Suppression of eukaryotic translation termination by selected RNAs

Carnes, Jason; Frolova, Ludmila; Zinnen, Shawn; Drugeon, Gabrièle; Phillippe, Michel; Justesen, Just; Haenni, Anne‐Lise; Leinwand, Leslie A.; Kisselev, Lev L.; Yarus, Michael

doi:10.1017/s1355838200001242

Cited by 24 publications

(17 citation statements)

References 50 publications

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“…Rather, our results suggest that eukaryotic release factors, eRF1/eRF3, terminate translation more efficiently at the opal and ochre codons as opposed to the amber codons. Consistent with this hypothesis, addition of an RNA aptamer previously shown to inhibit eRF1/eRF3 (Carnes et al 2000) improved NBD-[ 14 C]Cys incorporation efficiency by twofold at the opal codon and fourfold at the ochre codon (Fig. 4B).…”

Section: Incorporation Of Nbd-labeled Cys Using Cys-derived Suppressosupporting

confidence: 77%

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In vitro incorporation of nonnatural amino acids into protein using tRNA^Cys-derived opal, ochre, and amber suppressor tRNAs

Gubbens¹,

Kim²,

Yang³

et al. 2010

RNA

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Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNA Cys that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies.

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Section: Incorporation Of Nbd-labeled Cys Using Cys-derived Suppressosupporting

confidence: 77%

“…The RNA aptamer was generated from synthetic overlapping DNA oligonucleotides encoding aptamer-12 (Carnes et al 2000), T7 promoter, and EcoRI and HindIII sites. The PCR products were ligated into pSP64, and transcription and aptamer purification were performed as for tRNAs.…”

Section: Plasmids and Transcriptionmentioning

confidence: 99%

In vitro incorporation of nonnatural amino acids into protein using tRNA^Cys-derived opal, ochre, and amber suppressor tRNAs

Gubbens¹,

Kim²,

Yang³

et al. 2010

RNA

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“…Experiments that address these issues of delivery and persistence in cells may manifest new possibilities for treatment of diseases caused by premature stop codons. We have now shown that, given delivery and persistence, therapeutic oligonucleotide suppression could rely on aptamers (Carnes et al 2000), antisense sequences or RNA interference (this work), or perhaps a more effective combination thereof.…”

Section: Effect Of Sirnas and Antisense Oligonucleotides On Erf1 Protmentioning

confidence: 85%

“…Nevertheless, aminoglycosides are being tested in clinical trials to treat diseases caused by premature stop codons (Wagner et al 2001). Another approach to promote readthrough uses eRF1-binding RNAs, called aptamers, which have been shown to inhibit eRF1 function and affect suppression during translation in vitro (Carnes et al 2000). The efficacy of these eRF1 aptamers, however, has not yet been demonstrated in living cells.…”

Section: Introductionmentioning

confidence: 99%

Stop codon suppression via inhibition of eRF1 expression

Carnes

Jacobson²,

Leinwand³

et al. 2003

RNA

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In humans, recognition of a stop codon by protein release factor eRF1 leads to release of the nascent peptide from the ribosome. Although efficient eRF1 activity is usually desirable, numerous pathologies result from eRF1 recognition of premature stop mutations in essential genes. In these cases, decreased eRF1 activity could increase readthrough of the premature stop codon, thereby making full-length protein. To broaden the means available to beneficially decrease eRF1 activity, we have targeted eRF1 mRNA using siRNAs and antisense oligonucleotides. We show that both eRF1-targeted siRNA and antisense oligonucleotides decrease eRF1 mRNA and eRF1 protein concentrations, and increase UAG readthrough in cultured human cells.

