George; Onyebujoh, Philip; Nkengasong, John N

2017-03-01

Viral load measurement is a key indicator that determines patients' response to treatment and risk for disease progression. Efforts are ongoing in different countries to scale-up access to viral load testing to meet the Joint United Nations Programme on HIV and AIDS target of achieving 90% viral suppression among HIV-infected patients receiving antiretroviral therapy. However, the impact of these initiatives may be challenged by increased inefficiencies along the viral load testing spectrum. This will translate to increased costs and ineffectiveness of scale-up approaches. This review describes different parameters that could be addressed across the viral load testing spectrum aimed at improving efficiencies and utilizing test results for patient management. Though progress is being made in some countries to scale-up viral load, many others still face numerous challenges that may affect scale-up efficiencies: weak demand creation, ineffective supply chain management systems; poor specimen referral systems; inadequate data and quality management systems; and weak laboratory-clinical interface leading to diminished uptake of test results. In scaling up access to viral load testing, there should be a renewed focus to address efficiencies across the entire spectrum, including factors related to access, uptake, and impact of test results.

Decapping activators in Saccharomyces cerevisiae act by multiple mechanisms.

PubMed

Nissan, Tracy; Rajyaguru, Purusharth; She, Meipei; Song, Haiwei; Parker, Roy

2010-09-10

Eukaryotic mRNA degradation often occurs in a process whereby translation initiation is inhibited and the mRNA is targeted for decapping. In yeast cells, Pat1, Scd6, Edc3, and Dhh1 all function to promote decapping by an unknown mechanism(s). We demonstrate that purified Scd6 and a region of Pat1 directly repress translation in vitro by limiting the formation of a stable 48S preinitiation complex. Moreover, while Pat1, Edc3, Dhh1, and Scd6 all bind the decapping enzyme, only Pat1 and Edc3 enhance its activity. We also identify numerous direct interactions between Pat1, Dcp1, Dcp2, Dhh1, Scd6, Edc3, Xrn1, and the Lsm1-7 complex. These observations identify three classes of decapping activators that function to directly repress translation initiation and/or stimulate Dcp1/2. Moreover, Pat1 is identified as critical in mRNA decay by first inhibiting translation initiation, then serving as a scaffold to recruit components of the decapping complex, and finally activating Dcp2. Copyright © 2010 Elsevier Inc. All rights reserved.

Identification of Conflicting Selective Effects on Highly Expressed Genes

PubMed Central

Higgs, Paul G.; Hao, Weilong; Golding, G. Brian

2007-01-01

Many different selective effects on DNA and proteins influence the frequency of codons and amino acids in coding sequences. Selection is often stronger on highly expressed genes. Hence, by comparing high- and low-expression genes it is possible to distinguish the factors that are selected by evolution. It has been proposed that highly expressed genes should (i) preferentially use codons matching abundant tRNAs (translational efficiency), (ii) preferentially use amino acids with low cost of synthesis, (iii) be under stronger selection to maintain the required amino acid content, and (iv) be selected for translational robustness. These effects act simultaneously and can be contradictory. We develop a model that combines these factors, and use Akaike’s Information Criterion for model selection. We consider pairs of paralogues that arose by whole-genome duplication in Saccharmyces cerevisiae. A codon-based model is used that includes asymmetric effects due to selection on highly expressed genes. The largest effect is translational efficiency, which is found to strongly influence synonymous, but not non-synonymous rates. Minimization of the cost of amino acid synthesis is implicated. However, when a more general measure of selection for amino acid usage is used, the cost minimization effect becomes redundant. Small effects that we attribute to selection for translational robustness can be identified as an improvement in the model fit on top of the effects of translational efficiency and amino acid usage. PMID:19430600

Rembrandt: Helping Personalized Medicine Become a Reality Through Integrative Translational Research

PubMed Central

Madhavan, Subha; Zenklusen, Jean-Claude; Kotliarov, Yuri; Sahni, Himanso; Fine, Howard A.; Buetow, Kenneth

2009-01-01

Finding better therapies for the treatment of brain tumors is hampered by the lack of consistently obtained molecular data in a large sample set, and ability to integrate biomedical data from disparate sources enabling translation of therapies from bench to bedside. Hence, a critical factor in the advancement of biomedical research and clinical translation is the ease with which data can be integrated, redistributed and analyzed both within and across functional domains. Novel biomedical informatics infrastructure and tools are essential for developing individualized patient treatment based on the specific genomic signatures in each patient’s tumor. Here we present Rembrandt, Repository of Molecular BRAin Neoplasia DaTa, a cancer clinical genomics database and a web-based data mining and analysis platform aimed at facilitating discovery by connecting the dots between clinical information and genomic characterization data. To date, Rembrandt contains data generated through the Glioma Molecular Diagnostic Initiative from 874 glioma specimens comprising nearly 566 gene expression arrays, 834 copy number arrays and 13,472 clinical phenotype data points. Data can be queried and visualized for a selected gene across all data platforms or for multiple genes in a selected platform. Additionally, gene sets can be limited to clinically important annotations including secreted, kinase, membrane, and known gene-anomaly pairs to facilitate the discovery of novel biomarkers and therapeutic targets. We believe that REMBRANDT represents a prototype of how high throughput genomic and clinical data can be integrated in a way that will allow expeditious and efficient translation of laboratory discoveries to the clinic. PMID:19208739

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove

PubMed Central

Filbin, Megan E.; Kieft, Jeffrey S.

2011-01-01

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem–loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit's decoding groove. This configuration depends on the sequence and structure of a different stem–loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis. PMID:21606179

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove.

PubMed

Filbin, Megan E; Kieft, Jeffrey S

2011-07-01

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem-loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit's decoding groove. This configuration depends on the sequence and structure of a different stem-loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis.

Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them.

PubMed

Leppek, Kathrin; Das, Rhiju; Barna, Maria

2018-03-01

RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5' untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5' UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms.

Entrapping ribosomes for viral translation: tRNA mimicry as a molecular Trojan horse.

PubMed

Barends, Sharief; Bink, Hugo H J; van den Worm, Sjoerd H E; Pleij, Cornelis W A; Kraal, Barend

2003-01-10

Turnip yellow mosaic virus (TYMV) has a genomic plus-strand RNA with a 5' cap followed by overlapping and different reading frames for the movement protein and polyprotein, while the distal coat protein cistron is translated from a subgenomic RNA. The 3'-untranslated region harbors a tRNA-like structure (TLS) to which a valine moiety can be added and it is indispensable for virus viability. Here, we report about a surprising interaction between TYMV-RNA-programmed ribosomes and 3'-valylated TLS that yields polyprotein with the valine N terminally incorporated by a translation mechanism resistant to regular initiation inhibitors. Disruption of the TLS exclusively abolishes polyprotein synthesis, which can be restored by adding excess TLS in trans. Our observations imply a novel eukaryotic mechanism for internal initiation of mRNA translation.

Machine Translation-Assisted Language Learning: Writing for Beginners

ERIC Educational Resources Information Center

Garcia, Ignacio; Pena, Maria Isabel

2011-01-01

The few studies that deal with machine translation (MT) as a language learning tool focus on its use by advanced learners, never by beginners. Yet, freely available MT engines (i.e. Google Translate) and MT-related web initiatives (i.e. Gabble-on.com) position themselves to cater precisely to the needs of learners with a limited command of a…

Translation efficiency of heterologous proteins is significantly affected by the genetic context of RBS sequences in engineered cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Thiel, Kati; Mulaku, Edita; Dandapani, Hariharan; Nagy, Csaba; Aro, Eva-Mari; Kallio, Pauli

2018-03-02

Photosynthetic cyanobacteria have been studied as potential host organisms for direct solar-driven production of different carbon-based chemicals from CO 2 and water, as part of the development of sustainable future biotechnological applications. The engineering approaches, however, are still limited by the lack of comprehensive information on most optimal expression strategies and validated species-specific genetic elements which are essential for increasing the intricacy, predictability and efficiency of the systems. This study focused on the systematic evaluation of the key translational control elements, ribosome binding sites (RBS), in the cyanobacterial host Synechocystis sp. PCC 6803, with the objective of expanding the palette of tools for more rigorous engineering approaches. An expression system was established for the comparison of 13 selected RBS sequences in Synechocystis, using several alternative reporter proteins (sYFP2, codon-optimized GFPmut3 and ethylene forming enzyme) as quantitative indicators of the relative translation efficiencies. The set-up was shown to yield highly reproducible expression patterns in independent analytical series with low variation between biological replicates, thus allowing statistical comparison of the activities of the different RBSs in vivo. While the RBSs covered a relatively broad overall expression level range, the downstream gene sequence was demonstrated in a rigorous manner to have a clear impact on the resulting translational profiles. This was expected to reflect interfering sequence-specific mRNA-level interaction between the RBS and the coding region, yet correlation between potential secondary structure formation and observed translation levels could not be resolved with existing in silico prediction tools. The study expands our current understanding on the potential and limitations associated with the regulation of protein expression at translational level in engineered cyanobacteria. The acquired information can be used for selecting appropriate RBSs for optimizing over-expression constructs or multicistronic pathways in Synechocystis, while underlining the complications in predicting the activity due to gene-specific interactions which may reduce the translational efficiency for a given RBS-gene combination. Ultimately, the findings emphasize the need for additional characterized insulator sequence elements to decouple the interaction between the RBS and the coding region for future engineering approaches.

Cdc20 hypomorphic mice fail to counteract de novo synthesis of cyclin B1 in mitosis

PubMed Central

Malureanu, Liviu; Jeganathan, Karthik B.; Jin, Fang; Baker, Darren J.; van Ree, Janine H.; Gullon, Oliver; Chen, Zheyan; Henley, John R.

2010-01-01

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome–microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element–binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset. PMID:20956380

Developing a Collection of Composable Data Translation Software Units to Improve Efficiency and Reproducibility in Ecohydrologic Modeling Workflows

NASA Astrophysics Data System (ADS)

Olschanowsky, C.; Flores, A. N.; FitzGerald, K.; Masarik, M. T.; Rudisill, W. J.; Aguayo, M.

2017-12-01

Dynamic models of the spatiotemporal evolution of water, energy, and nutrient cycling are important tools to assess impacts of climate and other environmental changes on ecohydrologic systems. These models require spatiotemporally varying environmental forcings like precipitation, temperature, humidity, windspeed, and solar radiation. These input data originate from a variety of sources, including global and regional weather and climate models, global and regional reanalysis products, and geostatistically interpolated surface observations. Data translation measures, often subsetting in space and/or time and transforming and converting variable units, represent a seemingly mundane, but critical step in the application workflows. Translation steps can introduce errors, misrepresentations of data, slow execution time, and interrupt data provenance. We leverage a workflow that subsets a large regional dataset derived from the Weather Research and Forecasting (WRF) model and prepares inputs to the Parflow integrated hydrologic model to demonstrate the impact translation tool software quality on scientific workflow results and performance. We propose that such workflows will benefit from a community approved collection of data transformation components. The components should be self-contained composable units of code. This design pattern enables automated parallelization and software verification, improving performance and reliability. Ensuring that individual translation components are self-contained and target minute tasks increases reliability. The small code size of each component enables effective unit and regression testing. The components can be automatically composed for efficient execution. An efficient data translation framework should be written to minimize data movement. Composing components within a single streaming process reduces data movement. Each component will typically have a low arithmetic intensity, meaning that it requires about the same number of bytes to be read as the number of computations it performs. When several components' executions are coordinated the overall arithmetic intensity increases, leading to increased efficiency.

Eukaryotic Translation Initiation Factor 4E Is a Feed-Forward Translational Coactivator of Transforming Growth Factor β Early Protransforming Events in Breast Epithelial Cells

PubMed Central

Decarlo, Lindsey; Mestel, Celine; Barcellos-Hoff, Mary-Helen

2015-01-01

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed early in breast cancers in association with disease progression and reduced survival. Much remains to be understood regarding the role of eIF4E in human cancer. We determined, using immortalized human breast epithelial cells, that elevated expression of eIF4E translationally activates the transforming growth factor β (TGF-β) pathway, promoting cell invasion, a loss of cell polarity, increased cell survival, and other hallmarks of early neoplasia. Overexpression of eIF4E is shown to facilitate the selective translation of integrin β1 mRNA, which drives the translationally controlled assembly of a TGF-β receptor signaling complex containing α3β1 integrins, β-catenin, TGF-β receptor I, E-cadherin, and phosphorylated Smad2/3. This receptor complex acutely sensitizes nonmalignant breast epithelial cells to activation by typically substimulatory levels of activated TGF-β. TGF-β can promote cellular differentiation or invasion and transformation. As a translational coactivator of TGF-β, eIF4E confers selective mRNA translation, reprogramming nonmalignant cells to an invasive phenotype by reducing the set point for stimulation by activated TGF-β. Overexpression of eIF4E may be a proinvasive facilitator of TGF-β activity. PMID:25986608

Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection

PubMed Central

Au, Hilda H.; Cornilescu, Gabriel; Mouzakis, Kathryn D.; Ren, Qian; Burke, Jordan E.; Lee, Seonghoon; Butcher, Samuel E.; Jan, Eric

2015-01-01

The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the three-helical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame. PMID:26554019

Translational science: past, present, and future.

PubMed

Curry, Stephen H

2008-02-01

The concept of translational science is at least 15 years old. However, in its most recent incarnation, it represents the identification of a funding category designed to encourage academic participation in a critical stage of the drug discovery and product development process. It is hoped that this will make the process both shorter and more efficient. In this review, the author first considers the historical development of the pharmaceutical R&D process. The place of translational science in the process, the scientific techniques involved, and aspects of the business environment necessary for its success are then considered. Translational science does not displace preclinical development. Both concepts are relevant to the paramount importance of successfully and expeditiously bridging the gap between preclinical science and clinical testing, "from bench to bedside." Translational science is particularly likely to stimulate biomarker research in the universities and related business community and will probably give a modest boost to early clinical testing and commercialization of discoveries within the academic setting. Whether there will be a consequent improvement in the quality and efficiency of the overall process remains to be seen.

Teachers' and Learners' Perceptions of Applying Translation as a Method, Strategy, or Technique in an Iranian EFL Setting

ERIC Educational Resources Information Center

Mollaei, Fatemeh; Taghinezhad, Ali; Sadighi, Firooz

2017-01-01

It has been found that translation is an efficient means to teach/learn grammar, syntax, and lexis of a foreign language. Meanwhile, translation is good for beginners who do not still enjoy the critical level of proficiency in their target language for expression. This study was conducted to examine the teachers' and learners' perceptions of…

A novel mechanism for Bcr-Abl action: Bcr-Abl-mediated induction of the eIF4F translation initiation complex and mRNA translation.

PubMed

Prabhu, S; Saadat, D; Zhang, M; Halbur, L; Fruehauf, J P; Ong, S T

2007-02-22

The oncogenic kinase Bcr-Abl is thought to cause chronic myelogenous leukemia (CML) by altering the transcription of specific genes with growth- and survival-promoting functions. Recently, Bcr-Abl has also been shown to activate an important regulator of protein synthesis, the mammalian target of rapamycin (mTOR), which suggests that dysregulated translation may also contribute to CML pathogenesis. In this study, we found that both Bcr-Abl and the rapamycin-sensitive mTORC1 complex contribute to the phosphorylation (inactivation) of 4E-BP1, an inhibitor of the eIF4E translation initiation factor. Experiments with rapamycin and the Bcr-Abl inhibitor, imatinib mesylate, in Bcr-Abl-expressing cell lines and primary CML cells indicated that Bcr-Abl and mTORC1 induced formation of the translation initiation complex, eIF4F. This was characterized by reduced 4E-BP1 binding and increased eIF4G binding to eIF4E, two events that lead to the assembly of eIF4F. One target transcript is cyclin D3, which is regulated in Bcr-Abl-expressing cells by both Bcr-Abl and mTORC1 in a translational manner. In addition, the combination of imatinib and rapamycin was found to act synergistically against committed CML progenitors from chronic and blast phase patients. These experiments establish a novel mechanism of action for Bcr-Abl, and they provide insights into the modes of action of imatinib mesylate and rapamycin in treatment of CML. They also suggest that aberrant cap-dependent mRNA translation may be a therapeutic target in Bcr-Abl-driven malignancies.

The NS1 Protein from Influenza Virus Stimulates Translation Initiation by Enhancing Ribosome Recruitment to mRNAs.

PubMed

Panthu, Baptiste; Terrier, Olivier; Carron, Coralie; Traversier, Aurélien; Corbin, Antoine; Balvay, Laurent; Lina, Bruno; Rosa-Calatrava, Manuel; Ohlmann, Théophile

2017-10-27

The non-structural protein NS1 of influenza A viruses exerts pleiotropic functions during infection. Among these functions, NS1 was shown to be involved in the control of both viral and cellular translation; however, the mechanism by which this occurs remains to be determined. Thus, we have revisited the role of NS1 in translation by using a combination of influenza infection, mRNA reporter transfection, and in vitro functional and biochemical assays. Our data show that the NS1 protein is able to enhance the translation of virtually all tested mRNAs with the exception of constructs bearing the Dicistroviruses Internal ribosome entry segment (IRESes) (DCV and CrPV), suggesting a role at the level of translation initiation. The domain of NS1 required for translation stimulation was mapped to the RNA binding amino-terminal motif of the protein with residues R38 and K41 being critical for activity. Although we show that NS1 can bind directly to mRNAs, it does not correlate with its ability to stimulate translation. This activity rather relies on the property of NS1 to associate with ribosomes and to recruit them to target mRNAs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Upstream open reading frames regulate the expression of the nuclear Wnt13 isoforms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang Tao; Rector, Kyle; Barnett, Corey D.

2008-02-22

Wnt proteins control cell survival and cell fate during development. Although Wnt expression is tightly regulated in a spatio-temporal manner, the mechanisms involved both at the transcriptional and translational levels are poorly defined. We have identified a downstream translation initiation codon, AUG(+74), in Wnt13B and Wnt13C mRNAs responsible for the expression of Wnt13 nuclear forms. In this report, we demonstrate that the expression of the nuclear Wnt13C form is translationally regulated in response to stress and apoptosis. Though the 5'-leaders of both Wnt13C and Wnt13B mRNAs have an inhibitory effect on translation, they did not display an internal ribosome entrymore » site activity as demonstrated by dicistronic reporter assays. However, mutations or deletions of the upstream AUG(-99) and AUG(+1) initiation codons abrogate these translation inhibitory effects, demonstrating that Wnt13C expression is controlled by upstream open reading frames. Since long 5'-untranslated region with short upstream open reading frames characterize other Wnt transcripts, our present data on the translational control of Wnt13 expression open the way to further studies on the translation control of Wnt expression as a modulator of their subcellular localization and activity.« less

Translation, adaptation and validation the contents of the Diabetes Medical Management Plan for the Brazilian context

PubMed Central

Torres, Heloísa de Carvalho; Chaves, Fernanda Figueredo; da Silva, Daniel Dutra Romualdo; Bosco, Adriana Aparecida; Gabriel, Beatriz Diniz; Reis, Ilka Afonso; Rodrigues, Júlia Santos Nunes; Pagano, Adriana Silvina

2016-01-01

ABSTRACT Objective: to translate, adapt and validate the contents of the Diabetes Medical Management Plan for the Brazilian context. This protocol was developed by the American Diabetes Association and guides the procedure of educators for the care of children and adolescents with diabetes in schools. Method: this methodological study was conducted in four stages: initial translation, synthesis of initial translation, back translation and content validation by an expert committee, composed of 94 specialists (29 applied linguists and 65 health professionals), for evaluation of the translated version through an online questionnaire. The concordance level of the judges was calculated based on the Content Validity Index. Data were exported into the R program for statistical analysis: Results: the evaluation of the instrument showed good concordance between the judges of the Health and Applied Linguistics areas, with a mean content validity index of 0.9 and 0.89, respectively, and slight variability of the index between groups (difference of less than 0.01). The items in the translated version, evaluated as unsatisfactory by the judges, were reformulated based on the considerations of the professionals of each group. Conclusion: a Brazilian version of Diabetes Medical Management Plan was constructed, called the Plano de Manejo do Diabetes na Escola. PMID:27508911

The applicability of Lean and Six Sigma techniques to clinical and translational research.

PubMed

Schweikhart, Sharon A; Dembe, Allard E

2009-10-01

Lean and Six Sigma are business management strategies commonly used in production industries to improve process efficiency and quality. During the past decade, these process improvement techniques increasingly have been applied outside the manufacturing sector, for example, in health care and in software development. This article concerns the potential use of Lean and Six Sigma in improving the processes involved in clinical and translational research. Improving quality, avoiding delays and errors, and speeding up the time to implementation of biomedical discoveries are prime objectives of the National Institutes of Health (NIH) Roadmap for Medical Research and the NIH's Clinical and Translational Science Award program. This article presents a description of the main principles, practices, and methods used in Lean and Six Sigma. Available literature involving applications of Lean and Six Sigma to health care, laboratory science, and clinical and translational research is reviewed. Specific issues concerning the use of these techniques in different phases of translational research are identified. Examples of Lean and Six Sigma applications that are being planned at a current Clinical and Translational Science Award site are provided, which could potentially be replicated elsewhere. We describe how different process improvement approaches are best adapted for particular translational research phases. Lean and Six Sigma process improvement methods are well suited to help achieve NIH's goal of making clinical and translational research more efficient and cost-effective, enhancing the quality of the research, and facilitating the successful adoption of biomedical research findings into practice.

The 5'-poly(A) leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation

PubMed Central

Dhungel, Pragyesh; Cao, Shuai

2017-01-01

The poly(A) leader at the 5’-untranslated region (5’-UTR) is an unusually striking feature of all poxvirus mRNAs transcribed after viral DNA replication (post-replicative mRNAs). These poly(A) leaders are non-templated and of heterogeneous lengths; and their function during poxvirus infection remains a long-standing question. Here, we discovered that a 5’-poly(A) leader conferred a selective translational advantage to mRNA in poxvirus-infected cells. A constitutive and uninterrupted 5’-poly(A) leader with 12 residues was optimal. Because the most frequent lengths of the 5’-poly(A) leaders are 8–12 residues, the result suggests that the poly(A) leader has been evolutionarily optimized to boost poxvirus protein production. A 5’-poly(A) leader also could increase protein production in the bacteriophage T7 promoter-based expression system of vaccinia virus, the prototypic member of poxviruses. Interestingly, although vaccinia virus post-replicative mRNAs do have 5’- methylated guanosine caps and can use cap-dependent translation, in vaccinia virus-infected cells, mRNA with a 5’-poly(A) leader could also be efficiently translated in cells with impaired cap-dependent translation. However, the translation was not mediated through an internal ribosome entry site (IRES). These results point to a fundamental mechanism poxvirus uses to efficiently translate its post-replicative mRNAs. PMID:28854224

The neuronal ceroid lipofuscinoses program: A translational research experience in Argentina.

PubMed

Kohan, Romina; Pesaola, Favio; Guelbert, Norberto; Pons, Patricia; Oller-Ramírez, Ana María; Rautenberg, Gisela; Becerra, Adriana; Sims, Katherine; Xin, Winnie; Cismondi, Inés Adriana; Noher de Halac, Inés

2015-10-01

The Argentinean program was initiated more than a decade ago as the first experience of systematic translational research focused on NCL in Latin America. The aim was to overcome misdiagnoses and underdiagnoses in the region. 216 NCL suspected individuals from 8 different countries and their direct family members. Clinical assessment, enzyme testing, electron microscopy, and DNA screening. 1) The study confirmed NCL disease in 122 subjects. Phenotypic studies comprised epileptic seizures and movement disorders, ophthalmology, neurophysiology, image analysis, rating scales, enzyme testing, and electron microscopy, carried out under a consensus algorithm; 2) DNA screening and validation of mutations in genes PPT1 (CLN1), TPP1 (CLN2), CLN3, CLN5, CLN6, MFSD8 (CLN7), and CLN8: characterization of variant types, novel/known mutations and polymorphisms; 3) Progress of the epidemiological picture in Latin America; and 4) NCL-like pathology studies in progress. The Translational Research Program was highly efficient in addressing the misdiagnosis/underdiagnosis in the NCL disorders. The study of "orphan diseases" in a public administrated hospital should be adopted by the health systems, as it positively impacts upon the family's quality of life, the collection of epidemiological data, and triggers research advances. This article is part of a Special Issue entitled: "Current Research on the Neuronal Ceroid Lipofuscinoses (Batten Disease)". Copyright © 2015 Elsevier B.V. All rights reserved.

The DaveMLTranslator: An Interface for DAVE-ML Aerodynamic Models

NASA Technical Reports Server (NTRS)

Hill, Melissa A.; Jackson, E. Bruce

2007-01-01

It can take weeks or months to incorporate a new aerodynamic model into a vehicle simulation and validate the performance of the model. The Dynamic Aerospace Vehicle Exchange Markup Language (DAVE-ML) has been proposed as a means to reduce the time required to accomplish this task by defining a standard format for typical components of a flight dynamic model. The purpose of this paper is to describe an object-oriented C++ implementation of a class that interfaces a vehicle subsystem model specified in DAVE-ML and a vehicle simulation. Using the DaveMLTranslator class, aerodynamic or other subsystem models can be automatically imported and verified at run-time, significantly reducing the elapsed time between receipt of a DAVE-ML model and its integration into a simulation environment. The translator performs variable initializations, data table lookups, and mathematical calculations for the aerodynamic build-up, and executes any embedded static check-cases for verification. The implementation is efficient, enabling real-time execution. Simple interface code for the model inputs and outputs is the only requirement to integrate the DaveMLTranslator as a vehicle aerodynamic model. The translator makes use of existing table-lookup utilities from the Langley Standard Real-Time Simulation in C++ (LaSRS++). The design and operation of the translator class is described and comparisons with existing, conventional, C++ aerodynamic models of the same vehicle are given.

Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor

PubMed Central

Simonetti, Angelita; Marzi, Stefano; Billas, Isabelle M. L.; Tsai, Albert; Fabbretti, Attilio; Myasnikov, Alexander G.; Roblin, Pierre; Vaiana, Andrea C.; Hazemann, Isabelle; Eiler, Daniel; Steitz, Thomas A.; Puglisi, Joseph D.; Gualerzi, Claudio O.; Klaholz, Bruno P.

2013-01-01

Translation initiation factor 2 (IF2) promotes 30S initiation complex (IC) formation and 50S subunit joining, which produces the 70S IC. The architecture of full-length IF2, determined by small angle X-ray diffraction and cryo electron microscopy, reveals a more extended conformation of IF2 in solution and on the ribosome than in the crystal. The N-terminal domain is only partially visible in the 30S IC, but in the 70S IC, it stabilizes interactions between IF2 and the L7/L12 stalk of the 50S, and on its deletion, proper N-formyl-methionyl(fMet)-tRNAfMet positioning and efficient transpeptidation are affected. Accordingly, fast kinetics and single-molecule fluorescence data indicate that the N terminus promotes 70S IC formation by stabilizing the productive sampling of the 50S subunit during 30S IC joining. Together, our data highlight the dynamics of IF2-dependent ribosomal subunit joining and the role played by the N terminus of IF2 in this process. PMID:24029017

TRIAD: The Translational Research Informatics and Data Management Grid

PubMed Central

Payne, P.; Ervin, D.; Dhaval, R.; Borlawsky, T.; Lai, A.

2011-01-01

Objective Multi-disciplinary and multi-site biomedical research programs frequently require infrastructures capable of enabling the collection, management, analysis, and dissemination of heterogeneous, multi-dimensional, and distributed data and knowledge collections spanning organizational boundaries. We report on the design and initial deployment of an extensible biomedical informatics platform that is intended to address such requirements. Methods A common approach to distributed data, information, and knowledge management needs in the healthcare and life science settings is the deployment and use of a service-oriented architecture (SOA). Such SOA technologies provide for strongly-typed, semantically annotated, and stateful data and analytical services that can be combined into data and knowledge integration and analysis “pipelines.” Using this overall design pattern, we have implemented and evaluated an extensible SOA platform for clinical and translational science applications known as the Translational Research Informatics and Data-management grid (TRIAD). TRIAD is a derivative and extension of the caGrid middleware and has an emphasis on supporting agile “working interoperability” between data, information, and knowledge resources. Results Based upon initial verification and validation studies conducted in the context of a collection of driving clinical and translational research problems, we have been able to demonstrate that TRIAD achieves agile “working interoperability” between distributed data and knowledge sources. Conclusion Informed by our initial verification and validation studies, we believe TRIAD provides an example instance of a lightweight and readily adoptable approach to the use of SOA technologies in the clinical and translational research setting. Furthermore, our initial use cases illustrate the importance and efficacy of enabling “working interoperability” in heterogeneous biomedical environments. PMID:23616879

TRIAD: The Translational Research Informatics and Data Management Grid.

PubMed

Payne, P; Ervin, D; Dhaval, R; Borlawsky, T; Lai, A

2011-01-01

Multi-disciplinary and multi-site biomedical research programs frequently require infrastructures capable of enabling the collection, management, analysis, and dissemination of heterogeneous, multi-dimensional, and distributed data and knowledge collections spanning organizational boundaries. We report on the design and initial deployment of an extensible biomedical informatics platform that is intended to address such requirements. A common approach to distributed data, information, and knowledge management needs in the healthcare and life science settings is the deployment and use of a service-oriented architecture (SOA). Such SOA technologies provide for strongly-typed, semantically annotated, and stateful data and analytical services that can be combined into data and knowledge integration and analysis "pipelines." Using this overall design pattern, we have implemented and evaluated an extensible SOA platform for clinical and translational science applications known as the Translational Research Informatics and Data-management grid (TRIAD). TRIAD is a derivative and extension of the caGrid middleware and has an emphasis on supporting agile "working interoperability" between data, information, and knowledge resources. Based upon initial verification and validation studies conducted in the context of a collection of driving clinical and translational research problems, we have been able to demonstrate that TRIAD achieves agile "working interoperability" between distributed data and knowledge sources. Informed by our initial verification and validation studies, we believe TRIAD provides an example instance of a lightweight and readily adoptable approach to the use of SOA technologies in the clinical and translational research setting. Furthermore, our initial use cases illustrate the importance and efficacy of enabling "working interoperability" in heterogeneous biomedical environments.

The scanning model for translation: an update

PubMed Central

1989-01-01

The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation. The first-AUG rule is not absolute, but there are rules for breaking the rule. Some anomalous observations that seemed to contradict the scanning mechanism now appear to be artifacts. A few genuine anomalies remain unexplained. PMID:2645293

Novel coding, translation, and gene expression of a replicating covalently closed circular RNA of 220 nt

PubMed Central

AbouHaidar, Mounir Georges; Venkataraman, Srividhya; Golshani, Ashkan; Liu, Bolin; Ahmad, Tauqeer

2014-01-01

The highly structured (64% GC) covalently closed circular (CCC) RNA (220 nt) of the virusoid associated with rice yellow mottle virus codes for a 16-kDa highly basic protein using novel modalities for coding, translation, and gene expression. This CCC RNA is the smallest among all known viroids and virusoids and the only one that codes proteins. Its sequence possesses an internal ribosome entry site and is directly translated through two (or three) completely overlapping ORFs (shifting to a new reading frame at the end of each round). The initiation and termination codons overlap UGAUGA (underline highlights the initiation codon AUG within the combined initiation-termination sequence). Termination codons can be ignored to obtain larger read-through proteins. This circular RNA with no noncoding sequences is a unique natural supercompact “nanogenome.” PMID:25253891

Design of a Temperature-Responsive Transcription Terminator.

PubMed

Roßmanith, Johanna; Weskamp, Mareen; Narberhaus, Franz

2018-02-16

RNA structures regulate various steps in gene expression. Transcription in bacteria is typically terminated by stable hairpin structures. Translation initiation can be modulated by metabolite- or temperature-sensitive RNA structures, called riboswitches or RNA thermometers (RNATs), respectively. RNATs control translation initiation by occlusion of the ribosome binding site at low temperatures. Increasing temperatures destabilize the RNA structure and facilitate ribosome access. In this study, we exploited temperature-responsive RNAT structures to design regulatory elements that control transcription termination instead of translation initiation in Escherichia coli. In order to mimic the structure of factor-independent intrinsic terminators, naturally occurring RNAT hairpins were genetically engineered to be followed by a U-stretch. Functional temperature-responsive terminators (thermoterms) prevented mRNA synthesis at low temperatures but resumed transcription after a temperature upshift. The successful design of temperature-controlled terminators highlights the potential of RNA structures as versatile gene expression control elements.

Frameshifting in the expression of the Escherichia coli trpR gene is modulated by translation initiation.

PubMed Central

Benhar, I; Miller, C; Engelberg-Kulka, H

1993-01-01

The Escherichia coli trpR gene encodes the 108-amino-acid-long Trp repressor. We have shown previously that a +1 frameshifting event occurs during the expression of trpR, resulting in the synthesis of an additional (+1 frame) polypeptide. Using trpR-lac'Z fusions, we have recently found that the transition from the 0 to the +1 frame occurs via the bypassing of a 55-nucleotide-long segment of the trpR+1-lac'Z mRNA (I. Benhar, and H. Engelberg-Kulka, Cell 72:121-130, 1993). Here we show that the frequency of trpR frameshifting (or bypassing) can be regulated both in vivo and in vitro. This frequency is inversely proportional to the rate of initiation of translation of the trpR gene. Hence, modulating the level of translation initiation affects the frequency of frameshifting. Images PMID:8491735

Integrating Bioethics into Clinical and Translational Science Research: A Roadmap

PubMed Central

Shapiro, Robyn S.; Layde, Peter M.

2008-01-01

Abstract Recent initiatives to improve human health emphasize the need to effectively and appropriately translate new knowledge gleaned from basic biomedical and behavioral research to clinical and community application. To maximize the beneficial impact of scientific advances in clinical practice and community health, and to guard against potential deleterious medical and societal consequences of such advances, incorporation of bioethics at each stage of clinical and translational science research is essential. At the earliest stage, bioethics input is critical to address issues such as whether to limit certain areas of scientific inquiry. Subsequently, bioethics input is important to assure not only that human subjects trials are conducted and reported responsibly, but also that results are incorporated into clinical and community practices in a way that promotes and protects bioethical principles. At the final stage of clinical and translational science research, bioethics helps to identify the need and approach for refining clinical practices when safety or other concerns arise. The framework we present depicts how bioethics interfaces with each stage of clinical and translational science research, and suggests an important research agenda for systematically and comprehensively assuring bioethics input into clinical and translational science initiatives. PMID:20443821

Translation suppression promotes stress granule formation and cell survival in response to cold shock

PubMed Central

Hofmann, Sarah; Cherkasova, Valeria; Bankhead, Peter; Bukau, Bernd; Stoecklin, Georg

2012-01-01

Cells respond to different types of stress by inhibition of protein synthesis and subsequent assembly of stress granules (SGs), cytoplasmic aggregates that contain stalled translation preinitiation complexes. Global translation is regulated through the translation initiation factor eukaryotic initiation factor 2α (eIF2α) and the mTOR pathway. Here we identify cold shock as a novel trigger of SG assembly in yeast and mammals. Whereas cold shock–induced SGs take hours to form, they dissolve within minutes when cells are returned to optimal growth temperatures. Cold shock causes eIF2α phosphorylation through the kinase PERK in mammalian cells, yet this pathway is not alone responsible for translation arrest and SG formation. In addition, cold shock leads to reduced mitochondrial function, energy depletion, concomitant activation of AMP-activated protein kinase (AMPK), and inhibition of mTOR signaling. Compound C, a pharmacological inhibitor of AMPK, prevents the formation of SGs and strongly reduces cellular survival in a translation-dependent manner. Our results demonstrate that cells actively suppress protein synthesis by parallel pathways, which induce SG formation and ensure cellular survival during hypothermia. PMID:22875991

Translation, cultural adaptation and validation of the Diabetes Attitudes Scale - third version into Brazilian Portuguese 1

PubMed Central

Vieira, Gisele de Lacerda Chaves; Pagano, Adriana Silvino; Reis, Ilka Afonso; Rodrigues, Júlia Santos Nunes; Torres, Heloísa de Carvalho

2018-01-01

ABSTRACT Objective: to perform the translation, adaptation and validation of the Diabetes Attitudes Scale - third version instrument into Brazilian Portuguese. Methods: methodological study carried out in six stages: initial translation, synthesis of the initial translation, back-translation, evaluation of the translated version by the Committee of Judges (27 Linguists and 29 health professionals), pre-test and validation. The pre-test and validation (test-retest) steps included 22 and 120 health professionals, respectively. The Content Validity Index, the analyses of internal consistency and reproducibility were performed using the R statistical program. Results: in the content validation, the instrument presented good acceptance among the Judges with a mean Content Validity Index of 0.94. The scale presented acceptable internal consistency (Cronbach’s alpha = 0.60), while the correlation of the total score at the test and retest moments was considered high (Polychoric Correlation Coefficient = 0.86). The Intra-class Correlation Coefficient, for the total score, presented a value of 0.65. Conclusion: the Brazilian version of the instrument (Escala de Atitudes dos Profissionais em relação ao Diabetes Mellitus) was considered valid and reliable for application by health professionals in Brazil. PMID:29319739

Translation and cultural adaptation of the Aguado Syntax Test (AST) into Brazilian Portuguese.

PubMed

Baggio, Gustavo Inheta; Hage, Simone Rocha de Vasconcellos

2017-12-07

To perform the translation and cultural adaptation of the Aguado Syntax Test (AST) into Brazilian Portuguese considering the linguistic and cultural reality of the language. The AST assesses the early morphosyntactic development in children aged 3 to 7 in terms of understanding and expression of various types of structures such as sentences, pronouns, verbal voices, comparisons, prepositions and verbal desinence as to number, mode and tense. The process of translation and cultural adaptation followed four steps: 1) preparation of two translations; 2) synthesis of consensual translations; 3) backtranslation; and 4) verification of equivalence between the initial translations and backtranslations that resulted in the final translated version. The whole process of translation and cultural adaptation revealed the presence of equivalence and reconciliation of the translated items and an almost complete semantic equivalence between the two translations and the absence of consistent translation difficulties. The AST was translated and culturally adapted into Brazilian Portuguese, constituting the first step towards validation and standardization of the test.

75 FR 12243 - Disease, Disability, and Injury Prevention and Control Special Emphasis Panel (SEP): Translating...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-15

... Eye and Vision Loss Prevention, Funding Opportunity Announcement (FOA) DP 10-004, Initial Review In... of ``Translating Research into Healthy Eye and Vision Loss Prevention, RFA DP 10- 004.'' Contact...

76 FR 13414 - Disease, Disability, and Injury Prevention and Control Special Emphasis Panel: Continuation of...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-03-11

... Mesothelioma Virtual Bank for Translational Research, Program Announcement PAR 11-002, Initial Review In... ``Continuation of the National Mesothelioma Virtual Bank for Translational Research, PAR 11-002.'' Contact Person...

A growth-dependent transcription initiation factor (TIF-IA) interacting with RNA polymerase I regulates mouse ribosomal RNA synthesis.

PubMed

Schnapp, A; Pfleiderer, C; Rosenbauer, H; Grummt, I

1990-09-01

Control of mouse ribosomal RNA synthesis in response to extracellular signals is mediated by TIF-IA, a regulatory factor whose amount or activity correlates with cell proliferation. Factor TIF-IA interacts with RNA polymerase I (pol I), thus converting it into a transcriptionally active holoenzyme, which is able to initiate specifically at the rDNA promoter in the presence of the other auxiliary transcription initiation factors, designated TIF-IB, TIF-IC and UBF. With regard to several criteria, the growth-dependent factor TIF-IA behaves like a bacterial sigma factor: (i) it associates physically with pol I, (ii) it is required for initiation of transcription, (iii) it is present in limiting amounts and (iv) under certain salt conditions, it is chromatographically separable from the polymerase. In addition, evidence is presented that dephosphorylation of pol I abolishes in vitro transcription initiation from the ribosomal gene promoter without significantly affecting the polymerizing activity of the enzyme at nonspecific templates. The involvement of both a regulatory factor and post-translational modification of the transcribing enzyme provides an efficient and versatile mechanism of rDNA transcription regulation which enables the cell to adapt ribosome synthesis rapidly to a variety of extracellular signals.

LOCAL TRANSLATION. Comment on "Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling".

PubMed

Reid, David W; Nicchitta, Christopher V

2015-06-12

Jan et al. (Research Articles, 7 November 2014, p. 716) propose that ribosomes translating secretome messenger RNAs (mRNAs) traffic from the cytosol to the endoplasmic reticulum (ER) upon emergence of the signal peptide and return to the cytosol after termination. An accounting of controls demonstrates that mRNAs initiate translation on ER-bound ribosomes and that ribosomes are retained on the ER through many cycles of translation. Copyright © 2015, American Association for the Advancement of Science.

Cap-independent protein synthesis is enhanced by betaine under hypertonic conditions.

PubMed

Carnicelli, Domenica; Arfilli, Valentina; Onofrillo, Carmine; Alfieri, Roberta R; Petronini, Pier Giorgio; Montanaro, Lorenzo; Brigotti, Maurizio

2017-02-12

Protein synthesis is one of the main cellular functions inhibited during hypertonic challenge. The subsequent accumulation of the compatible osmolyte betaine during the later adaptive response allows not only recovery of translation but also its stimulation. In this paper, we show that betaine modulates translation by enhancing the formation of cap-independent 48 S pre-initiation complexes, leaving cap-dependent 48 S pre-initiation complexes basically unchanged. In the presence of betaine, CrPV IRES- and sodium-dependent neutral amino acid transporter-2 (SNAT2) 5'-UTR-driven translation is 2- and 1.5-fold stimulated in MCF7 cells, respectively. Thus, betaine could provide an advantage in translation of messengers coding for proteins implicated in the response of cells to different stressors, which are often recognized by ribosomal 40 S subunit through simplified cap-independent mechanisms. Copyright © 2017 Elsevier Inc. All rights reserved.

Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them

PubMed Central

Leppek, Kathrin; Das, Rhiju; Barna, Maria

2017-01-01

RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5′ untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5′ UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms. PMID:29165424

The influence of viral coding sequences on pestivirus IRES activity reveals further parallels with translation initiation in prokaryotes.

PubMed Central

Fletcher, Simon P; Ali, Iraj K; Kaminski, Ann; Digard, Paul; Jackson, Richard J

2002-01-01

Classical swine fever virus (CSFV) is a member of the pestivirus family, which shares many features in common with hepatitis C virus (HCV). It is shown here that CSFV has an exceptionally efficient cis-acting internal ribosome entry segment (IRES), which, like that of HCV, is strongly influenced by the sequences immediately downstream of the initiation codon, and is optimal with viral coding sequences in this position. Constructs that retained 17 or more codons of viral coding sequence exhibited full IRES activity, but with only 12 codons, activity was approximately 66% of maximum in vitro (though close to maximum in transfected BHK cells), whereas with just 3 codons or fewer, the activity was only approximately 15% of maximum. The minimal coding region elements required for high activity were exchanged between HCV and CSFV. Although maximum activity was observed in each case with the homologous combination of coding region and 5' UTR, the heterologous combinations were sufficiently active to rule out a highly specific functional interplay between the 5' UTR and coding sequences. On the other hand, inversion of the coding sequences resulted in low IRES activity, particularly with the HCV coding sequences. RNA structure probing showed that the efficiency of internal initiation of these chimeric constructs correlated most closely with the degree of single-strandedness of the region around and immediately downstream of the initiation codon. The low activity IRESs could not be rescued by addition of supplementary eIF4A (the initiation factor with ATP-dependent RNA helicase activity). The extreme sensitivity to secondary structure around the initiation codon is likely to be due to the fact that the eIF4F complex (which has eIF4A as one of its subunits) is not required for and does not participate in initiation on these IRESs. PMID:12515388

Differential Properties of Cytomegalovirus pUL97 Kinase Isoforms Affect Viral Replication and Maribavir Susceptibility

PubMed Central

Webel, Rike; Hakki, Morgan; Prichard, Mark N.; Rawlinson, William D.; Marschall, Manfred

2014-01-01

ABSTRACT The human cytomegalovirus (HCMV)-encoded kinase pUL97 is required for efficient viral replication. Previous studies described two isoforms of pUL97, the full-length isoform (M1) and a smaller isoform likely resulting from translation initiation at codon 74 (M74). Here, we report the detection of a third pUL97 isoform during viral infection resulting from translation initiation at codon 157 (isoform M157). The consistent expression of isoform M157 as a minor component of pUL97 during infection with clinical and laboratory-adapted HCMV strains was suppressed when codon 157 was mutagenized. Viral mutants expressing specific isoforms were generated to compare their growth and drug susceptibility phenotypes, as well as pUL97 intracellular localization patterns and kinase activities. The exclusive expression of isoform M157 resulted in substantially reduced viral growth and resistance to the pUL97 inhibitor maribavir while retaining susceptibility to ganciclovir. Confocal imaging demonstrated reduced nuclear import of amino-terminal deletion isoforms compared to isoform M1. Isoform M157 showed reduced efficiency of various substrate protein interactions and autophosphorylation, whereas Rb phosphorylation was preserved. These results reveal differential properties of pUL97 isoforms that affect viral replication, with implications for the antiviral efficacy of maribavir. IMPORTANCE The HCMV UL97 kinase performs important functions in viral replication that are targeted by the antiviral drug maribavir. Here, we describe a naturally occurring short isoform of the kinase that when expressed by itself in a recombinant virus results in altered intracellular localization, impaired growth, and high-level resistance to maribavir compared to those of the predominant full-length counterpart. This is another factor to consider in explaining why maribavir appears to have variable antiviral activity in cell culture and in vivo. PMID:24522923

Characterisation of the biological response of Saccharomyces cerevisiae to the loss of an allele of the eukaryotic initiation factor 4A.

PubMed

Venturi, Veronica; Little, Richard; Bircham, Peter W; Rodigheri Brito, Juliana; Atkinson, Paul H; Maass, David R; Teesdale-Spittle, Paul H

2018-02-19

The translation initiation machinery is emerging as an important target for therapeutic intervention, with potential in the treatment of cancer, viral infections, and muscle wasting. Amongst the targets for pharmacological control of translation initiation is the eukaryotic initiation factor 4A (eIF4A), an RNA helicase that is essential for cap-dependent translation initiation. We set out to explore the system-wide impact of a reduction of functional eIF4A. To this end, we investigated the effect of deletion of TIF1, one of the duplicate genes that produce eIF4A in yeast, through synthetic genetic array interactions and system-wide changes in GFP-tagged protein abundances. We show that there is a biological response to deletion of the TIF1 gene that extends through the proteostasis network. Effects of the deletion are apparent in processes as distributed as chromatin remodelling, ribosome biogenesis, amino acid metabolism, and protein trafficking. The results from this study identify protein complexes and pathways that will make ideal targets for combination therapies with eIF4A inhibitors. Copyright © 2018 Elsevier Inc. All rights reserved.

Crossing the chasm: information technology to biomedical informatics.

PubMed

Fahy, Brenda G; Balke, C William; Umberger, Gloria H; Talbert, Jeffery; Canales, Denise Niles; Steltenkamp, Carol L; Conigliaro, Joseph

2011-06-01

Accelerating the translation of new scientific discoveries to improve human health and disease management is the overall goal of a series of initiatives integrated in the National Institutes of Health (NIH) "Roadmap for Medical Research." The Clinical and Translational Science Award (CTSA) program is, arguably, the most visible component of the NIH Roadmap providing resources to institutions to transform their clinical and translational research enterprises along the goals of the Roadmap. The CTSA program emphasizes biomedical informatics as a critical component for the accomplishment of the NIH's translational objectives. To be optimally effective, emerging biomedical informatics programs must link with the information technology platforms of the enterprise clinical operations within academic health centers.This report details one academic health center's transdisciplinary initiative to create an integrated academic discipline of biomedical informatics through the development of its infrastructure for clinical and translational science infrastructure and response to the CTSA mechanism. This approach required a detailed informatics strategy to accomplish these goals. This transdisciplinary initiative was the impetus for creation of a specialized biomedical informatics core, the Center for Biomedical Informatics (CBI). Development of the CBI codified the need to incorporate medical informatics including quality and safety informatics and enterprise clinical information systems within the CBI. This article describes the steps taken to develop the biomedical informatics infrastructure, its integration with clinical systems at one academic health center, successes achieved, and barriers encountered during these efforts.

Linking scientific discovery and better health for the nation: the first three years of the NIH's Clinical and Translational Science Awards.

PubMed

Califf, Robert M; Berglund, Lars

2010-03-01

A comprehensive system for translating basic biomedical research into useful and effectively implemented clinical diagnostic, preventive, and therapeutic practices is essential to the nation's health. The state of clinical and translational research (CTR) in the United States, however, has been characterized as fragmented, slow, expensive, and poorly coordinated. As part of its Roadmap Initiative, the National Institutes of Health instituted the Clinical and Translational Science Awards (CTSA), a sweeping and ambitious program designed to transform the conduct of biomedical research in the United States by speeding the translation of scientific discoveries into useful therapies and then developing methods to ensure that those therapies reach the patients who need them the most. The authors review the circumstances of the U.S. biomedical research enterprise that led to the creation of the CTSA and discuss the initial strategic plan of the CTSA, which was developed from the first three years of experience with the program and was designed to overcome organizational, methodological, and cultural barriers within and among research institutions. The authors also describe the challenges encountered during these efforts and discuss the promise of this vital national health care initiative, which is essential to creating a pipeline for the scientific workforce needed to conduct research that will, in turn, provide a rational evidence base for better health in the United States.

Linking Scientific Discovery and Better Health for the Nation: The First Three Years of the NIH’s Clinical and Translational Science Awards

PubMed Central

Califf, Robert M.; Berglund, Lars

2015-01-01

A comprehensive system for translating basic biomedical research into useful and effectively implemented clinical diagnostic, preventive, and therapeutic practices is essential to the nation’s health. The state of clinical and translational research (CTR) in the United States, however, has been characterized as fragmented, slow, expensive, and poorly coordinated. As part of its Roadmap Initiative, the National Institutes of Health instituted the Clinical and Translational Science Awards (CTSA), a sweeping and ambitious program designed to transform the conduct of biomedical research in the United States by speeding the translation of scientific discoveries into useful therapies and then developing methods to ensure that those therapies reach the patients who need them the most. The authors review the circumstances of the U.S. biomedical research enterprise that led to the creation of the CTSA and discuss the initial strategic plan of the CTSA, which was developed from the first 3 years of experience with the program and was designed to overcome organizational, methodological, and cultural barriers within and among research institutions. The authors also describe the challenges encountered during these efforts and discuss the promise of this vital national health care initiative, which is essential to creating a pipeline for the scientific workforce needed to conduct research that will in turn provide a rational evidence base for better health in the United States. PMID:20182118

Knowledge Translation Interventions to Improve the Timing of Dialysis Initiation: Protocol for a Cluster Randomized Trial.

PubMed

Chau, Elaine M T; Manns, Braden J; Garg, Amit X; Sood, Manish M; Kim, S Joseph; Naimark, David; Nesrallah, Gihad E; Soroka, Steven D; Beaulieu, Monica; Dixon, Stephanie; Alam, Ahsan; Tangri, Navdeep

2016-01-01

Early initiation of chronic dialysis (starting dialysis with higher vs lower kidney function) has risen rapidly in the past 2 decades in Canada and internationally, despite absence of established health benefits and higher costs. In 2014, a Canadian guideline on the timing of dialysis initiation, recommending an intent-to-defer approach, was published. The objective of this study is to evaluate the efficacy and safety of a knowledge translation intervention to promote the intent-to-defer approach in clinical practice. This study is a multicenter, 2-arm parallel, cluster randomized trial. The study involves 55 advanced chronic kidney disease clinics across Canada. Patients older than 18 years who are managed by nephrologists for more than 3 months, and initiate dialysis in the follow-up period are included in the study. Outcomes will be measured at the patient-level and enumerated within a cluster. Data on characteristics of each dialysis start will be determined by linkages with the Canadian Organ Replacement Register. Primary outcomes include the proportion of patients who start dialysis early with an estimated glomerular filtration rate greater than 10.5 mL/min/1.73 m 2 and start dialysis in hospital as inpatients or in an emergency room setting. Secondary outcomes include the rate of change in early dialysis starts; rates of hospitalizations, deaths, and cost of predialysis care (wherever available); quarterly proportion of new starts; and acceptability of the knowledge translation materials. We randomized 55 multidisciplinary chronic disease clinics (clusters) in Canada to receive either an active knowledge translation intervention or no intervention for the uptake of the guideline on the timing of dialysis initiation. The active knowledge translation intervention consists of audit and feedback as well as patient- and provider-directed educational tools delivered at a comprehensive in-person medical detailing visit. Control clinics are only exposed to guideline release without active dissemination. We hypothesize that the clinics randomized to the intervention group will have a lower proportion of early dialysis starts. Limitations include passive dissemination of the guideline through publication, and lead-time and survivor bias, which favors delayed dialysis initiation. If successful, this active knowledge translation intervention will reduce early dialysis starts, lead to health and economic benefits, and provide a successful framework for evaluating and disseminating future guidelines. ClinicalTrials.gov, NCT02183987.

Modifying the 5'-Cap for Click Reactions of Eukaryotic mRNA and To Tune Translation Efficiency in Living Cells.

PubMed

Holstein, Josephin M; Anhäuser, Lea; Rentmeister, Andrea

2016-08-26

The 5'-cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5'-cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7-modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N(2) -modified 5'-caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live-cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Translational Research from an Informatics Perspective

NASA Technical Reports Server (NTRS)

Bernstam, Elmer; Meric-Bernstam, Funda; Johnson-Throop, Kathy A.; Turley, James P.; Smith, Jack W.

2007-01-01

Clinical and translational research (CTR) is an essential part of a sustainable global health system. Informatics is now recognized as an important en-abler of CTR and informaticians are increasingly called upon to help CTR efforts. The US National Institutes of Health mandated biomedical informatics activity as part of its new national CTR grant initiative, the Clinical and Translational Science Award (CTSA). Traditionally, translational re-search was defined as the translation of laboratory discoveries to patient care (bench to bedside). We argue, however, that there are many other kinds of translational research. Indeed, translational re-search requires the translation of knowledge dis-covered in one domain to another domain and is therefore an information-based activity. In this panel, we will expand upon this view of translational research and present three different examples of translation to illustrate the point: 1) bench to bedside, 2) Earth to space and 3) academia to community. We will conclude with a discussion of our local translational research efforts that draw on each of the three examples.

Serum-deprivation stimulates cap-binding by PARN at the expense of eIF4E, consistent with the observed decrease in mRNA stability

PubMed Central

Seal, Ruth; Temperley, Richard; Wilusz, Jeffrey; Lightowlers, Robert N.; Chrzanowska-Lightowlers, Zofia M. A.

2005-01-01

PARN, a poly(A)-specific ribonuclease, binds the 5′ cap-structure of mRNA and initiates deadenylation-dependent decay. Eukaryotic initiation factor 4E (eIF4E) also binds to the cap structure, an interaction that is critical for initiating cap-dependent translation. The stability of various mRNA transcripts in human cell lines is reduced under conditions of serum starvation as determined by both functional and chemical half-lives. Serum starvation also leads to enhanced cap association by PARN. In contrast, the 5′ cap occupancy by eIF4E decreases under serum-deprivation, as does the translation of reporter transcripts. Further, we show that PARN is a phosphoprotein and that this modification can be modulated by serum status. Taken together, these data are consistent with a natural competition existing at the 5′ cap structure between PARN and eIF4E that may be regulated by changes in post-translational modifications. These phosphorylation-induced changes in the interplay of PARN and eIF4E may determine whether the mRNA is translated or decayed. PMID:15653638

Doxorubicin induces apoptosis by targeting Madcam1 and AKT and inhibiting protein translation initiation in hepatocellular carcinoma cells

PubMed Central

Tang, Xun; Zhang, Xiao; Qiao, Yongxia; Shi, Yuling; Xu, Yanfeng; Wang, Zhongyong; Yu, Yongchun; Sun, Fenyong

2015-01-01

Doxorubicin (Doxo) is one of the most widely used chemotherapeutic drugs for patients with hepatocellular carcinoma (HCC). Doxo is a DNA intercalating drug that inhibits topoisomerase II. Thereby Doxo has the ability to block DNA replication and induce apoptosis. However, the other targets and mechanisms through which Doxo induces apoptosis to treat HCC still remain unknown. Here, we identified Mucosal vascular addressin cell adhesion molecule 1 (Madcam1) as a potential Doxo target because Madcam1 overexpression suppressed, while Madcam1 depletion stimulated Doxo-induced apoptosis. Furthermore, we first revealed that Doxo can induce apoptosis by blocking protein translation initiation. In contrast, Madcam1 activated protein translation through an opposite mechanism. We also found de-phosphorylation of AKT may be an important pro-apoptotic event that is triggered by Doxo-induced Madcam1 down-regulation. Finally, we revealed that Madcam1 promoted increased AKT phosphorylation, which is essential for maintaining the sensitivity of HCC cells to Doxo treatment. Taken together, we uncovered a potential mechanism for Doxo-induced apoptosis in HCC treatment through targeting Madcam1 and AKT and blocking protein translation initiation. PMID:26124182

Public-private partnerships in translational medicine: concepts and practical examples.

PubMed

Luijten, Peter R; van Dongen, Guus A M S; Moonen, Chrit T; Storm, Gert; Crommelin, Daan J A

2012-07-20

The way forward in multidisciplinary research according to former NIH's director Elias Zerhouni is to engage in predictive, personalized, preemptive and participatory medicine. For the creation of the optimal innovation climate that would allow for such a strategy, public-private partnerships have been widely proposed as an important instrument. Public-private partnerships have become an important instrument to expedite translational research in medicine. The Netherlands have initiated three large public-private partnerships in the life sciences and health area to facilitate the translation of valuable basic scientific concepts to new products and services in medicine. The focus of these partnerships has been on drug development, improved diagnosis and regenerative medicine. The Dutch model of public-private partnership forms the blueprint of a much larger European initiative called EATRIS. This paper will provide practical examples of public-private partnerships initiated to expedite the translation of new technology for drug development towards the clinic. Three specific technologies are in focus: companion diagnostics using nuclear medicine, the use of ultra high field MRI to generate sensitive surrogate endpoints based on endogenous contrast, and MRI guidance for High Intensity Focused Ultrasound mediated drug delivery. Copyright © 2012 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murooka, Thomas T.; Rahbar, Ramtin; Department of Immunology, University of Toronto, Ont.

The proliferative capacity of cancer cells is regulated by factors intrinsic to cancer cells and by secreted factors in the microenvironment. Here, we investigated the proto-oncogenic potential of the chemokine receptor, CCR5, in MCF-7 breast cancer cell lines. At physiological levels, CCL5, a ligand for CCR5, enhanced MCF-7.CCR5 proliferation. Treatment with the mTOR inhibitor, rapamycin, inhibited this CCL5-inducible proliferation. Because mTOR directly modulates mRNA translation, we investigated whether CCL5 activation of CCR5 leads to increased translation. CCL5 induced the formation of the eIF4F translation initiation complex through an mTOR-dependent process. Indeed, CCL5 initiated mRNA translation, shown by an increase inmore » high-molecular-weight polysomes. Specifically, we show that CCL5 mediated a rapid up-regulation of protein expression for cyclin D1, c-Myc and Dad-1, without affecting their mRNA levels. Taken together, we describe a mechanism by which CCL5 influences translation of rapamycin-sensitive mRNAs, thereby providing CCR5-positive breast cancer cells with a proliferative advantage.« less

Molecular Architecture of the 40S⋅eIF1⋅eIF3 Translation Initiation Complex

PubMed Central

Erzberger, Jan P.; Stengel, Florian; Pellarin, Riccardo; Zhang, Suyang; Schaefer, Tanja; Aylett, Christopher H.S.; Cimermančič, Peter; Boehringer, Daniel; Sali, Andrej; Aebersold, Ruedi; Ban, Nenad

2014-01-01

Summary Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA. PMID:25171412

Clinical translation of human microRNA 21 as a potential biomarker for exposure to ionizing radiation.

PubMed

Halimi, Mohammad; Parsian, Hadi; Asghari, S Mohsen; Sariri, Reyhaneh; Moslemi, Dariush; Yeganeh, Farshid; Zabihi, Ebrahim

2014-06-01

This study investigated to what extent the serum microRNA 21 (miR-21) level alters in response to ionizing radiation (IR). Initially, we evaluated the appropriateness of our RNA extraction efficiency and microRNA assay in serum, and then investigated the serum miR-21 level in 4 patients with breast cancer in 4 stages: pre- and postoperation, at the beginning radiotherapy, and after 25 sessions of radiotherapy with a total of 50 Gy irradiation, as well as in 20 healthy volunteers. The initial analysis showed the appropriateness of our RNA extraction efficiency and microRNA assay in serum for identifying people exposed to IR. We then analyzed the serum miR-21 level in another group of 40 patients with breast cancer before and after radiotherapy. During our large-scale analysis, the miR-21 level before radiotherapy was comparable with healthy volunteers (P = 0.10) and increased significantly after radiotherapy (P < 0.001)-an indication that this could discriminate irradiated patients from nonirradiated ones with high specificity (75%) and sensitivity (80%). According to this study, serum miR-21 has the potential to be used as a biomarker for the identification of people exposed to ionizing radiation. Copyright © 2014 Mosby, Inc. All rights reserved.

Remodeling of the pioneer translation initiation complex involves translation and the karyopherin importin β

PubMed Central

Sato, Hanae; Maquat, Lynne E.

2009-01-01

Mammalian mRNAs lose and acquire proteins throughout their life span while undergoing processing, transport, translation, and decay. How translation affects messenger RNA (mRNA)–protein interactions is largely unknown. The pioneer round of translation uses newly synthesized mRNA that is bound by cap-binding protein 80 (CBP80)–CBP20 (also known as the cap-binding complex [CBC]) at the cap, poly(A)-binding protein N1 (PABPN1) and PABPC1 at the poly(A) tail, and, provided biogenesis involves pre-mRNA splicing, exon junction complexes (EJCs) at exon–exon junctions. Subsequent rounds of translation engage mRNA that is bound by eukaryotic translation initiation factor 4E (eIF4E) at the cap and PABPC1 at the poly(A) tail, but that lacks detectable EJCs and PABPN1. Using the level of intracellular iron to regulate the translation of specific mRNAs, we show that translation promotes not only removal of EJC constituents, including the eIF4AIII anchor, but also replacement of PABPN1 by PABPC1. Remarkably, translation does not affect replacement of CBC by eIF4E. Instead, replacement of CBC by eIF4E is promoted by importin β (IMPβ): Inhibiting the binding of IMPβ to the complex of CBC–IMPα at an mRNA cap using the IMPα IBB (IMPβ-binding) domain or a RAN variant increases the amount of CBC-bound mRNA and decreases the amount of eIF4E-bound mRNA. Our studies uncover a previously unappreciated role for IMPβ and a novel paradigm for how newly synthesized messenger ribonucleoproteins (mRNPs) are matured. PMID:19884259

Demystifying knowledge translation: learning from the community.

PubMed

Bowen, Sarah; Martens, Patricia

2005-10-01

While there is increasing interest in research related to so-called Knowledge Translation, much of this research is undertaken from the perspective of researchers. The objective of this paper is to explore, through the participatory evaluation of Manitoba's The Need to Know Project, the characteristics of effective knowledge translation initiatives from the perspective of community partners. The multi-method evaluation adopted a utilization-focused approach, where stakeholders participated in identifying evaluation questions, and methods were made transparent to participants. Over 100 open-ended, semi-structured interviews were conducted with project stakeholders over the first three years of the project. These interviews explored the perspectives of participants on all aspects of project development. Formal feedback processes allowed further refinement of emerging theory. This research suggests that there has been insufficient emphasis on personal factors in knowledge translation. The themes of 'quality of relationships' and 'trust' connected many different components of knowledge translation, and were essential for collaborative research. Organizational barriers and lack of confidence in researchers present greater challenges to knowledge translation than individual interest or community capacity. The costs of participation in collaborative research for community partners and the benefits for researchers, also require greater attention. Participation of community partners in The Need to Know Project has provided unique perspectives on knowledge translation theory. It has identified limitations to the common interpretations of knowledge translation principles and highlighted the characteristics of collaborative research initiatives that are of greatest importance to community partners.

Applying Process Improvement Methods to Clinical and Translational Research: Conceptual Framework and Case Examples.

PubMed

Daudelin, Denise H; Selker, Harry P; Leslie, Laurel K

2015-12-01

There is growing appreciation that process improvement holds promise for improving quality and efficiency across the translational research continuum but frameworks for such programs are not often described. The purpose of this paper is to present a framework and case examples of a Research Process Improvement Program implemented at Tufts CTSI. To promote research process improvement, we developed online training seminars, workshops, and in-person consultation models to describe core process improvement principles and methods, demonstrate the use of improvement tools, and illustrate the application of these methods in case examples. We implemented these methods, as well as relational coordination theory, with junior researchers, pilot funding awardees, our CTRC, and CTSI resource and service providers. The program focuses on capacity building to address common process problems and quality gaps that threaten the efficient, timely and successful completion of clinical and translational studies. © 2015 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc.

A fast Fourier transform on multipoles (FFTM) algorithm for solving Helmholtz equation in acoustics analysis.

PubMed

Ong, Eng Teo; Lee, Heow Pueh; Lim, Kian Meng

2004-09-01

This article presents a fast algorithm for the efficient solution of the Helmholtz equation. The method is based on the translation theory of the multipole expansions. Here, the speedup comes from the convolution nature of the translation operators, which can be evaluated rapidly using fast Fourier transform algorithms. Also, the computations of the translation operators are accelerated by using the recursive formulas developed recently by Gumerov and Duraiswami [SIAM J. Sci. Comput. 25, 1344-1381(2003)]. It is demonstrated that the algorithm can produce good accuracy with a relatively low order of expansion. Efficiency analyses of the algorithm reveal that it has computational complexities of O(Na), where a ranges from 1.05 to 1.24. However, this method requires substantially more memory to store the translation operators as compared to the fast multipole method. Hence, despite its simplicity in implementation, this memory requirement issue may limit the application of this algorithm to solving very large-scale problems.

Global analysis of translation termination in E. coli.

PubMed

Baggett, Natalie E; Zhang, Yan; Gross, Carol A

2017-03-01

Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 variant of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins.

mRNA Translation Gone Awry: Translation Fidelity and Neurological Disease.

PubMed

Kapur, Mridu; Ackerman, Susan L

2018-03-01

Errors during mRNA translation can lead to a reduction in the levels of functional proteins and an increase in deleterious molecules. Advances in next-generation sequencing have led to the discovery of rare genetic disorders, many caused by mutations in genes encoding the mRNA translation machinery, as well as to a better understanding of translational dynamics through ribosome profiling. We discuss here multiple neurological disorders that are linked to errors in tRNA aminoacylation and ribosome decoding. We draw on studies from genetic models, including yeast and mice, to enhance our understanding of the translational defects observed in these diseases. Finally, we emphasize the importance of tRNA, their associated enzymes, and the inextricable link between accuracy and efficiency in the maintenance of translational fidelity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins.

PubMed

Gan, Rui; Perez, Jessica G; Carlson, Erik D; Ntai, Ioanna; Isaacs, Farren J; Kelleher, Neil L; Jewett, Michael C

2017-05-01

The ability to site-specifically incorporate non-canonical amino acids (ncAAs) into proteins has made possible the study of protein structure and function in fundamentally new ways, as well as the bio synthesis of unnatural polymers. However, the task of site-specifically incorporating multiple ncAAs into proteins with high purity and yield continues to present a challenge. At the heart of this challenge lies the lower efficiency of engineered orthogonal translation system components compared to their natural counterparts (e.g., translation elements that specifically use a ncAA and do not interact with the cell's natural translation apparatus). Here, we show that evolving and tuning expression levels of multiple components of an engineered translation system together as a whole enhances ncAA incorporation efficiency. Specifically, we increase protein yield when incorporating multiple p-azido-phenylalanine(pAzF) residues into proteins by (i) evolving the Methanocaldococcus jannaschii p-azido-phenylalanyl-tRNA synthetase anti-codon binding domain, (ii) evolving the elongation factor Tu amino acid-binding pocket, and (iii) tuning the expression of evolved translation machinery components in a single vector. Use of the evolved translation machinery in a genomically recoded organism lacking release factor one enabled enhanced multi-site ncAA incorporation into proteins. We anticipate that our approach to orthogonal translation system development will accelerate and expand our ability to site-specifically incorporate multiple ncAAs into proteins and biopolymers, advancing new horizons for synthetic and chemical biotechnology. Biotechnol. Bioeng. 2017;114: 1074-1086. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

An MSC2 Promoter-lacZ Fusion Gene Reveals Zinc-Responsive Changes in Sites of Transcription Initiation That Occur across the Yeast Genome

PubMed Central

Wu, Yi-Hsuan; Taggart, Janet; Song, Pamela Xiyao; MacDiarmid, Colin; Eide, David J.

2016-01-01

The Msc2 and Zrg17 proteins of Saccharomyces cerevisiae form a complex to transport zinc into the endoplasmic reticulum. ZRG17 is transcriptionally induced in zinc-limited cells by the Zap1 transcription factor. In this report, we show that MSC2 mRNA also increases (~1.5 fold) in zinc-limited cells. The MSC2 gene has two in-frame ATG codons at its 5’ end, ATG1 and ATG2; ATG2 is the predicted initiation codon. When the MSC2 promoter was fused at ATG2 to the lacZ gene, we found that unlike the chromosomal gene this reporter showed a 4-fold decrease in lacZ mRNA in zinc-limited cells. Surprisingly, β-galactosidase activity generated by this fusion gene increased ~7 fold during zinc deficiency suggesting the influence of post-transcriptional factors. Transcription of MSC2ATG2-lacZ was found to start upstream of ATG1 in zinc-replete cells. In zinc-limited cells, transcription initiation shifted to sites just upstream of ATG2. From the results of mutational and polysome profile analyses, we propose the following explanation for these effects. In zinc-replete cells, MSC2ATG2-lacZ mRNA with long 5’ UTRs fold into secondary structures that inhibit translation. In zinc-limited cells, transcripts with shorter unstructured 5’ UTRs are generated that are more efficiently translated. Surprisingly, chromosomal MSC2 did not show start site shifts in response to zinc status and only shorter 5’ UTRs were observed. However, the shifts that occur in the MSC2ATG2-lacZ construct led us to identify significant transcription start site changes affecting the expression of ~3% of all genes. Therefore, zinc status can profoundly alter transcription initiation across the yeast genome. PMID:27657924

Prolonged Exposure of Primary Human Muscle Cells to Plasma Fatty Acids Associated with Obese Phenotype Induces Persistent Suppression of Muscle Mitochondrial ATP Synthase β Subunit.

PubMed

Tran, Lee; Hanavan, Paul D; Campbell, Latoya E; De Filippis, Elena; Lake, Douglas F; Coletta, Dawn K; Roust, Lori R; Mandarino, Lawrence J; Carroll, Chad C; Katsanos, Christos S

2016-01-01

Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of β-F1-ATPase in obese (BMI, 36±1 kg/m2) and lean (BMI, 22±1 kg/m2) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h-1) and translation efficiency of β-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P<0.05). We then evaluated these same responses in a primary cell culture model, and tested the specific hypothesis that circulating non-esterified fatty acids (NEFA) in obesity play a role in the responses observed in humans. The findings on total protein synthesis and translation efficiency of β-F1-ATPase in primary myotubes cultured from a lean subject, and after exposure to NEFA extracted from serum of an obese subject, were similar to those obtained in humans. Among candidate microRNAs (i.e., non-coding RNAs regulating gene expression), we identified miR-127-5p in preventing the production of β-F1-ATPase. Muscle expression of miR-127-5p negatively correlated with β-F1-ATPase protein translation efficiency in humans (r = - 0.6744; P<0.01), and could be modeled in vitro by prolonged exposure of primary myotubes derived from the lean subject to NEFA extracted from the obese subject. On the other hand, locked nucleic acid inhibitor synthesized to target miR-127-5p significantly increased β-F1-ATPase translation efficiency in myotubes (0.6±0.1 vs 1.3±0.3, in control vs exposure to 50 nM inhibitor; P<0.05). Our experiments implicate circulating NEFA in obesity in suppressing muscle protein metabolism, and establish impaired β-F1-ATPase translation as an important consequence of obesity.

Signalling to eIF4E in cancer

PubMed Central

Siddiqui, Nadeem; Sonenberg, Nahum

2015-01-01

Translational control plays a critical role in the regulation of gene expression in eukaryotes and affects many essential cellular processes, including proliferation, apoptosis and differentiation. Under most circumstances, translational control occurs at the initiation step at which the ribosome is recruited to the mRNA. The eukaryotic translation initiation factor 4E (eIF4E), as part of the eIF4F complex, interacts first with the mRNA and facilitates the recruitment of the 40S ribosomal subunit. The activity of eIF4E is regulated at many levels, most profoundly by two major signalling pathways: PI3K (phosphoinositide 3-kinase)/Akt (also known and Protein Kinase B, PKB)/mTOR (mechanistic/mammalian target of rapamycin) and Ras (rat sarcoma)/MAPK (mitogen-activated protein kinase)/Mnk (MAPK-interacting kinases). mTOR directly phosphorylates the 4E-BPs (eIF4E-binding proteins), which are inhibitors of eIF4E, to relieve translational suppression, whereas Mnk phosphorylates eIF4E to stimulate translation. Hyperactivation of these pathways occurs in the majority of cancers, which results in increased eIF4E activity. Thus, translational control via eIF4E acts as a convergence point for hyperactive signalling pathways to promote tumorigenesis. Consequently, recent works have aimed to target these pathways and ultimately the translational machinery for cancer therapy. PMID:26517881

Training the Translational Research Teams of the Future: UC Davis—HHMI Integrating Medicine into Basic Science Program

PubMed Central

Rainwater, Julie A.; Chiamvimonvat, Nipavan; Bonham, Ann C.; Robbins, John A.; Henderson, Stuart; Meyers, Frederick J.

2013-01-01

Abstract There is a need for successful models of how to recruit, train, and retain bench scientists at the earliest stages of their careers into translational research. One recent, promising model is the University of California Davis Howard Hughes Medical Institute Integrating Medicine into Basic Science (HHMI‐IMBS) program, part of the HHMI Med into Grad initiative. This paper outlines the HHMI‐IMBS program's logic, design, and curriculum that guide the goal of research that moves from bedside to bench. That is, a curriculum that provides graduate students with guided translational training, clinical exposure, team science competencies, and mentors from diverse disciplines that will advance the students careers in clinical translational research and re‐focusing of research to answer clinical dilemmas. The authors have collected data on 55 HHMI‐IMBS students to date. Many of these students are still completing their graduate work. In the current study the authors compare the initial two cohorts (15 students) with a group of 29 control students to examine the program success and outcomes. The data indicate that this training program provides an effective, adaptable model for training future translational researchers. HHMI‐IMBS students showed improved confidence in conducting translational research, greater interest in a future translational career, and higher levels of research productivity and collaborations than a comparable group of predoctoral students. PMID:24127920

Translation Initiation Factor eIF4B Interacts with a Picornavirus Internal Ribosome Entry Site in both 48S and 80S Initiation Complexes Independently of Initiator AUG Location

PubMed Central

Ochs, Kerstin; Rust, René C.; Niepmann, Michael

1999-01-01

Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation. PMID:10438840

mRNA-Selective Translation Induced by FSH in Primary Sertoli Cells

PubMed Central

Musnier, Astrid; León, Kelly; Morales, Julia; Reiter, Eric; Boulo, Thomas; Costache, Vlad; Vourc'h, Patrick; Heitzler, Domitille; Oulhen, Nathalie; Poupon, Anne; Boulben, Sandrine; Cormier, Patrick

2012-01-01

FSH is a key hormonal regulator of Sertoli cell secretory activity, required to optimize sperm production. To fulfil its biological function, FSH binds a G protein-coupled receptor, the FSH-R. The FSH-R-transduced signaling network ultimately leads to the transcription or down-regulation of numerous genes. In addition, recent evidence has suggested that FSH might also regulate protein translation. However, this point has never been demonstrated conclusively yet. Here we have addressed this issue in primary rat Sertoli cells endogenously expressing physiological levels of FSH-R. We observed that, within 90 min of stimulation, FSH not only enhanced overall protein synthesis in a mammalian target of rapamycin-dependent manner but also increased the recruitment of mRNA to polysomes. m7GTP pull-down experiments revealed the functional recruitment of mammalian target of rapamycin and p70 S6 kinase to the 5′cap, further supported by the enhanced phosphorylation of one of p70 S6 kinase targets, the eukaryotic initiation factor 4B. Importantly, the scaffolding eukaryotic initiation factor 4G was also recruited, whereas eukaryotic initiation factor 4E-binding protein, the eukaryotic initiation factor 4E generic inhibitor, appeared to play a minor role in translational regulations induced by FSH, in contrast to what is generally observed in response to anabolic factors. This particular regulation of the translational machinery by FSH stimulation might support mRNA-selective translation, as shown here by quantitative RT-PCR amplification of the c-fos and vascular endothelial growth factor mRNA but not of all FSH target mRNA, in polysomal fractions. These findings add a new level of complexity to FSH biological roles in its natural target cells, which has been underappreciated so far. PMID:22383463

Structure and dynamics of translation initiation factor aIF-1A from the archaeon Methanococcus jannaschii determined by NMR spectroscopy

PubMed Central

Li, Wei; Hoffman, David W.

2001-01-01

Translation initiation factor 1A (aIF-1A) from the archaeon Methanococcus jannaschii was expressed in Escherichia coli, purified, and characterized in terms of its structure and dynamics using multidimensional NMR methods. The protein was found to be a member of the OB-fold family of RNA-associated proteins, containing a barrel of five beta-strands, a feature that is shared with the homologous eukaryotic translation initiation factor 1A (eIF-1A), as well as the prokaryotic translation initiation factor IF1. External to the β barrel, aIF-1A contains an α-helix at its C-terminal and a flexible loop at its N-terminal, features that are qualitatively similar to those found in eIF-1A, but not present in prokaryotic IF1. The structural model of aIF-1A, when used in combination with primary sequence information for aIF-1A in divergent species, permitted the most-conserved residues on the protein surface to be identified, including the most likely candidates for direct interaction with the 16S ribosomal RNA and other components of the translational apparatus. Several of the conserved surface residues appear to be unique to the archaea. Nitrogen-15 relaxation and amide exchange rate data were used to characterize the internal motions within aIF-1A, providing evidence that the protein surfaces that are most likely to participate in intermolecular interactions are relatively flexible. A model is proposed, suggesting some specific interactions that may occur between aIF-1A and the small subunit of the archaeal ribosome. PMID:11714910

Inhibition of Group I Metabotropic Glutamate Receptors Reverses Autistic-Like Phenotypes Caused by Deficiency of the Translation Repressor eIF4E Binding Protein 2

PubMed Central

Aguilar-Valles, Argel; Matta-Camacho, Edna; Khoutorsky, Arkady; Gkogkas, Christos; Nader, Karim

2015-01-01

Exacerbated mRNA translation during brain development has been linked to autism spectrum disorders (ASDs). Deletion of the eukaryotic initiation factor 4E (eIF4E)-binding protein 2 gene (Eif4ebp2), encoding the suppressor of mRNA translation initiation 4E-BP2, leads to an imbalance in excitatory-to-inhibitory neurotransmission and ASD-like behaviors. Inhibition of group I metabotropic glutamate receptors (mGluRs) mGluR1 and mGluR5 reverses the autistic phenotypes in several ASD mouse models. Importantly, these receptors control synaptic physiology via activation of mRNA translation. We investigated the potential reversal of autistic-like phenotypes in Eif4ebp2−/− mice by using antagonists of mGluR1 (JNJ16259685) or mGluR5 (fenobam). Augmented hippocampal mGluR-induced long-term depression (LTD; or chemically induced mGluR-LTD) in Eif4ebp2−/− mice was rescued by mGluR1 or mGluR5 antagonists. While rescue by mGluR5 inhibition occurs through the blockade of a protein synthesis-dependent component of LTD, normalization by mGluR1 antagonists requires the activation of protein synthesis. Synaptically induced LTD was deficient in Eif4ebp2−/− mice, and this deficit was not rescued by group I mGluR antagonists. Furthermore, a single dose of mGluR1 (0.3 mg/kg) or mGluR5 (3 mg/kg) antagonists in vivo reversed the deficits in social interaction and repetitive behaviors (marble burying) in Eif4ebp2−/− mice. Our results demonstrate that Eif4ebp2−/− mice serve as a relevant model to test potential therapies for ASD symptoms. In addition, we provide substantive evidence that the inhibition of mGluR1/mGluR5 is an effective treatment for physiological and behavioral alterations caused by exacerbated mRNA translation initiation. SIGNIFICANCE STATEMENT Exacerbated mRNA translation during brain development is associated with several autism spectrum disorders (ASDs). We recently demonstrated that the deletion of a negative regulator of mRNA translation initiation, the eukaryotic initiation factor 4E-binding protein 2, leads to ASD-like behaviors and increased excitatory synaptic activity. Here we demonstrated that autistic behavioral and electrophysiological phenotypes can be treated in adult mice with antagonists of group I metabotropic glutamate receptors (mGluRs), which have been previously used in other ASD models (i.e., fragile X syndrome). These findings support the use of group I mGluR antagonists as a potential therapy that extends to autism models involving exacerbated mRNA translation initiation. PMID:26245973

Inhibition of Group I Metabotropic Glutamate Receptors Reverses Autistic-Like Phenotypes Caused by Deficiency of the Translation Repressor eIF4E Binding Protein 2.

PubMed

Aguilar-Valles, Argel; Matta-Camacho, Edna; Khoutorsky, Arkady; Gkogkas, Christos; Nader, Karim; Lacaille, Jean-Claude; Sonenberg, Nahum

2015-08-05

Exacerbated mRNA translation during brain development has been linked to autism spectrum disorders (ASDs). Deletion of the eukaryotic initiation factor 4E (eIF4E)-binding protein 2 gene (Eif4ebp2), encoding the suppressor of mRNA translation initiation 4E-BP2, leads to an imbalance in excitatory-to-inhibitory neurotransmission and ASD-like behaviors. Inhibition of group I metabotropic glutamate receptors (mGluRs) mGluR1 and mGluR5 reverses the autistic phenotypes in several ASD mouse models. Importantly, these receptors control synaptic physiology via activation of mRNA translation. We investigated the potential reversal of autistic-like phenotypes in Eif4ebp2(-/-) mice by using antagonists of mGluR1 (JNJ16259685) or mGluR5 (fenobam). Augmented hippocampal mGluR-induced long-term depression (LTD; or chemically induced mGluR-LTD) in Eif4ebp2(-/-) mice was rescued by mGluR1 or mGluR5 antagonists. While rescue by mGluR5 inhibition occurs through the blockade of a protein synthesis-dependent component of LTD, normalization by mGluR1 antagonists requires the activation of protein synthesis. Synaptically induced LTD was deficient in Eif4ebp2(-/-) mice, and this deficit was not rescued by group I mGluR antagonists. Furthermore, a single dose of mGluR1 (0.3 mg/kg) or mGluR5 (3 mg/kg) antagonists in vivo reversed the deficits in social interaction and repetitive behaviors (marble burying) in Eif4ebp2(-/-) mice. Our results demonstrate that Eif4ebp2(-/-) mice serve as a relevant model to test potential therapies for ASD symptoms. In addition, we provide substantive evidence that the inhibition of mGluR1/mGluR5 is an effective treatment for physiological and behavioral alterations caused by exacerbated mRNA translation initiation. Exacerbated mRNA translation during brain development is associated with several autism spectrum disorders (ASDs). We recently demonstrated that the deletion of a negative regulator of mRNA translation initiation, the eukaryotic initiation factor 4E-binding protein 2, leads to ASD-like behaviors and increased excitatory synaptic activity. Here we demonstrated that autistic behavioral and electrophysiological phenotypes can be treated in adult mice with antagonists of group I metabotropic glutamate receptors (mGluRs), which have been previously used in other ASD models (i.e., fragile X syndrome). These findings support the use of group I mGluR antagonists as a potential therapy that extends to autism models involving exacerbated mRNA translation initiation. Copyright © 2015 the authors 0270-6474/15/3511126-08$15.00/0.

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

PubMed Central

Dever, Thomas E.; Kinzy, Terri Goss; Pavitt, Graham D.

2016-01-01

In this review, we provide an overview of protein synthesis in the yeast Saccharomyces cerevisiae. The mechanism of protein synthesis is well conserved between yeast and other eukaryotes, and molecular genetic studies in budding yeast have provided critical insights into the fundamental process of translation as well as its regulation. The review focuses on the initiation and elongation phases of protein synthesis with descriptions of the roles of translation initiation and elongation factors that assist the ribosome in binding the messenger RNA (mRNA), selecting the start codon, and synthesizing the polypeptide. We also examine mechanisms of translational control highlighting the mRNA cap-binding proteins and the regulation of GCN4 and CPA1 mRNAs. PMID:27183566

Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

PubMed Central

Tsotakos, Nikolaos; Silveyra, Patricia; Lin, Zhenwu; Thomas, Neal; Vaid, Mudit

2014-01-01

Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5′-untranslated regions (5′-UTR) due to differential splicing. The 5′-UTR variant ACD′ is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/SP-A2 ratio, with potential for clinical implication. PMID:25326576

The Applicability of Lean and Six Sigma Techniques to Clinical and Translational Research

PubMed Central

Schweikhart, Sharon A.; Dembe, Allard E

2010-01-01

Background Lean and Six Sigma are business management strategies commonly used in production industries to improve process efficiency and quality. During the past decade, these process improvement techniques increasingly have been applied outside of the manufacturing sector, for example, in health care and in software development. This article concerns the potential use of Lean and Six Sigma to improve the processes involved in clinical and translational research. Improving quality, avoiding delays and errors, and speeding up the time to implementation of biomedical discoveries are prime objectives of the NIH Roadmap for Biomedical Research and the NIH Clinical and Translational Science Award (CTSA) program. Methods This article presents a description of the main principles, practices, and methodologies used in Lean and Six Sigma. Available literature involving applications of Lean and Six Sigma to health care, laboratory science, and clinical and translational research is reviewed. Specific issues concerning the use of these techniques in different phases of translational research are identified. Results Examples are provided of Lean and Six Sigma applications that are being planned at a current CTSA site, which could potentially be replicated elsewhere. We describe how different process improvement approaches are best adapted for particularly translational research phases. Conclusions Lean and Six Sigma process improvement methodologies are well suited to help achieve NIH’s goal of making clinical and translational research more efficient and cost-effective, enhancing the quality of the research, and facilitating the successful adoption of biomedical research findings into practice. PMID:19730130

How Messenger RNA and Nascent Chain Sequences Regulate Translation Elongation.

PubMed

Choi, Junhong; Grosely, Rosslyn; Prabhakar, Arjun; Lapointe, Christopher P; Wang, Jinfan; Puglisi, Joseph D

2018-06-20

Translation elongation is a highly coordinated, multistep, multifactor process that ensures accurate and efficient addition of amino acids to a growing nascent-peptide chain encoded in the sequence of translated messenger RNA (mRNA). Although translation elongation is heavily regulated by external factors, there is clear evidence that mRNA and nascent-peptide sequences control elongation dynamics, determining both the sequence and structure of synthesized proteins. Advances in methods have driven experiments that revealed the basic mechanisms of elongation as well as the mechanisms of regulation by mRNA and nascent-peptide sequences. In this review, we highlight how mRNA and nascent-peptide elements manipulate the translation machinery to alter the dynamics and pathway of elongation.

The mutant vasopressin gene from diabetes insipidus (Brattleboro) rats is transcribed but the message is not efficiently translated.

PubMed Central

Schmale, H; Ivell, R; Breindl, M; Darmer, D; Richter, D

1984-01-01

The vasopressin gene from normal and diabetes insipidus (Brattleboro) rats has been isolated and sequenced. Except for a single deletion of a G residue in region coding for the neurophysin carrier protein the approximately 2300 nucleotides of both genes are identical. Blot analysis of hypothalamic RNA as well as transfection and microinjection experiments indicate that the mutant gene is correctly transcribed and spliced, however the resulting mRNA is not efficiently translated. Images Fig. 2. Fig. 3. PMID:6526016

ImTK: an open source multi-center information management toolkit

NASA Astrophysics Data System (ADS)

Alaoui, Adil; Ingeholm, Mary Lou; Padh, Shilpa; Dorobantu, Mihai; Desai, Mihir; Cleary, Kevin; Mun, Seong K.

2008-03-01

The Information Management Toolkit (ImTK) Consortium is an open source initiative to develop robust, freely available tools related to the information management needs of basic, clinical, and translational research. An open source framework and agile programming methodology can enable distributed software development while an open architecture will encourage interoperability across different environments. The ISIS Center has conceptualized a prototype data sharing network that simulates a multi-center environment based on a federated data access model. This model includes the development of software tools to enable efficient exchange, sharing, management, and analysis of multimedia medical information such as clinical information, images, and bioinformatics data from multiple data sources. The envisioned ImTK data environment will include an open architecture and data model implementation that complies with existing standards such as Digital Imaging and Communications (DICOM), Health Level 7 (HL7), and the technical framework and workflow defined by the Integrating the Healthcare Enterprise (IHE) Information Technology Infrastructure initiative, mainly the Cross Enterprise Document Sharing (XDS) specifications.

Box-Counting Dimension Revisited: Presenting an Efficient Method of Minimizing Quantization Error and an Assessment of the Self-Similarity of Structural Root Systems

PubMed Central

Bouda, Martin; Caplan, Joshua S.; Saiers, James E.

2016-01-01

Fractal dimension (FD), estimated by box-counting, is a metric used to characterize plant anatomical complexity or space-filling characteristic for a variety of purposes. The vast majority of published studies fail to evaluate the assumption of statistical self-similarity, which underpins the validity of the procedure. The box-counting procedure is also subject to error arising from arbitrary grid placement, known as quantization error (QE), which is strictly positive and varies as a function of scale, making it problematic for the procedure's slope estimation step. Previous studies either ignore QE or employ inefficient brute-force grid translations to reduce it. The goals of this study were to characterize the effect of QE due to translation and rotation on FD estimates, to provide an efficient method of reducing QE, and to evaluate the assumption of statistical self-similarity of coarse root datasets typical of those used in recent trait studies. Coarse root systems of 36 shrubs were digitized in 3D and subjected to box-counts. A pattern search algorithm was used to minimize QE by optimizing grid placement and its efficiency was compared to the brute force method. The degree of statistical self-similarity was evaluated using linear regression residuals and local slope estimates. QE, due to both grid position and orientation, was a significant source of error in FD estimates, but pattern search provided an efficient means of minimizing it. Pattern search had higher initial computational cost but converged on lower error values more efficiently than the commonly employed brute force method. Our representations of coarse root system digitizations did not exhibit details over a sufficient range of scales to be considered statistically self-similar and informatively approximated as fractals, suggesting a lack of sufficient ramification of the coarse root systems for reiteration to be thought of as a dominant force in their development. FD estimates did not characterize the scaling of our digitizations well: the scaling exponent was a function of scale. Our findings serve as a caution against applying FD under the assumption of statistical self-similarity without rigorously evaluating it first. PMID:26925073

IFLA General Conference, 1987. Division of General Research Libraries. National Libraries Section. University Libraries and Other General Research Libraries Section. Papers.

ERIC Educational Resources Information Center

International Federation of Library Associations, The Hague (Netherlands).

Of the 11 papers in this collection, three are in German and in English translation; one is in French with an English translation and two are in French only; one is in Spanish with an English translation; and four are in English. The papers include: (1) "Europaische Bibliotheksinitiativen = European Library Initiatives" (Gunther Pflug); (2) "Die…

Translational control by eIF2α phosphorylation regulates vulnerability to the synaptic and behavioral effects of cocaine

PubMed Central

Huang, Wei; Placzek, Andon N; Viana Di Prisco, Gonzalo; Khatiwada, Sanjeev; Sidrauski, Carmela; Krnjević, Krešimir; Walter, Peter; Dani, John A; Costa-Mattioli, Mauro

2016-01-01

Adolescents are especially prone to drug addiction, but the underlying biological basis of their increased vulnerability remains unknown. We reveal that translational control by phosphorylation of the translation initiation factor eIF2α (p-eIF2α) accounts for adolescent hypersensitivity to cocaine. In adolescent (but not adult) mice, a low dose of cocaine reduced p-eIF2α in the ventral tegmental area (VTA), potentiated synaptic inputs to VTA dopaminergic neurons, and induced drug-reinforced behavior. Like adolescents, adult mice with reduced p-eIF2α-mediated translational control were more susceptible to cocaine-induced synaptic potentiation and behavior. Conversely, like adults, adolescent mice with increased p-eIF2α became more resistant to cocaine's effects. Accordingly, metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD)—whose disruption is postulated to increase vulnerability to drug addiction—was impaired in both adolescent mice and adult mice with reduced p-eIF2α mediated translation. Thus, during addiction, cocaine hijacks translational control by p-eIF2α, initiating synaptic potentiation and addiction-related behaviors. These insights may hold promise for new treatments for addiction. DOI: http://dx.doi.org/10.7554/eLife.12052.001 PMID:26928234

75 FR 62543 - Proposed Collection; Comment Request; National Evaluation of the Clinical and Translational...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-12

... of the Clinical and Translational Science Awards (CTSA) Initiative SUMMARY: In compliance with the... comment on proposed data collection projects, the National Center for Research Resources (NCRR), the... National Evaluation of the Clinical and [[Page 62544

Crossing the Chasm: Information Technology to Biomedical Informatics

PubMed Central

Fahy, Brenda G.; Balke, C. William; Umberger, Gloria H.; Talbert, Jeffery; Canales, Denise Niles; Steltenkamp, Carol L.; Conigliaro, Joseph

2011-01-01

Accelerating the translation of new scientific discoveries to improve human health and disease management is the overall goal of a series of initiatives integrated in the National Institutes of Health (NIH) “Roadmap for Medical Research.” The Clinical and Translational Research Award (CTSA) program is, arguably, the most visible component of the NIH Roadmap providing resources to institutions to transform their clinical and translational research enterprises along the goals of the Roadmap. The CTSA program emphasizes biomedical informatics as a critical component for the accomplishment of the NIH’s translational objectives. To be optimally effective, emerging biomedical informatics programs must link with the information technology (IT) platforms of the enterprise clinical operations within academic health centers. This report details one academic health center’s transdisciplinary initiative to create an integrated academic discipline of biomedical informatics through the development of its infrastructure for clinical and translational science infrastructure and response to the CTSA mechanism. This approach required a detailed informatics strategy to accomplish these goals. This transdisciplinary initiative was the impetus for creation of a specialized biomedical informatics core, the Center for Biomedical Informatics (CBI). Development of the CBI codified the need to incorporate medical informatics including quality and safety informatics and enterprise clinical information systems within the CBI. This paper describes the steps taken to develop the biomedical informatics infrastructure, its integration with clinical systems at one academic health center, successes achieved, and barriers encountered during these efforts. PMID:21383632

Man-machine analysis of translation and work tasks of Skylab films

NASA Technical Reports Server (NTRS)

Morrow, J. R.; Boelter, J.

1978-01-01

Selected film segments were digitized. An efficiency of translation scale was developed, and each of 200 segments of film were rated with regard to the astronauts translation characteristics. Results indicated that in general the astronauts were able to acclimate themselves to the zero g environment quite well. Results also indicated that astronauts tended to translate in 1 g orientations when in the experimental compartment and the wardroom which were architecturally 1 g. However, when the astronauts were in the forward compartment, which was zero g oriented, they began to translate more frequently in a zero g manner. There appeared to be improvements in translation across time. These improvements appeared more so in the forward compartment than in the wardroom or the experimental compartment. Possible changes in the architecture of the wardroom and the experimental compartment were suggested in order to improve translation within these compartments.

Performance limitations of translationally symmetric nonimaging devices

NASA Astrophysics Data System (ADS)

Bortz, John C.; Shatz, Narkis E.; Winston, Roland

2001-11-01

The component of the optical direction vector along the symmetry axis is conserved for all rays propagated through a translationally symmetric optical device. This quality, referred to herein as the translational skew invariant, is analogous to the conventional skew invariant, which is conserved in rotationally symmetric optical systems. The invariance of both of these quantities is a consequence of Noether's theorem. We show how performance limits for translationally symmetric nonimaging optical devices can be derived from the distributions of the translational skew invariant for the optical source and for the target to which flux is to be transferred. Examples of computed performance limits are provided. In addition, we show that a numerically optimized non-tracking solar concentrator utilizing symmetry-breaking surface microstructure can overcome the performance limits associated with translational symmetry. The optimized design provides a 47.4% increase in efficiency and concentration relative to an ideal translationally symmetric concentrator.

Cryo-EM structure of Hepatitis C virus IRES bound to the human ribosome at 3.9-Å resolution

NASA Astrophysics Data System (ADS)

Quade, Nick; Boehringer, Daniel; Leibundgut, Marc; van den Heuvel, Joop; Ban, Nenad

2015-07-01

Hepatitis C virus (HCV), a widespread human pathogen, is dependent on a highly structured 5'-untranslated region of its mRNA, referred to as internal ribosome entry site (IRES), for the translation of all of its proteins. The HCV IRES initiates translation by directly binding to the small ribosomal subunit (40S), circumventing the need for many eukaryotic translation initiation factors required for mRNA scanning. Here we present the cryo-EM structure of the human 40S ribosomal subunit in complex with the HCV IRES at 3.9 Å resolution, determined by focused refinement of an 80S ribosome-HCV IRES complex. The structure reveals the molecular details of the interactions between the IRES and the 40S, showing that expansion segment 7 (ES7) of the 18S rRNA acts as a central anchor point for the HCV IRES. The structural data rationalizes previous biochemical and genetic evidence regarding the initiation mechanism of the HCV and other related IRESs.

IRES-mediated translation of foot-and-mouth disease virus (FMDV) in cultured cells derived from FMDV-susceptible and -insusceptible animals.

PubMed

Kanda, Takehiro; Ozawa, Makoto; Tsukiyama-Kohara, Kyoko

2016-03-31

Foot-and-mouth disease virus (FMDV) possess a positive sense, single stranded RNA genome. Internal ribosomal entry site (IRES) element exists within its 5' untranslated region (5'UTR) of the viral RNA. Translation of the viral RNA is initiated by internal entry of the 40S ribosome within the IRES element. This process is facilitated by cellular factors known as IRES trans-acting factors (ITAFs). Foot-and-mouth disease (FMD) is host-restricted disease for cloven-hoofed animals such as cattle and pigs, but the factors determining the host range have not been identified yet. Although, ITAFs are known to promote IRES-mediated translation, these findings were confirmed only in cells derived from FMDV-insusceptible animals so far. We evaluated and compared the IRES-mediated translation activities among cell lines derived from four different animal species using bicistronic luciferase reporter plasmid, which possesses an FMDV-IRES element between Renilla and Firefly luciferase genes. Furthermore, we analyzed the effect of the cellular factors on IRES-mediated translation by silencing the cellular factors using siRNA in both FMDV-susceptible and -insusceptible animal cells. Our data indicated that IRES-mediated translational activity was not linked to FMDV host range. ITAF45 promoted IRES-mediated translation in all cell lines, and the effects of poly-pyrimidine tract binding protein (PTB) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) were observed only in FMDV-susceptible cells. Thus, PTB and 4E-BP1 may influence the host range of FMDV. IRES-mediated translation activity of FMDV was not predictive of its host range. ITAF45 promoted IRES-mediated translation in all cells, and the effects of PTB and 4E-BP1 were observed only in FMDV-susceptible cells.

sST2 translation is regulated by FGF2 via an hnRNP A1-mediated IRES-dependent mechanism.

PubMed

Kunze, Michael M; Benz, Fabienne; Brauß, Thilo F; Lampe, Sebastian; Weigand, Julia E; Braun, Johannes; Richter, Florian M; Wittig, Ilka; Brüne, Bernhard; Schmid, Tobias

2016-07-01

Translation is an energy-intensive process and tightly regulated. Generally, translation is initiated in a cap-dependent manner. Under stress conditions, typically found within the tumor microenvironment in association with e.g. nutrient deprivation or hypoxia, cap-dependent translation decreases, and alternative modes of translation initiation become more important. Specifically, internal ribosome entry sites (IRES) facilitate translation of specific mRNAs under otherwise translation-inhibitory conditions. This mechanism is controlled by IRES trans-acting factors (ITAF), i.e. by RNA-binding proteins, which interact with and determine the activity of selected IRESs. We aimed at characterizing the translational regulation of the IL-33 decoy receptor sST2, which was enhanced by fibroblast growth factor 2 (FGF2). We identified and verified an IRES within the 5'UTR of sST2. Furthermore, we found that MEK/ERK signaling contributes to FGF2-induced, sST2-IRES activation and translation. Determination of the sST2-5'UTR structure by in-line probing followed by deletion analyses identified 23 nucleotides within the sST2-5'UTR to be required for optimal IRES activity. Finally, we show that the RNA-binding protein heterogeneous ribonucleoprotein A1 (hnRNP A1) binds to the sST2-5'UTR, acts as an ITAF, and thus controls the activity of the sST2-IRES and consequently sST2 translation. Specifically, FGF2 enhances nuclear-cytoplasmic translocation of hnRNP A1, which requires intact MEK/ERK activity. In summary, we provide evidence that the sST2-5'UTR contains an IRES element, which is activated by a MEK/ERK-dependent increase in cytoplasmic localization of hnRNP A1 in response to FGF2, enhancing the translation of sST2. Copyright © 2016 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Damian, Luminita, E-mail: luminitadamian@microcal.eu.com; Universite de Toulouse, UPS, IPBS, F-31077 Toulouse; IUB, School of Engineering and Science, D-28727 Bremen

Is single-strand DNA translatable? Since the 60s, the question still remains whether or not DNA could be directly translated into protein. Some discrepancies in the results were reported about functional translation of single-strand DNA but all results converged on a similar behavior of RNA and ssDNA in the initiation step. Isothermal Titration Calorimetry method was used to determine thermodynamic constants of interaction between single-strand DNA and S30 extract of Escherichia coli. Our results showed that the binding was not affected by the nature of the template tested and the dissociation constants were in the same range when ssDNA (K{sub d}more » = 3.62 {+-} 2.1 x 10{sup -8} M) or the RNA corresponding sequence (K{sub d} = 2.7 {+-} 0.82 x 10{sup -8} M) bearing SD/ATG sequences were used. The binding specificity was confirmed by antibiotic interferences which block the initiation complex formation. These results suggest that the limiting step in translation of ssDNA is the elongation process.« less

Recognition of cyclic-di-GMP by a riboswitch conducts translational repression through masking the ribosome-binding site distant from the aptamer domain.

PubMed

Inuzuka, Saki; Kakizawa, Hitoshi; Nishimura, Kei-Ichiro; Naito, Takuto; Miyazaki, Katsushi; Furuta, Hiroyuki; Matsumura, Shigeyoshi; Ikawa, Yoshiya

2018-06-01

The riboswitch is a class of RNA-based gene regulatory machinery that is dependent on recognition of its target ligand by RNA tertiary structures. Ligand recognition is achieved by the aptamer domain, and ligand-dependent structural changes of the expression platform then usually mediate termination of transcription or translational initiation. Ligand-dependent structural changes of the aptamer domain and expression platform have been reported for several riboswitches with short (<40 nucleotides) expression platforms. In this study, we characterized structural changes of the Vc2 c-di-GMP riboswitch that represses translation of downstream open reading frames in a ligand-dependent manner. The Vc2 riboswitch has a long (97 nucleotides) expression platform, but its structure and function are largely unknown. Through mutational analysis and chemical probing, we identified its secondary structures that are possibly responsible for switch-OFF and switch-ON states of translational initiation. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis

PubMed Central

Qi, Lei; Yue, Lei; Feng, Deqin; Qi, Fengxia

2017-01-01

Abstract Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an ‘all-or-none’ pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry. PMID:28520982

Application of β-glucuronidase (GusA) as an effective reporter for extremely acidophilic Acidithiobacillus ferrooxidans.

PubMed

Wang, Huiyan; Fang, Liangyan; Wen, Qing; Lin, Jianqun; Liu, Xiangmei

2017-04-01

Acidithiobacillus ferrooxidans is a model organism for investigating metal sulfide bioleaching. The regulatory mechanism of gene expression by metabolizing various substrates is critical for understanding its role in bioleaching processes. However, no reporter has been successfully employed to study gene expression in A. ferrooxidans to date. In this study, a sensitive and robust reporter system based on β-glucuronidase (GusA) was described for feasible application in A. ferrooxidans. A set of vectors, which contained the transcriptional and translational fusions of gusA, were constructed and employed to analyze promoter activity and efficiency of translation initiation in A. ferrooxidans. Ptac and P2811 were screened out from ten tested promoters and could be used as strong promoters for gene overexpression in A. ferrooxidans. Among the four translational fusions of gusA with different start codons, ATG was most active, followed by TTG and GTG, while CTG showed the least activity. The transcriptional inhibition effect of an IclR-like transcription factor was also observed on its own encoding gene AFE_1668 as well as its neighboring AFE_1667. In addition, the specific chromogenic reaction of GusA could be detected and visualized by colonies of A. ferrooxidans containing gusA expression plasmids. Generally, the established GusA reporter system would be applied not only for quantitative analysis of promoter strength and its transcriptional regulation but also for qualitative colony screening in A. ferrooxidans in the future.

'2A-Like' Signal Sequences Mediating Translational Recoding: A Novel Form of Dual Protein Targeting.

PubMed

Roulston, Claire; Luke, Garry A; de Felipe, Pablo; Ruan, Lin; Cope, Jonathan; Nicholson, John; Sukhodub, Andriy; Tilsner, Jens; Ryan, Martin D

2016-08-01

We report the initial characterization of an N-terminal oligopeptide '2A-like' sequence that is able to function both as a signal sequence and as a translational recoding element. Owing to this translational recoding activity, two forms of nascent polypeptide are synthesized: (i) when 2A-mediated translational recoding has not occurred: the nascent polypeptide is fused to the 2A-like N-terminal signal sequence and the fusion translation product is targeted to the exocytic pathway, and, (ii) a translation product where 2A-mediated translational recoding has occurred: the 2A-like signal sequence is synthesized as a separate translation product and, therefore, the nascent (downstream) polypeptide lacks the 2A-like signal sequence and is localized to the cytoplasm. This type of dual-functional signal sequence results, therefore, in the partitioning of the translation products between the two sub-cellular sites and represents a newly described form of dual protein targeting. © 2016 The Authors. Traffic published by John Wiley & Sons Ltd.

Expanding the Toolkit and Resource Environment to Assist Translation (TREAT) and Its User Base

DTIC Science & Technology

2011-06-01

3 Figure 2. Screenshot of TREAT (translation of Arabic source into English target) and two corresponding markup tool windows on Arabic source...initial framework in place, we decided to expand TREAT to provide support to two new groups of users: students learning to be Arabic -language...translators and teachers training them. The students and the teachers are native English speakers, so the training includes learning how to read Arabic

An efficient, movable single-particle detector for use in cryogenic ultra-high vacuum environments.

PubMed

Spruck, Kaija; Becker, Arno; Fellenberger, Florian; Grieser, Manfred; von Hahn, Robert; Klinkhamer, Vincent; Novotný, Oldřich; Schippers, Stefan; Vogel, Stephen; Wolf, Andreas; Krantz, Claude

2015-02-01

A compact, highly efficient single-particle counting detector for ions of keV/u kinetic energy, movable by a long-stroke mechanical translation stage, has been developed at the Max-Planck-Institut für Kernphysik (Max Planck Institute for Nuclear Physics, MPIK). Both, detector and translation mechanics, can operate at ambient temperatures down to ∼10 K and consist fully of ultra-high vacuum compatible, high-temperature bakeable, and non-magnetic materials. The set-up is designed to meet the technical demands of MPIK's Cryogenic Storage Ring. We present a series of functional tests that demonstrate full suitability for this application and characterise the set-up with regard to its particle detection efficiency.

Editing the human genome: where ART and science intersect.

PubMed

Hershlag, Avner; Bristow, Sara L

2018-06-07

The rapid development of gene-editing technologies has led to an exponential rise in both basic and translational research initiatives studying molecular processes and investigating possible clinical applications. Early experiments using genome editing to study human embryo development have contradicted findings in studies on model organisms. Additionally, a series of four experiments over the past 2 years set out to investigate the possibilities of introducing genetic modifications to human embryos, each with varying levels of success. Here, we discuss the key findings of these studies, including the efficiency, the safety, the potential untoward effects, major flaws of the studies, and emerging alternative genome editing methods that may allow overcoming the hurdles encountered so far. Given these results, we also raise several questions about the clinical utilization of germline gene editing: For which indications is gene editing appropriate? How do gene-editing technologies compare with genetic testing methods currently used for screening embryos? What are the ethical considerations we should be concerned about? While further research is underway, and our understanding of how to implement this technology continues to evolve, it is critical to contemplate if and how it should be translated from the bench to clinical practice.

Dealing with an Unconventional Genetic Code in  Mitochondria: The Biogenesis and Pathogenic  Defects of the 5-Formylcytosine Modification in  Mitochondrial tRNAMet.

PubMed

Van Haute, Lindsey; Powell, Christopher A; Minczuk, Michal

2017-03-02

Human mitochondria contain their own genome, which uses an unconventional genetic code. In addition to the standard AUG methionine codon, the single mitochondrial tRNA Methionine (mt-tRNAMet) also recognises AUA during translation initiation and elongation. Post-transcriptional modifications of tRNAs are important for structure, stability, correct folding and aminoacylation as well as decoding. The unique 5-formylcytosine (f5C) modification of position 34 in mt-tRNAMet has been long postulated to be crucial for decoding of unconventional methionine codons and efficient mitochondrial translation. However, the enzymes responsible for the formation of mitochondrial f5C have been identified only recently. The first step of the f5C pathway consists of methylation of cytosine by NSUN3. This is followed by further oxidation by ABH1. Here, we review the role of f5C, the latest breakthroughs in our understanding of the biogenesis of this unique mitochondrial tRNA modification and its involvement in human disease.

Cotranslational Coat Protein-Mediated Inhibition of Potyviral RNA Translation

PubMed Central

Besong-Ndika, Jane; Ivanov, Konstantin I.; Hafrèn, Anders; Michon, Thierry

2015-01-01

ABSTRACT Potato virus A (PVA) is a single-stranded positive-sense RNA virus and a member of the family Potyviridae. The PVA coat protein (CP) has an intrinsic capacity to self-assemble into filamentous virus-like particles, but the mechanism responsible for the initiation of viral RNA encapsidation in vivo remains unclear. Apart from virion assembly, PVA CP is also involved in the inhibition of viral RNA translation. In this study, we show that CP inhibits PVA RNA translation in a dose-dependent manner, through a mechanism involving the CP-encoding region. Analysis of this region, however, failed to identify any RNA secondary structure(s) preferentially recognized by CP, suggesting that the inhibition depends on CP-CP rather than CP-RNA interactions. In agreement with this possibility, insertion of an in-frame stop codon upstream of the CP sequence led to a marked decrease in the inhibition of viral RNA translation. Based on these results, we propose a model in which the cotranslational interactions between excess CP accumulating in trans and CP translated from viral RNA in cis are required to initiate the translational repression. This model suggests a mechanism for how viral RNA can be sequestered from translation and specifically selected for encapsidation at the late stages of viral infection. IMPORTANCE The main functions of the CP during potyvirus infection are to protect viral RNA from degradation and to transport it locally, systemically, and from host to host. Although virion assembly is a key step in the potyviral infectious cycle, little is known about how it is initiated and how viral RNA is selected for encapsidation. The results presented here suggest that CP-CP rather than CP-RNA interactions are predominantly involved in the sequestration of viral RNA away from translation. We propose that the cotranslational nature of these interactions may represent a mechanism for the selection of viral RNA for encapsidation. A better understanding of the mechanism of virion assembly may lead to development of crops resistant to potyviruses at the level of viral RNA encapsidation, thereby reducing the detrimental effects of potyvirus infections on food production. PMID:25631087

Harmonization in preclinical epilepsy research: A joint AES/ILAE translational initiative.

PubMed

Galanopoulou, Aristea S; French, Jacqueline A; O'Brien, Terence; Simonato, Michele

2017-11-01

Among the priority next steps outlined during the first translational epilepsy research workshop in London, United Kingdom (2012), jointly organized by the American Epilepsy Society (AES) and the International League Against Epilepsy (ILAE), are the harmonization of research practices used in preclinical studies and the development of infrastructure that facilitates multicenter preclinical studies. The AES/ILAE Translational Task Force of the ILAE has been pursuing initiatives that advance these goals. In this supplement, we present the first reports of the working groups of the Task Force that aim to improve practices of performing rodent video-electroencephalography (vEEG) studies in experimental controls, generate systematic reviews of preclinical research data, and develop preclinical common data elements (CDEs) for epilepsy research in animals. Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.

uORFs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs

PubMed Central

Ivanov, Ivaylo P.; Loughran, Gary; Atkins, John F.

2008-01-01

In a minority of eukaryotic mRNAs, a small functional upstream ORF (uORF), often performing a regulatory role, precedes the translation start site for the main product(s). Here, conserved uORFs in numerous ornithine decarboxylase homologs are identified from yeast to mammals. Most have noncanonical evolutionarily conserved start codons, the main one being AUU, which has not been known as an initiator for eukaryotic chromosomal genes. The AUG-less uORF present in mouse antizyme inhibitor, one of the ornithine decarboxylase homologs in mammals, mediates polyamine-induced repression of the downstream main ORF. This repression is part of an autoregulatory circuit, and one of its sensors is the AUU codon, which suggests that translation initiation codon identity is likely used for regulation in eukaryotes. PMID:18626014

76 FR 66932 - The National Cancer Institute (NCI) Announces the Initiation of a Public Private Industry...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-28

...The Alliance for Nanotechnology in Cancer of the National Cancer Institute (NCI) is initiating a public private industry partnership called TONIC (Translation Of Nanotechnology In Cancer) to promote translational research and development opportunities of nanotechnology-based cancer solutions. An immediate consequence of this effort will be the formation of a consortium involving government and pharmaceutical, and biotechnology companies. This consortium will evaluate promising nanotechnology platforms and facilitate their successful translation from academic research to clinical environment, resulting in safe, timely, effective and novel diagnosis and treatment options for cancer patients. The purpose of this notice is to inform the community about the Alliance for Nanotechnology in Cancer of NCI's intention to form the consortium and to invite eligible companies (as defined in last paragraph) to participate.

Emerging Therapeutics Targeting mRNA Translation

PubMed Central

Malina, Abba; Mills, John R.; Pelletier, Jerry

2012-01-01

A defining feature of many cancers is deregulated translational control. Typically, this occurs at the level of recruitment of the 40S ribosomes to the 5′-cap of cellular messenger RNAs (mRNAs), the rate-limiting step of protein synthesis, which is controlled by the heterotrimeric eukaryotic initiation complex eIF4F. Thus, eIF4F in particular, and translation initiation in general, represent an exploitable vulnerability and unique opportunity for therapeutic intervention in many transformed cells. In this article, we discuss the development, mode of action and biological activity of a number of small-molecule inhibitors that interrupt PI3K/mTOR signaling control of eIF4F assembly, as well as compounds that more directly block eIF4F activity. PMID:22474009

Partnerships for knowledge translation and exchange in the context of continuing professional development.

PubMed

Légaré, France; Borduas, Francine; MacLeod, Tanya; Sketris, Ingrid; Campbell, Barbara; Jacques, André

2011-01-01

Continuing professional development (CPD) is an important vehicle for knowledge translation (KT); however, selecting CPD strategies that will impact health professionals' behavior and improve patient outcomes is complex. In response, we, KT researchers and CPD knowledge users, have recently formed a partnership known as the National Network for Patient-Centered Evidence-Based Continuing Professional Development. The partnership was initiated in 2006 with a series of CIHR Knowledge Translation: Planning, Meetings and Dissemination grants. The objectives of these grants were to bring members of the CPD and KT communities together, determine their interest in working together, identify similarities and differences in the fields of CPD and KT, and develop working groups to inform larger collaborative initiatives to support knowledge translation and exchange. The vision for this partnership is to become a premiere knowledge translation collaboration and a cutting-edge implementation network that informs the provision of CPD across Canada and abroad. This paper reports on the development and outcomes of this network to date. Copyright © 2010 The Alliance for Continuing Medical Education, the Society for Academic Continuing Medical Education, and the Council on CME, Association for Hospital Medical Education.

The eukaryotic translation initiation factor 3 subunit E binds to classical swine fever virus NS5A and facilitates viral replication.

PubMed

Liu, Xiaofeng; Wang, Xiaoyu; Wang, Qian; Luo, Mingyang; Guo, Huancheng; Gong, Wenjie; Tu, Changchun; Sun, Jinfu

2018-02-01

Classical swine fever virus (CSFV) NS5A protein is a multifunctional protein, playing critical roles in viral RNA replication, translation and assembly. To further explore its functions in viral replication, interaction of NS5A with host factors was assayed using a his-tag "pull down" assay coupled with shotgun LC-MS/MS. Host protein translation initiation factor 3 subunit E was identified as a binding partner of NS5A, and confirmed by co-immunoprecipitation and co-localization analysis. Overexpression of eIF3E markedly enhanced CSFV genomic replication, viral protein expression and production of progeny virus, and downregulation of eIF3E by siRNA significantly decreased viral proliferation in PK-15 cells. Luciferase reporter assay showed an enhancement of translational activity of the internal ribosome entry site of CSFV by eIF3E and a decrease in cellular translation by NS5A. These data indicate that eIF3E plays an important role in CSFV replication, thereby identifying it as a potential target for inhibition of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

Phosphoproteomics reveals the effect of ethylene in soybean root under flooding stress.

PubMed

Yin, Xiaojian; Sakata, Katsumi; Komatsu, Setsuko

2014-12-05

Flooding has severe negative effects on soybean growth. To explore the flooding-responsive mechanisms in early-stage soybean, a phosphoproteomic approach was used. Two-day-old soybean plants were treated without or with flooding for 3, 6, 12, and 24 h, and root tip proteins were then extracted and analyzed at each time point. After 3 h of flooding exposure, the fresh weight of soybeans increased, whereas the ATP content of soybean root tips decreased. Using a gel-free proteomic technique, a total of 114 phosphoproteins were identified in the root tip samples, and 34 of the phosphoproteins were significantly changed with respect to phosphorylation status after 3 h of flooding stress. Among these phosphoproteins, eukaryotic translation initiation factors were dephosphorylated, whereas several protein synthesis-related proteins were phosphorylated. The mRNA expression levels of sucrose phosphate synthase 1F and eukaryotic translation initiation factor 4 G were down-regulated, whereas UDP-glucose 6-dehydrogenase mRNA expression was up-regulated during growth but down-regulated under flooding stress. Furthermore, bioinformatic protein interaction analysis of flooding-responsive proteins based on temporal phosphorylation patterns indicated that eukaryotic translation initiation factor 4 G was located in the center of the network during flooding. Soybean eukaryotic translation initiation factor 4 G has homology to programmed cell death 4 protein and is implicated in ethylene signaling. The weight of soybeans was increased with treatment by an ethylene-releasing agent under flooding condition, but it was decreased when plants were exposed to an ethylene receptor antagonist. These results suggest that the ethylene signaling pathway plays an important role, via the protein phosphorylation, in mechanisms of plant tolerance to the initial stages of flooding stress in soybean root tips.

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

PubMed Central

Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann

2017-01-01

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5′ terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. PMID:28338950

The structure of clinical translation: efficiency, information, and ethics.

PubMed

Kimmelman, Jonathan; London, Alex John

2015-01-01

The so-called drug pipeline is not really about drugs and not much like a pipeline. It is really about the production and dissemination of information, and it is much more like a web. The misunderstanding leads to a poor understanding of what's wrong with clinical translation and how it can be improved.

Beyond Theory and Practice: Towards an Ethics of Translation

ERIC Educational Resources Information Center

Schwimmer, Marina

2017-01-01

In this article, I will discuss the idea of teachers as knowledge translators, not in a pedagogical or didactical sense, but in a "professional" one. A professional practice is supposed to be theoretically informed by academic research. In the name of effectiveness and efficiency, current policies in teaching and higher education…

The chloroplast signal recognition particle (CpSRP) pathway as a tool to minimize chlorophyll antenna size and maximize photosynthetic productivity.

PubMed

Kirst, Henning; Melis, Anastasios

2014-01-01

The concept of the Truncated Light-harvesting chlorophyll Antenna (TLA) size, as a tool by which to maximize sunlight utilization and photosynthetic productivity in microalgal mass cultures or high-density plant canopies, is discussed. TLA technology is known to improve sunlight-to-product energy conversion efficiencies and is hereby exemplified by photosynthetic productivity estimates of wild type and a TLA strain under simulated mass culture conditions. Recent advances in the generation of TLA-type mutants by targeting genes of the chloroplast signal-recognition particle (CpSRP) pathway, affecting the thylakoid membrane assembly of light-harvesting proteins, are also summarized. Two distinct CpSRP assembly pathways are recognized, one entailing post-translational, the other a co-translational mechanism. Differences between the post-translational and co-translational integration mechanisms are outlined, as these pertain to the CpSRP-mediated assembly of thylakoid membrane protein complexes in higher plants and green microalgae. The applicability of the CpSRP pathway genes in efforts to generate TLA-type strains with enhanced solar energy conversion efficiency in photosynthesis is evaluated. © 2013.

Fully Flexible Docking of Medium Sized Ligand Libraries with RosettaLigand

PubMed Central

DeLuca, Samuel; Khar, Karen; Meiler, Jens

2015-01-01

RosettaLigand has been successfully used to predict binding poses in protein-small molecule complexes. However, the RosettaLigand docking protocol is comparatively slow in identifying an initial starting pose for the small molecule (ligand) making it unfeasible for use in virtual High Throughput Screening (vHTS). To overcome this limitation, we developed a new sampling approach for placing the ligand in the protein binding site during the initial ‘low-resolution’ docking step. It combines the translational and rotational adjustments to the ligand pose in a single transformation step. The new algorithm is both more accurate and more time-efficient. The docking success rate is improved by 10–15% in a benchmark set of 43 protein/ligand complexes, reducing the number of models that typically need to be generated from 1000 to 150. The average time to generate a model is reduced from 50 seconds to 10 seconds. As a result we observe an effective 30-fold speed increase, making RosettaLigand appropriate for docking medium sized ligand libraries. We demonstrate that this improved initial placement of the ligand is critical for successful prediction of an accurate binding position in the ‘high-resolution’ full atom refinement step. PMID:26207742

Translation and adaptation of the Competencias Esenciales en Salud Pública para los recursos humanos en salud 1

PubMed Central

de Almeida, Maria de Lourdes; Peres, Aida Maris; Ferreira, Maria Manuela Frederico; Mantovani, Maria de Fátima

2017-01-01

ABSTRACT Objective: to perform the translation and cultural adaptation of the document named Marco Regional de Competencias Esenciales en Salud Pública para los Recursos Humanos en Salud de la Región de las Américas (Regional Framework of Core Competencies in Public Health for Health Human Resources in the Region of Americas) from Spanish to Brazilian Portuguese. Method: a methodological study comprising the following phases: authorization for translation; initial translation; synthesis of translations and consensus; back-translation and formation of an expert committee. Result: in the translation of domain names, there was no difference in 66.7% (N = 4); in the translation of domain description and competencies there were divergences in 100% of them (N = 6, N = 56). A consensus of more than 80% was obtained in the translation and improvement in the expert committee by the change of words and expressions for approximation of meanings to the Brazilian context. Conclusion: the translated and adapted document has the potential of application in research, and use in the practice of collective/public health care in Brazil. PMID:28591302

The Innovative Medicines Initiative moves translational immunology forward.

PubMed

Goldman, Michel; Wittelsberger, Angela; De Magistris, Maria-Teresa

2013-02-01

The Innovative Medicines Initiative (IMI) was established in 2008 as a public-private partnership between the European Union and the European Federation of Pharmaceutical Industries and Associations with the mission to promote the development of novel therapies through collaborative efforts based on the concept of pre-competitive research. Several consortia supported by IMI are dedicated to immuno-inflammatory disorders, immune-based biopharmaceuticals and vaccines. Herein, we present the key principles underlying IMI, briefly review the status of projects related to translational immunology, and present future topics of interest to immunologists.

Efficient production and purification of functional bacteriorhodopsin with a wheat-germ cell-free system and a combination of Fos-choline and CHAPS detergents.

PubMed

Genji, Takahisa; Nozawa, Akira; Tozawa, Yuzuru

2010-10-01

Cell-free translation is one potential approach to the production of functional transmembrane proteins. We have now examined various detergents as supplements to a wheat-germ cell-free system in order to optimize the production and subsequent purification of a functional model transmembrane protein, bacteriorhodopsin. We found that Fos-choline and CHAPS detergents counteracted each other's inhibitory effects on cell-free translation activity and thereby allowed the efficient production and subsequent purification of functional bacteriorhodopsin in high yield. Copyright © 2010 Elsevier Inc. All rights reserved.

NIPTE: a multi-university partnership supporting academic drug development.

PubMed

Gurvich, Vadim J; Byrn, Stephen R

2013-10-01

The strategic goal of academic translational research is to accelerate translational science through the improvement and development of resources for moving discoveries across translational barriers through 'first in humans' studies. To achieve this goal, access to drug discovery resources and preclinical IND-enabling infrastructure is crucial. One potential approach of research institutions for coordinating preclinical development, based on a model from the National Institute for Pharmaceutical Technology and Education (NIPTE), can provide academic translational and medical centers with access to a wide variety of enabling infrastructure for developing small molecule clinical candidates in an efficient, cost-effective manner. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mechanism of Cytoplasmic mRNA Translation

PubMed Central

2015-01-01

Protein synthesis is a fundamental process in gene expression that depends upon the abundance and accessibility of the mRNA transcript as well as the activity of many protein and RNA-protein complexes. Here we focus on the intricate mechanics of mRNA translation in the cytoplasm of higher plants. This chapter includes an inventory of the plant translational apparatus and a detailed review of the translational processes of initiation, elongation, and termination. The majority of mechanistic studies of cytoplasmic translation have been carried out in yeast and mammalian systems. The factors and mechanisms of translation are for the most part conserved across eukaryotes; however, some distinctions are known to exist in plants. A comprehensive understanding of the complex translational apparatus and its regulation in plants is warranted, as the modulation of protein production is critical to development, environmental plasticity and biomass yield in diverse ecosystems and agricultural settings. PMID:26019692

Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells

PubMed Central

Lee, Jinwoo; Tong, Tiegang; Duan, Haichuan; Foong, Yee Hoon; Musaitif, Ibrahim; Yamazaki, Takeshi; Jefcoate, Colin

2016-01-01

The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb StAR mRNA molecules via dual hybridization at the 3′- and 5′-ends and reveals an unexpectedly high frequency of 1:1 pairing with mitochondria marked by the matrix StAR protein. This pairing may be central to translation-coupled cholesterol transfer. Altogether, our results show that adrenal cells exhibit high-efficiency StAR activity that needs to integrate rapid cholesterol transfer with homeostasis and pulsatile hormonal stimulation. StAR NBD, the extended 3.5-kb mRNA, SIK1, and Tis11b play important roles. PMID:27531991

Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells.

PubMed

Lee, Jinwoo; Tong, Tiegang; Duan, Haichuan; Foong, Yee Hoon; Musaitif, Ibrahim; Yamazaki, Takeshi; Jefcoate, Colin

2016-01-01

The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb StAR mRNA molecules via dual hybridization at the 3'- and 5'-ends and reveals an unexpectedly high frequency of 1:1 pairing with mitochondria marked by the matrix StAR protein. This pairing may be central to translation-coupled cholesterol transfer. Altogether, our results show that adrenal cells exhibit high-efficiency StAR activity that needs to integrate rapid cholesterol transfer with homeostasis and pulsatile hormonal stimulation. StAR NBD, the extended 3.5-kb mRNA, SIK1, and Tis11b play important roles.

75 FR 81611 - Submission for OMB Review; Comment Request; National Evaluation of the Clinical and Translational...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-28

... Patricia Newman, Program Analyst, Office of Science Policy, National Center for Research Resources, 6701...: December 20, 2010. Meryl Sufian, Supervisory Health Science Policy Analyst, Office of Science Policy, NCRR... Evaluation of the Clinical and Translational Science Awards (CTSA) Initiative SUMMARY: Under the provisions...

AtLa1 protein initiates IRES-dependent translation of WUSCHEL mRNA and regulates the stem cell homeostasis of Arabidopsis in response to environmental hazards.

PubMed

Cui, Yuchao; Rao, Shaofei; Chang, Beibei; Wang, Xiaoshuang; Zhang, Kaidian; Hou, Xueliang; Zhu, Xueyi; Wu, Haijun; Tian, Zhaoxia; Zhao, Zhong; Yang, Chengwei; Huang, Tao

2015-10-01

Plant stem cells are hypersensitive to environmental hazards throughout their life cycle, but the mechanism by which plants safeguard stem cell homeostasis in response to environmental hazards is largely unknown. The homeodomain transcription factor WUSCHEL (WUS) protein maintains the stem cell pool in the shoot apical meristem of Arabidopsis. Here, we demonstrate that the translation of WUS mRNA is directed by an internal ribosomal entry site (IRES) located in the 5'-untranslated region. The AtLa1 protein, an RNA-binding factor, binds to the 5'-untranslated region and initiates the IRES-dependent translation of WUS mRNA. Knockdown of AtLa1 expression represses the WUS IRES-dependent translation and leads to the arrest of growth and development. The AtLa1 protein is mainly located in the nucleoplasm. However, environmental hazards promote the nuclear-to-cytoplasmic translocation of the AtLa1 protein, which further enhances the IRES-dependent translation of WUS mRNA. Genetic evidence indicates that the WUS protein increases the tolerance of the shoot apical meristem to environmental hazards. Based on these results, we conclude that the stem cell niche in Arabidopsis copes with environmental hazards by enhancing the IRES-dependent translation of WUS mRNA under the control of the AtLa1 protein. © 2015 John Wiley & Sons Ltd.

The Frustrated Host Response to Legionella pneumophila Is Bypassed by MyD88-Dependent Translation of Pro-inflammatory Cytokines

PubMed Central

Asrat, Seblewongel; Dugan, Aisling S.; Isberg, Ralph R.

2014-01-01

Many pathogens, particularly those that require their host for survival, have devised mechanisms to subvert the host immune response in order to survive and replicate intracellularly. Legionella pneumophila, the causative agent of Legionnaires' disease, promotes intracellular growth by translocating proteins into its host cytosol through its type IV protein secretion machinery. At least 5 of the bacterial translocated effectors interfere with the function of host cell elongation factors, blocking translation and causing the induction of a unique host cell transcriptional profile. In addition, L. pneumophila also interferes with translation initiation, by preventing cap-dependent translation in host cells. We demonstrate here that protein translation inhibition by L. pneumophila leads to a frustrated host MAP kinase response, where genes involved in the pathway are transcribed but fail to be translated due to the bacterium-induced protein synthesis inhibition. Surprisingly, few pro-inflammatory cytokines, such as IL-1α and IL-1β, bypass this inhibition and get synthesized in the presence of Legionella effectors. We show that the selective synthesis of these genes requires MyD88 signaling and takes place in both infected cells that harbor bacteria and neighboring bystander cells. Our findings offer a perspective of how host cells are able to cope with pathogen-encoded activities that disrupt normal cellular process and initiate a successful inflammatory response. PMID:25058342

Correlation between Ribosome Biogenesis and the Magnitude of Hypertrophy in Overloaded Skeletal Muscle.

PubMed

Nakada, Satoshi; Ogasawara, Riki; Kawada, Shigeo; Maekawa, Takahiro; Ishii, Naokata

2016-01-01

External loads applied to skeletal muscle cause increases in the protein translation rate, which leads to muscle hypertrophy. Although some studies have demonstrated that increases in the capacity and efficiency of translation are involved in this process, it remains unclear how these two factors are related to the magnitude of muscle hypertrophy. The present study aimed to clarify the roles played by the capacity and efficiency of translation in muscle hypertrophy. We used an improved synergist ablation in which the magnitude of compensatory hypertrophy could be controlled by partial removal of synergist muscles. Male rats were assigned to four groups in which the plantaris muscle was unilaterally subjected to weak (WK), moderate (MO), middle (MI), and strong (ST) overloading by four types of synergist ablation. Fourteen days after surgery, the weight of the plantaris muscle per body weight increased by 8%, 22%, 32% and 45%, in the WK, MO, MI and ST groups, respectively. Five days after surgery, 18+28S rRNA content (an indicator of translational capacity) increased with increasing overload, with increases of 1.8-fold (MO), 2.2-fold (MI), and 2.5-fold (ST), respectively, relative to non-overloaded muscle (NL) in the WK group. rRNA content showed a strong correlation with relative muscle weight measured 14 days after surgery (r = 0.98). The phosphorylated form of p70S6K (a positive regulator of translational efficiency) showed a marked increase in the MO group, but no further increase was observed with further increase in overload (increases of 22.6-fold (MO), 17.4-fold (MI), and 18.2-fold (ST), respectively, relative to NL in the WK group). These results indicate that increases in ribosome biogenesis at the early phase of overloading are strongly dependent on the amount of overloading, and may play an important role in increasing the translational capacity for further gain of muscular size.

Prolonged Exposure of Primary Human Muscle Cells to Plasma Fatty Acids Associated with Obese Phenotype Induces Persistent Suppression of Muscle Mitochondrial ATP Synthase β Subunit

PubMed Central

Tran, Lee; Hanavan, Paul D.; Campbell, Latoya E.; De Filippis, Elena; Lake, Douglas F.; Coletta, Dawn K.; Roust, Lori R.; Mandarino, Lawrence J.; Carroll, Chad C.; Katsanos, Christos S.

2016-01-01

Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of β-F1-ATPase in obese (BMI, 36±1 kg/m2) and lean (BMI, 22±1 kg/m2) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h-1) and translation efficiency of β-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P<0.05). We then evaluated these same responses in a primary cell culture model, and tested the specific hypothesis that circulating non-esterified fatty acids (NEFA) in obesity play a role in the responses observed in humans. The findings on total protein synthesis and translation efficiency of β-F1-ATPase in primary myotubes cultured from a lean subject, and after exposure to NEFA extracted from serum of an obese subject, were similar to those obtained in humans. Among candidate microRNAs (i.e., non-coding RNAs regulating gene expression), we identified miR-127-5p in preventing the production of β-F1-ATPase. Muscle expression of miR-127-5p negatively correlated with β-F1-ATPase protein translation efficiency in humans (r = – 0.6744; P<0.01), and could be modeled in vitro by prolonged exposure of primary myotubes derived from the lean subject to NEFA extracted from the obese subject. On the other hand, locked nucleic acid inhibitor synthesized to target miR-127-5p significantly increased β-F1-ATPase translation efficiency in myotubes (0.6±0.1 vs 1.3±0.3, in control vs exposure to 50 nM inhibitor; P<0.05). Our experiments implicate circulating NEFA in obesity in suppressing muscle protein metabolism, and establish impaired β-F1-ATPase translation as an important consequence of obesity. PMID:27532680

Molecular Mechanism of Signal Perception and Integration by the Innate Immune Sensor Retinoic Acid-inducible Gene-I (RIG-I)*

PubMed Central

Binder, Marco; Eberle, Florian; Seitz, Stefan; Mücke, Norbert; Hüber, Christian M.; Kiani, Narsis; Kaderali, Lars; Lohmann, Volker; Dalpke, Alexander; Bartenschlager, Ralf

2011-01-01

RIG-I is a major innate immune sensor for viral infection, triggering an interferon (IFN)-mediated antiviral response upon cytosolic detection of viral RNA. Double-strandedness and 5′-terminal triphosphates were identified as motifs required to elicit optimal immunological signaling. However, very little is known about the response dynamics of the RIG-I pathway, which is crucial for the ability of the cell to react to diverse classes of viral RNA while maintaining self-tolerance. In the present study, we addressed the molecular mechanism of RIG-I signal detection and its translation into pathway activation. By employing highly quantitative methods, we could establish the length of the double-stranded RNA (dsRNA) to be the most critical determinant of response strength. Size exclusion chromatography and direct visualization in scanning force microscopy suggested that this was due to cooperative oligomerization of RIG-I along dsRNA. The initiation efficiency of this oligomerization process critically depended on the presence of high affinity motifs, like a 5′-triphosphate. It is noteworthy that for dsRNA longer than 200 bp, internal initiation could effectively compensate for a lack of terminal triphosphates. In summary, our data demonstrate a very flexible response behavior of the RIG-I pathway, in which sensing and integration of at least two distinct signals, initiation efficiency and double strand length, allow the host cell to mount an antiviral response that is tightly adjusted to the type of the detected signal, such as viral genomes, replication intermediates, or small by-products. PMID:21659521

A Novel Method for Gene-Specific Enhancement of Protein Translation by Targeting 5’UTRs of Selected Tumor Suppressors

PubMed Central

Master, Adam; Wójcicka, Anna; Giżewska, Kamilla; Popławski, Piotr; Williams, Graham R.; Nauman, Alicja

2016-01-01

Background Translational control is a mechanism of protein synthesis regulation emerging as an important target for new therapeutics. Naturally occurring microRNAs and synthetic small inhibitory RNAs (siRNAs) are the most recognized regulatory molecules acting via RNA interference. Surprisingly, recent studies have shown that interfering RNAs may also activate gene transcription via the newly discovered phenomenon of small RNA-induced gene activation (RNAa). Thus far, the small activating RNAs (saRNAs) have only been demonstrated as promoter-specific transcriptional activators. Findings We demonstrate that oligonucleotide-based trans-acting factors can also specifically enhance gene expression at the level of protein translation by acting at sequence-specific targets within the messenger RNA 5’-untranslated region (5’UTR). We designed a set of short synthetic oligonucleotides (dGoligos), specifically targeting alternatively spliced 5’UTRs in transcripts expressed from the THRB and CDKN2A suppressor genes. The in vitro translation efficiency of reporter constructs containing alternative TRβ1 5’UTRs was increased by up to more than 55-fold following exposure to specific dGoligos. Moreover, we found that the most folded 5’UTR has higher translational regulatory potential when compared to the weakly folded TRβ1 variant. This suggests such a strategy may be especially applied to enhance translation from relatively inactive transcripts containing long 5’UTRs of complex structure. Significance This report represents the first method for gene-specific translation enhancement using selective trans-acting factors designed to target specific 5’UTR cis-acting elements. This simple strategy may be developed further to complement other available methods for gene expression regulation including gene silencing. The dGoligo-mediated translation-enhancing approach has the potential to be transferred to increase the translation efficiency of any suitable target gene and may have future application in gene therapy strategies to enhance expression of proteins including tumor suppressors. PMID:27171412

Global analysis of translation termination in E. coli

PubMed Central

Baggett, Natalie E.

2017-01-01

Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 variant of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins. PMID:28301469

High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling

PubMed Central

Jones, Joshua D.; Chung, Betty Y.-W.; Siddell, Stuart G.; Brierley, Ian

2016-01-01

Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus. PMID:26919232

Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation

PubMed Central

2010-01-01

Over 50 years of biological research with bacteriophage T4 includes notable discoveries in post-transcriptional control, including the genetic code, mRNA, and tRNA; the very foundations of molecular biology. In this review we compile the past 10 - 15 year literature on RNA-protein interactions with T4 and some of its related phages, with particular focus on advances in mRNA decay and processing, and on translational repression. Binding of T4 proteins RegB, RegA, gp32 and gp43 to their cognate target RNAs has been characterized. For several of these, further study is needed for an atomic-level perspective, where resolved structures of RNA-protein complexes are awaiting investigation. Other features of post-transcriptional control are also summarized. These include: RNA structure at translation initiation regions that either inhibit or promote translation initiation; programmed translational bypassing, where T4 orchestrates ribosome bypass of a 50 nucleotide mRNA sequence; phage exclusion systems that involve T4-mediated activation of a latent endoribonuclease (PrrC) and cofactor-assisted activation of EF-Tu proteolysis (Gol-Lit); and potentially important findings on ADP-ribosylation (by Alt and Mod enzymes) of ribosome-associated proteins that might broadly impact protein synthesis in the infected cell. Many of these problems can continue to be addressed with T4, whereas the growing database of T4-related phage genome sequences provides new resources and potentially new phage-host systems to extend the work into a broader biological, evolutionary context. PMID:21129205

Applying Process Improvement Methods to Clinical and Translational Research: Conceptual Framework and Case Examples

PubMed Central

Selker, Harry P.; Leslie, Laurel K.

2015-01-01

Abstract There is growing appreciation that process improvement holds promise for improving quality and efficiency across the translational research continuum but frameworks for such programs are not often described. The purpose of this paper is to present a framework and case examples of a Research Process Improvement Program implemented at Tufts CTSI. To promote research process improvement, we developed online training seminars, workshops, and in‐person consultation models to describe core process improvement principles and methods, demonstrate the use of improvement tools, and illustrate the application of these methods in case examples. We implemented these methods, as well as relational coordination theory, with junior researchers, pilot funding awardees, our CTRC, and CTSI resource and service providers. The program focuses on capacity building to address common process problems and quality gaps that threaten the efficient, timely and successful completion of clinical and translational studies. PMID:26332869

La-related Protein 1 (LARP1) Represses Terminal Oligopyrimidine (TOP) mRNA Translation Downstream of mTOR Complex 1 (mTORC1).

PubMed

Fonseca, Bruno D; Zakaria, Chadi; Jia, Jian-Jun; Graber, Tyson E; Svitkin, Yuri; Tahmasebi, Soroush; Healy, Danielle; Hoang, Huy-Dung; Jensen, Jacob M; Diao, Ilo T; Lussier, Alexandre; Dajadian, Christopher; Padmanabhan, Niranjan; Wang, Walter; Matta-Camacho, Edna; Hearnden, Jaclyn; Smith, Ewan M; Tsukumo, Yoshinori; Yanagiya, Akiko; Morita, Masahiro; Petroulakis, Emmanuel; González, Jose L; Hernández, Greco; Alain, Tommy; Damgaard, Christian K

2015-06-26

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5'TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

La-related Protein 1 (LARP1) Represses Terminal Oligopyrimidine (TOP) mRNA Translation Downstream of mTOR Complex 1 (mTORC1)*

PubMed Central

Fonseca, Bruno D.; Zakaria, Chadi; Jia, Jian-Jun; Graber, Tyson E.; Svitkin, Yuri; Tahmasebi, Soroush; Healy, Danielle; Hoang, Huy-Dung; Jensen, Jacob M.; Diao, Ilo T.; Lussier, Alexandre; Dajadian, Christopher; Padmanabhan, Niranjan; Wang, Walter; Matta-Camacho, Edna; Hearnden, Jaclyn; Smith, Ewan M.; Tsukumo, Yoshinori; Yanagiya, Akiko; Morita, Masahiro; Petroulakis, Emmanuel; González, Jose L.; Hernández, Greco; Alain, Tommy; Damgaard, Christian K.

2015-01-01

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5′TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1. PMID:25940091

Digital voice recording: An efficient alternative for data collection

Treesearch

Mark A. Rumble; Thomas M. Juntti; Thomas W. Bonnot; Joshua J. Millspaugh

2009-01-01

Study designs are usually constrained by logistical and budgetary considerations that can affect the depth and breadth of the research. Little attention has been paid to increasing the efficiency of data recording. Digital voice recording and translation may offer improved efficiency of field personnel. Using this technology, we increased our data collection by 55...

Restoration of chemosensitivity in cancer cells with MDR phenotype by deoxyribozyme, compared with ribozyme.

PubMed

Xing, Ai-Yan; Shi, Duan-bo; Liu, Wei; Chen, Xu; Sun, Yan-Lin; Wang, Xiao; Zhang, Jian-ping; Gao, Peng

2013-06-01

One of the main mechanisms for multidrug resistance (MDR) involves multidrug resistance gene 1 (MDR1) which encodes P-glycoprotein (Pgp). Pgp acts as a drug efflux pump and exports chemotherapeutic agents from cancer cells. Specific inhibition of Pgp expression by gene therapy is considered a well-respective strategy having less innate toxicities. At present, the investigation of DRz in reversal MDR is scarce. In the study, phosphorothioate DRz that targets to the translation initiation codon AUG was synthesized and transfected into breast cancer cells and leukemia cells with MDR phenotype. ASODN (antisense oligonucleotide) and ribozyme targets to the same region were also synthesized for comparison analysis. Alterations in MDR1 mRNA and Pgp were determined by RT-PCR, Northern blot, flow cytometry and Rh123 retention tests. Chemosensitivity of the treated cells was determined by MTT assay. The results showed that DRz could significantly suppress expression of MDR1 mRNA and inhibit synthesis of Pgp. The efflux activity of Pgp was inhibited accordingly. Chemosensitivity assay showed that a 21-fold reduction in drug resistance for Adriamycin and a 45-fold reduction in drug resistance for Vinblastine were found in the treated cells 36h after transfection. These data suggest that DRz targeted to the translation initiation codon AUG can reverse MDR phenotype in cancer cells and restore their chemosensitivity. Moreover, the reversal efficiency of DRz is better than that of ribozyme and ASODN targets to the same region of MDR1 mRNA. Copyright © 2013 Elsevier Inc. All rights reserved.

A knowledge infrastructure for occupational safety and health.

PubMed

van Dijk, Frank J H; Verbeek, Jos H; Hoving, Jan L; Hulshof, Carel T J

2010-12-01

Occupational Safety and Health (OSH) professionals should use scientific evidence to support their decisions in policy and practice. Although examples from practice show that progress has been made in evidence-based decision making, there is a challenge to improve and extend the facilities that support knowledge translation in practice. A knowledge infrastructure that supports OSH practice should include scientific research, systematic reviews, practice guidelines, and other tools for professionals such as well accessible virtual libraries and databases providing knowledge, quality tools, and good learning materials. A good infrastructure connects facilities with each other and with practice. Training and education is needed for OSH professionals in the use of evidence to improve effectiveness and efficiency. New initiatives show that occupational health can profit from intensified international collaboration to establish a good functioning knowledge infrastructure.

Translational plant proteomics: a perspective.

PubMed

Agrawal, Ganesh Kumar; Pedreschi, Romina; Barkla, Bronwyn J; Bindschedler, Laurence Veronique; Cramer, Rainer; Sarkar, Abhijit; Renaut, Jenny; Job, Dominique; Rakwal, Randeep

2012-08-03

Translational proteomics is an emerging sub-discipline of the proteomics field in the biological sciences. Translational plant proteomics aims to integrate knowledge from basic sciences to translate it into field applications to solve issues related but not limited to the recreational and economic values of plants, food security and safety, and energy sustainability. In this review, we highlight the substantial progress reached in plant proteomics during the past decade which has paved the way for translational plant proteomics. Increasing proteomics knowledge in plants is not limited to model and non-model plants, proteogenomics, crop improvement, and food analysis, safety, and nutrition but to many more potential applications. Given the wealth of information generated and to some extent applied, there is the need for more efficient and broader channels to freely disseminate the information to the scientific community. This article is part of a Special Issue entitled: Translational Proteomics. Copyright © 2012 Elsevier B.V. All rights reserved.

Computing the Baker-Campbell-Hausdorff series and the Zassenhaus product

NASA Astrophysics Data System (ADS)

Weyrauch, Michael; Scholz, Daniel

2009-09-01

The Baker-Campbell-Hausdorff (BCH) series and the Zassenhaus product are of fundamental importance for the theory of Lie groups and their applications in physics and physical chemistry. Standard methods for the explicit construction of the BCH and Zassenhaus terms yield polynomial representations, which must be translated into the usually required commutator representation. We prove that a new translation proposed recently yields a correct representation of the BCH and Zassenhaus terms. This representation entails fewer terms than the well-known Dynkin-Specht-Wever representation, which is of relevance for practical applications. Furthermore, various methods for the computation of the BCH and Zassenhaus terms are compared, and a new efficient approach for the calculation of the Zassenhaus terms is proposed. Mathematica implementations for the most efficient algorithms are provided together with comparisons of efficiency.

Easing the transition between hospital and home: translating knowledge into action.

PubMed

Baumbusch, Jennifer; Semeniuk, Pat; McDonald, Heather; Khan, Koushambhi Basu; Reimer Kirkham, Sheryl; Tan, Elsie; Anderson, Joan M

2007-10-01

Knowledge translation is an interactive, dynamic approach to the uptake of evidence-based knowledge. In this article, the authors present a collaborative model for knowledge translation that grew out of a program of research focusing on the experiences of patients from ethnoculturally diverse groups as they were discharged home from hospital. Research findings highlight issues around gaps in the continuity of services and language and communication. The authors discuss a number of knowledge translation initiatives that were developed to address these gaps. Key to the success of this process has been a collaborative relationship between researchers and practitioners that is grounded in the shared goal of knowledge translation to support ethically sound decision-making in the delivery of health-care services.

Alterations of the three short open reading frames in the Rous sarcoma virus leader RNA modulate viral replication and gene expression.

PubMed Central

Moustakas, A; Sonstegard, T S; Hackett, P B

1993-01-01

The Rous sarcoma virus (RSV) leader RNA has three short open reading frames (ORF1 to ORF3) which are conserved in all avian sarcoma-leukosis retroviruses. Effects on virus propagation were determined following three types of alterations in the ORFs: (i) replacement of AUG initiation codons in order to prohibit ORF translation, (ii) alterations of the codon context around the AUG initiation codon to enhance translation of the normally silent ORF3, and (iii) elongation of the ORF coding sequences. Mutagenesis of the AUG codons for ORF1 and ORF2 (AUG1 and AUG2) singly or together delayed the onset of viral replication and cell transformation. In contrast, mutagenesis of AUG3 almost completely suppressed these viral activities. Mutagenesis of ORF3 to enhance its translation inhibited viral propagation. When the mutant ORF3 included an additional frameshift mutation which extended the ORF beyond the initiation site for the gag, gag-pol, and env proteins, host cells were initially transformed but died soon thereafter. Elongation of ORF1 from 7 to 62 codons led to the accumulation of transformation-defective virus with a delayed onset of replication. In contrast, viruses with elongation of ORF1 from 7 to 30 codons, ORF2 from 16 to 48 codons, or ORF3 from 9 to 64 codons, without any alterations in the AUG context, exhibited wild-type phenotypes. These results are consistent with a model that translation of the ORFs is necessary to facilitate virus production. Images PMID:7685415

Development of a mentorship strategy: a knowledge translation case study.

PubMed

Straus, Sharon E; Graham, Ian D; Taylor, Mark; Lockyer, Jocelyn

2008-01-01

There are many theories and frameworks for achieving knowledge translation, and the assortment can be confusing to those responsible for planning, evaluation, or policymaking in knowledge translation. A conceptual framework developed by Graham and colleagues provides an approach that builds on the commonalities found in an assessment of planned-action theories. This article describes the application of this knowledge to action framework to a mentorship initiative in academic medicine. Mentorship influences career success but is threatened in academia by increased clinical, research, and administrative demands. A case study review was undertaken of the role of mentors, the experiences of mentors and mentees, and mentorship initiatives in developing and retaining clinician scientists at two universities in Alberta, Canada. This project involved relevant stakeholders including researchers, university administrators, and research funders. The knowledge to action framework was used to develop a strategy for mentorship for clinician researchers. The framework highlights the need to identify and engage stakeholders in the process of knowledge implementation. A series of initiatives were selected and tailored to barriers and facilitators to implementation of the mentorship initiative; strategies for evaluating the knowledge use and its impact on outcomes were developed. The knowledge to action framework can be used to develop a mentorship initiative for clinician researchers. Future work to evaluate the impact of this intervention on recruitment and retention is planned.

Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pelletier, J.; Kaplan, G.; Racaniello, V.R.

1988-03-01

Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, the authors determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. They found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element mapsmore » between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. They also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation.« less

Efficient Embedded Decoding of Neural Network Language Models in a Machine Translation System.

PubMed

Zamora-Martinez, Francisco; Castro-Bleda, Maria Jose

2018-02-22

Neural Network Language Models (NNLMs) are a successful approach to Natural Language Processing tasks, such as Machine Translation. We introduce in this work a Statistical Machine Translation (SMT) system which fully integrates NNLMs in the decoding stage, breaking the traditional approach based on [Formula: see text]-best list rescoring. The neural net models (both language models (LMs) and translation models) are fully coupled in the decoding stage, allowing to more strongly influence the translation quality. Computational issues were solved by using a novel idea based on memorization and smoothing of the softmax constants to avoid their computation, which introduces a trade-off between LM quality and computational cost. These ideas were studied in a machine translation task with different combinations of neural networks used both as translation models and as target LMs, comparing phrase-based and [Formula: see text]-gram-based systems, showing that the integrated approach seems more promising for [Formula: see text]-gram-based systems, even with nonfull-quality NNLMs.

NRF2: Translating the Redox Code

PubMed Central

Tummala, Krishna S.; Kottakis, Filippos; Bardeesy, Nabeel

2016-01-01

Cancer requires mechanisms to mitigate reactive oxygen species (ROS) generated during rapid growth, such as induction of the antioxidant transcription factor, Nrf2. However, the targets of ROS-mediated cytotoxicity are unclear. Recent studies in pancreatic cancer show that redox control by Nrf2 prevents cysteine oxidation of the mRNA translational machinery, thereby supporting efficient protein synthesis. PMID:27555347

Positioning Continuing Education: Boundaries and Intersections between the Domains Continuing Education, Knowledge Translation, Patient Safety and Quality Improvement

ERIC Educational Resources Information Center

Kitto, Simon; Bell, Mary; Peller, Jennifer; Sargeant, Joan; Etchells, Edward; Reeves, Scott; Silver, Ivan

2013-01-01

Public and professional concern about health care quality, safety and efficiency is growing. Continuing education, knowledge translation, patient safety and quality improvement have made concerted efforts to address these issues. However, a coordinated and integrated effort across these domains is lacking. This article explores and discusses the…

Maternal immune activation dysregulation of the fetal brain transcriptome and relevance to the pathophysiology of autism spectrum disorder.

PubMed

Lombardo, M V; Moon, H M; Su, J; Palmer, T D; Courchesne, E; Pramparo, T

2018-04-01

Maternal immune activation (MIA) via infection during pregnancy is known to increase risk for autism spectrum disorder (ASD). However, it is unclear how MIA disrupts fetal brain gene expression in ways that may explain this increased risk. Here we examine how MIA dysregulates rat fetal brain gene expression (at a time point analogous to the end of the first trimester of human gestation) in ways relevant to ASD-associated pathophysiology. MIA downregulates expression of ASD-associated genes, with the largest enrichments in genes known to harbor rare highly penetrant mutations. MIA also downregulates expression of many genes also known to be persistently downregulated in the ASD cortex later in life and which are canonically known for roles in affecting prenatally late developmental processes at the synapse. Transcriptional and translational programs that are downstream targets of highly ASD-penetrant FMR1 and CHD8 genes are also heavily affected by MIA. MIA strongly upregulates expression of a large number of genes involved in translation initiation, cell cycle, DNA damage and proteolysis processes that affect multiple key neural developmental functions. Upregulation of translation initiation is common to and preserved in gene network structure with the ASD cortical transcriptome throughout life and has downstream impact on cell cycle processes. The cap-dependent translation initiation gene, EIF4E, is one of the most MIA-dysregulated of all ASD-associated genes and targeted network analyses demonstrate prominent MIA-induced transcriptional dysregulation of mTOR and EIF4E-dependent signaling. This dysregulation of translation initiation via alteration of the Tsc2-mTor-Eif4e axis was further validated across MIA rodent models. MIA may confer increased risk for ASD by dysregulating key aspects of fetal brain gene expression that are highly relevant to pathophysiology affecting ASD.

Cap-proximal nucleotides via differential eIF4E binding and alternative promoter usage mediate translational response to energy stress.

PubMed

Tamarkin-Ben-Harush, Ana; Vasseur, Jean-Jacques; Debart, Françoise; Ulitsky, Igor; Dikstein, Rivka

2017-02-08

Transcription start-site (TSS) selection and alternative promoter (AP) usage contribute to gene expression complexity but little is known about their impact on translation. Here we performed TSS mapping of the translatome following energy stress. Assessing the contribution of cap-proximal TSS nucleotides, we found dramatic effect on translation only upon stress. As eIF4E levels were reduced, we determined its binding to capped-RNAs with different initiating nucleotides and found the lowest affinity to 5'cytidine in correlation with the translational stress-response. In addition, the number of differentially translated APs was elevated following stress. These include novel glucose starvation-induced downstream transcripts for the translation regulators eIF4A and Pabp, which are also translationally-induced despite general translational inhibition. The resultant eIF4A protein is N-terminally truncated and acts as eIF4A inhibitor. The induced Pabp isoform has shorter 5'UTR removing an auto-inhibitory element. Our findings uncovered several levels of coordination of transcription and translation responses to energy stress.

Structure of a human cap-dependent 48S translation pre-initiation complex

PubMed Central

Eliseev, Boris; Yeramala, Lahari; Leitner, Alexander; Karuppasamy, Manikandan; Raimondeau, Etienne; Huard, Karine; Alkalaeva, Elena; Aebersold, Ruedi

2018-01-01

Abstract Eukaryotic translation initiation is tightly regulated, requiring a set of conserved initiation factors (eIFs). Translation of a capped mRNA depends on the trimeric eIF4F complex and eIF4B to load the mRNA onto the 43S pre-initiation complex comprising 40S and initiation factors 1, 1A, 2, 3 and 5 as well as initiator-tRNA. Binding of the mRNA is followed by mRNA scanning in the 48S pre-initiation complex, until a start codon is recognised. Here, we use a reconstituted system to prepare human 48S complexes assembled on capped mRNA in the presence of eIF4B and eIF4F. The highly purified h-48S complexes are used for cross-linking/mass spectrometry, revealing the protein interaction network in this complex. We report the electron cryo-microscopy structure of the h-48S complex at 6.3 Å resolution. While the majority of eIF4B and eIF4F appear to be flexible with respect to the ribosome, additional density is detected at the entrance of the 40S mRNA channel which we attribute to the RNA-recognition motif of eIF4B. The eight core subunits of eIF3 are bound at the 40S solvent-exposed side, as well as the subunits eIF3d, eIF3b and eIF3i. elF2 and initiator-tRNA bound to the start codon are present at the 40S intersubunit side. This cryo-EM structure represents a molecular snap-shot revealing the h-48S complex following start codon recognition. PMID:29401259

Does short-term virologic failure translate to clinical events in antiretroviral-naïve patients initiating antiretroviral therapy in clinical practice?

PubMed

Mugavero, Michael J; May, Margaret; Harris, Ross; Saag, Michael S; Costagliola, Dominique; Egger, Matthias; Phillips, Andrew; Günthard, Huldrych F; Dabis, Francois; Hogg, Robert; de Wolf, Frank; Fatkenheuer, Gerd; Gill, M John; Justice, Amy; D'Arminio Monforte, Antonella; Lampe, Fiona; Miró, Jose M; Staszewski, Schlomo; Sterne, Jonathan A C

2008-11-30

To determine whether differences in short-term virologic failure among commonly used antiretroviral therapy (ART) regimens translate to differences in clinical events in antiretroviral-naïve patients initiating ART. Observational cohort study of patients initiating ART between January 2000 and December 2005. The Antiretroviral Therapy Cohort Collaboration (ART-CC) is a collaboration of 15 HIV cohort studies from Canada, Europe, and the United States. A total of 13 546 antiretroviral-naïve HIV-positive patients initiating ART with efavirenz, nevirapine, lopinavir/ritonavir, nelfinavir, or abacavir as third drugs in combination with a zidovudine and lamivudine nucleoside reverse transcriptase inhibitor backbone. Short-term (24-week) virologic failure (>500 copies/ml) and clinical events within 2 years of ART initiation (incident AIDS-defining event, death, and a composite measure of these two outcomes). Compared with efavirenz as initial third drug, short-term virologic failure was more common with all other third drugs evaluated; nevirapine (adjusted odds ratio = 1.87, 95% confidence interval (CI) = 1.58-2.22), lopinavir/ritonavir (1.32, 95% CI = 1.12-1.57), nelfinavir (3.20, 95% CI = 2.74-3.74), and abacavir (2.13, 95% CI = 1.82-2.50). However, the rate of clinical events within 2 years of ART initiation appeared higher only with nevirapine (adjusted hazard ratio for composite outcome measure 1.27, 95% CI = 1.04-1.56) and abacavir (1.22, 95% CI = 1.00-1.48). Among antiretroviral-naïve patients initiating therapy, between-ART regimen, differences in short-term virologic failure do not necessarily translate to differences in clinical outcomes. Our results should be interpreted with caution because of the possibility of residual confounding by indication.

PRMT1-Mediated Translation Regulation is a Crucial Vulnerability of Cancer | Office of Cancer Genomics

Cancer.gov

Through an shRNA screen, we identified the protein arginine methyltransferase Prmt1 as a vulnerable intervention point in murine p53/Rb-null osteosarcomas, the human counterpart of which lacks effective therapeutic options. Depletion of Prmt1 in p53-deficient cells impaired tumor initiation and maintenance in vitro and in vivo Mechanistic studies reveal that translation-associated pathways were enriched for Prmt1 downstream targets, implicating Prmt1 in translation control.

Epidemiology to public health intervention for preventing cardiovascular diseases: The role of translational research

PubMed Central

Krishnan, Anand; Yadav, Kapil; Kaur, Manmeet; Kumar, Rajesh

2010-01-01

Despite significant progress in medical research, cardiovascular diseases (CVDs) continue to be the largest contributors of morbidity and mortality both in developed and developing countries. The status of public health interventions related to CVDs prevention was reviewed to identify actions that are required to bridge the existing gap between the evidence and the policy. We used a framework comprising two steps - “bench to bedside” and from “bedside to community” to evaluate translational research. Available literature was reviewed to document the current status of CVD prevention and control at national level in India. Case studies of risk factor surveillance, tobacco control and blood pressure measurement were used to understand different aspects of translational research. National level initiatives in non-communicable diseases surveillance, prevention and control are a recent phenomena in India. The delay in translation of research to policy has occurred primarily at the second level, i.e., from ‘bedside to community’. The possible reasons for this were: inappropriate perception of the problem by policy makers and programme managers, lack of global public health guidelines and tools, and inadequate nationally relevant research related to operationalization and cost of public health interventions. Public health fraternity, both nationally and internationally, needs to establish institutional mechanisms to strengthen human resource capacity to initiate and monitor the process of translational research in India. Larger public interest demands that focus should shift to overcoming the barriers at community level translation. Only this will ensure that the extraordinary scientific advances of this century are rapidly translated for the benefit of more than one billion Indians. PMID:21150018

World-Record Solar Cell a Step Closer to Cheap Solar Energy

Science.gov Websites

envelope of solar-cell efficiency, we can begin to visualize the day when energy from the sun will be in efficiency translates into lower costs for harnessing energy from the sun. The cell's excellent

Use of Discussion Board and PaperShow in Translation Class

ERIC Educational Resources Information Center

Takewa, Mika

2013-01-01

This report presents how the learning technologies Discussion Board and PaperShow are incorporated into postgraduate translation modules by means of observing their impact on students' learning. Initially and mainly for administrative reasons as well as being encouraged to make use of the university's virtual learning environment (VLE), I set up…

Changes in Polysome Association of mRNA Throughout Growth and Development in Arabidopsis thaliana.

PubMed

Yamasaki, Shotaro; Matsuura, Hideyuki; Demura, Taku; Kato, Ko

2015-11-01

Translational control is a key regulatory step in the expression of genes as proteins. In plant cells, the translational efficiency of mRNAs differs for different mRNA species, and the efficiency dynamically changes in various conditions. To gain a global view of translational control throughout growth and development, we performed genome-wide analysis of polysome association of mRNA during growth and leaf development in Arabidopsis thaliana by subjecting the mRNAs in polysomes to DNA microarray. This analysis revealed that the degree of polysome association of mRNA was different depending on the mRNA species, and the polysome association changed greatly throughout growth and development for each. In the growth stage, transcripts showed varying changes in polysome association from strongly depressed to unchanged, with the majority of transcripts showing dissociation from ribosomes. On the other hand, during leaf development, the polysome association of transcripts showed a normal distribution from repressed to activated mRNAs when comparing expanding and expanded leaves. In addition, functional category analysis of the microarray data suggested that translational control has a physiological significance in the plant growth and development process, especially in the categories of signaling and protein synthesis. In addition to this, we compared changes in polysome association of mRNAs between various conditions and characterized translational controls in each. This result suggested that mRNA translation might be controlled by complicated mechanisms for response to each condition. Our results highlight the importance of dynamic changes in mRNA translation in plant development and growth. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins.

PubMed

Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann; Becher, Paul

2017-04-01

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5' terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Molecular mechanism and structure of Trigger Factor bound to the translating ribosome

PubMed Central

Merz, Frieder; Boehringer, Daniel; Schaffitzel, Christiane; Preissler, Steffen; Hoffmann, Anja; Maier, Timm; Rutkowska, Anna; Lozza, Jasmin; Ban, Nenad; Bukau, Bernd; Deuerling, Elke

2008-01-01

Ribosome-associated chaperone Trigger Factor (TF) initiates folding of newly synthesized proteins in bacteria. Here, we pinpoint by site-specific crosslinking the sequence of molecular interactions of Escherichia coli TF and nascent chains during translation. Furthermore, we provide the first full-length structure of TF associated with ribosome–nascent chain complexes by using cryo-electron microscopy. In its active state, TF arches over the ribosomal exit tunnel accepting nascent chains in a protective void. The growing nascent chain initially follows a predefined path through the entire interior of TF in an unfolded conformation, and even after folding into a domain it remains accommodated inside the protective cavity of ribosome-bound TF. The adaptability to accept nascent chains of different length and folding states may explain how TF is able to assist co-translational folding of all kinds of nascent polypeptides during ongoing synthesis. Moreover, we suggest a model of how TF's chaperoning function can be coordinated with the co-translational processing and membrane targeting of nascent polypeptides by other ribosome-associated factors. PMID:18497744

Translatome analysis at the egg-to-embryo transition in sea urchin

PubMed Central

Chassé, Héloïse; Aubert, Julie; Boulben, Sandrine; Le Corguillé, Gildas; Corre, Erwan; Cormier, Patrick

2018-01-01

Abstract Early embryogenesis relies on the translational regulation of maternally stored mRNAs. In sea urchin, fertilization triggers a dramatic rise in translation activity, necessary for the onset of cell division. Here, the full spectrum of the mRNAs translated upon fertilization was investigated by polysome profiling and sequencing. The translatome of the early sea urchin embryo gave a complete picture of the polysomal recruitment dynamics following fertilization. Our results indicate that only a subset of maternal mRNAs were selectively recruited onto polysomes, with over-represented functional categories in the translated set. The increase in translation upon fertilization depends on the formation of translation initiation complexes following mTOR pathway activation. Surprisingly, mTOR pathway inhibition differentially affected polysomal recruitment of the newly translated mRNAs, which thus appeared either mTOR-dependent or mTOR-independent. Therefore, our data argue for an alternative to the classical cap-dependent model of translation in early development. The identification of the mRNAs translated following fertilization helped assign translational activation events to specific mRNAs. This translatome is the first step to a comprehensive analysis of the molecular mechanisms governing translation upon fertilization and the translational regulatory networks that control the egg-to-embryo transition as well as the early steps of embryogenesis. PMID:29660001

Translatome analysis at the egg-to-embryo transition in sea urchin.

PubMed

Chassé, Héloïse; Aubert, Julie; Boulben, Sandrine; Le Corguillé, Gildas; Corre, Erwan; Cormier, Patrick; Morales, Julia

2018-05-18

Early embryogenesis relies on the translational regulation of maternally stored mRNAs. In sea urchin, fertilization triggers a dramatic rise in translation activity, necessary for the onset of cell division. Here, the full spectrum of the mRNAs translated upon fertilization was investigated by polysome profiling and sequencing. The translatome of the early sea urchin embryo gave a complete picture of the polysomal recruitment dynamics following fertilization. Our results indicate that only a subset of maternal mRNAs were selectively recruited onto polysomes, with over-represented functional categories in the translated set. The increase in translation upon fertilization depends on the formation of translation initiation complexes following mTOR pathway activation. Surprisingly, mTOR pathway inhibition differentially affected polysomal recruitment of the newly translated mRNAs, which thus appeared either mTOR-dependent or mTOR-independent. Therefore, our data argue for an alternative to the classical cap-dependent model of translation in early development. The identification of the mRNAs translated following fertilization helped assign translational activation events to specific mRNAs. This translatome is the first step to a comprehensive analysis of the molecular mechanisms governing translation upon fertilization and the translational regulatory networks that control the egg-to-embryo transition as well as the early steps of embryogenesis.

Translations on Near East and North Africa, Number 1647

DTIC Science & Technology

1977-04-28

Production capacity of the two plants is to amount to 220,000 tons of ammonia and 800,000 tons of uric acid . Improving operations efficiency at the...JPRS 69019 28 April 1977 TRANSLATIONS’ON NEAR EAST AND NORTH AFRICA No, 1647 < 2 w ÜJCEE »- .Q-5 ^ Q. C «MM H— —• £2 a« ES.Q...3. Recipient’s Accession No. 4. Title and Subtitle TRANSLATIONS ON NEAR EAST AND NORTH AFRICA , No. 164 7 5. Report Date 28 April 1977 6

NIa-Pro of Papaya ringspot virus interacts with Carica papaya eukaryotic translation initiation factor 3 subunit G (CpeIF3G).

PubMed

Gao, Le; Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

2015-02-01

The interaction of papaya eukaryotic translation initiation factor 3 subunit G (CpeIF3G) with Papaya ringspot virus (PRSV) NIa-Pro was validated using a bimolecular fluorescence complementation assay in papaya protoplasts based on the previous yeast two-hybrid assay results. The C-terminal (residues 133-239) fragment of PRSV NIa-Pro and the central domain (residues 59-167) of CpeIF3G were required for effective interaction between NIa-Pro and CpeIF3G as shown by a Sos recruitment yeast two-hybrid system with several deletion mutants of NIa-Pro and CpeIF3G. The central domain of CpeIF3G, which contains a C2HC-type zinc finger motif, is required to bind to other eIFs of the translational machinery. In addition, quantitative real-time reverse transcription PCR assay confirmed that PRSV infection leads to a 2- to 4.5-fold up-regulation of CpeIF3G mRNA in papaya. Plant eIF3G is involved in various stress response by enhancing the translation of resistance-related proteins. It is proposed that the NIa-Pro-CpeIF3G interaction may impair translation preinitiation complex assembly of defense proteins and interfere with host defense.

Viral factor TAV recruits TOR/S6K1 signalling to activate reinitiation after long ORF translation

PubMed Central

Schepetilnikov, Mikhail; Kobayashi, Kappei; Geldreich, Angèle; Caranta, Carole; Robaglia, Christophe; Keller, Mario; Ryabova, Lyubov A

2011-01-01

The protein kinase TOR (target-of-rapamycin) upregulates translation initiation in eukaryotes, but initiation restart after long ORF translation is restricted by largely unknown pathways. The plant viral reinitiation factor transactivator–viroplasmin (TAV) exceptionally promotes reinitiation through a mechanism involving retention on 80S and reuse of eIF3 and the host factor reinitiation-supporting protein (RISP) to regenerate reinitiation-competent ribosomal complexes. Here, we show that TAV function in reinitiation depends on physical association with TOR, with TAV–TOR binding being critical for both translation reinitiation and viral fitness. Consistently, TOR-deficient plants are resistant to viral infection. TAV triggers TOR hyperactivation and S6K1 phosphorylation in planta. When activated, TOR binds polyribosomes concomitantly with polysomal accumulation of eIF3 and RISP—a novel and specific target of TOR/S6K1—in a TAV-dependent manner, with RISP being phosphorylated. TAV mutants defective in TOR binding fail to recruit TOR, thereby abolishing RISP phosphorylation in polysomes and reinitiation. Thus, activation of reinitiation after long ORF translation is more complex than previously appreciated, with TOR/S6K1 upregulation being the key event in the formation of reinitiation-competent ribosomal complexes. PMID:21343906