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“…Translation termination factors may serve as a target for the virus. It has been demonstrated that certain isolated RNAs have an affinity for eukaryotic translation termination factors, eRF1, and eRF1⅐eRF3 complexes; to which they not only bind but also inhibit eFR1-mediated release of protein precursor chains from ribosomes (35).…”

Section: Discussionmentioning

confidence: 99%

Human Rhinovirus Selectively Modulates Membranous and Soluble Forms of Its Intercellular Adhesion Molecule–1 (ICAM-1) Receptor to Promote Epithelial Cell Infectivity

Whiteman

Bianco

Knight

et al. 2003

Journal of Biological Chemistry

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Human rhinoviruses are responsible for many upper respiratory tract infections. 90% of rhinoviruses utilize intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor, which also plays a critical role in recruitment of immune effector cells. Two forms of this receptor exist; membrane-bound (mICAM-1) and soluble ICAM-1 (sICAM-1). The soluble receptor may be produced independently from the membrane-bound form or it may be the product of proteolytic cleavage of mICAM-1. The ratio of airway epithelial cell expression of mICAM-1 to the sICAM-1 form may influence cell infectivity and outcome of rhinovirus infection. We therefore investigated the effect of rhinovirus on expression of both ICAM-1 receptors in normal human bronchial epithelial cells. We observed separate distinct messenger RNA transcripts coding for mICAM-1 and sICAM-1 in these cells, which were modulated by virus. Rhinovirus induced mICAM-1 expression on epithelial cells while simultaneously down-regulating sICAM-1 release, with consequent increase in target cell infectivity. The role of protein tyrosine kinases was investigated as a potential mechanistic pathway. Rhinovirus infection induced rapid phosphorylation of intracellular tyrosine kinase, which may be critical in up-regulation of mICAM-1. Elucidation of the underlying molecular mechanisms involved in differential modulation of both ICAM-1 receptors may lead to novel therapeutic strategies. Human rhinoviruses (HRV)1 are the most frequent cause of upper respiratory tract infections known as the "common cold." Although these infections are generally mild and self-limiting, they inflict a heavy economical burden due to high loss of productivity and medical costs (1). Currently, there is no effective treatment for HRV infections; over the counter cold remedies only alleviate the symptoms but do not eradicate the virus.Primarily, HRV target epithelial cells for attachment and entry. These cells express intercellular adhesion molecule 1 (ICAM-1), the receptor for 90% of HRV serotypes (2). Both this major group of HRV and the 10% of HRV that use alternative receptors for cell attachment enhance cell surface ICAM-1 expression (3). This glycoprotein, belonging to the immunoglobulin supergene family, consists of five Ig-like domains (4); domains 1 and 2 have been shown to fit snugly in a key-lock relationship into reciprocal canyons on the HRV shell (5). In addition, to this critical role as a docking molecule during HRV infection, ICAM-1 through separate domains with its cognate ligand LFA-1 (CD18/CD11a) drives the migration of immuneeffector cells to sites of inflammation (6). While most studies refer to the membranous form of ICAM-1, a soluble form (sICAM-1) has also been described (7). The molecular mass of sICAM-1 is similar to the molecular mass of the extracellular domain of ICAM-1 (80 -114 kDa) depending on the level of glycosylation, suggesting that this soluble circulating form of ICAM-1 consists of most of the extracellular domain of membranous ICAM-1 (7). Several circulating isoform...

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Suppression of eukaryotic translation termination by selected RNAs

Cited by 24 publications

References 50 publications

In vitro incorporation of nonnatural amino acids into protein using tRNA^Cys-derived opal, ochre, and amber suppressor tRNAs

In vitro incorporation of nonnatural amino acids into protein using tRNA^Cys-derived opal, ochre, and amber suppressor tRNAs

Stop codon suppression via inhibition of eRF1 expression

Human Rhinovirus Selectively Modulates Membranous and Soluble Forms of Its Intercellular Adhesion Molecule–1 (ICAM-1) Receptor to Promote Epithelial Cell Infectivity

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Suppression of eukaryotic translation termination by selected RNAs

Cited by 24 publications

References 50 publications

In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal, ochre, and amber suppressor tRNAs

In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal, ochre, and amber suppressor tRNAs

Stop codon suppression via inhibition of eRF1 expression

Human Rhinovirus Selectively Modulates Membranous and Soluble Forms of Its Intercellular Adhesion Molecule–1 (ICAM-1) Receptor to Promote Epithelial Cell Infectivity

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In vitro incorporation of nonnatural amino acids into protein using tRNA^Cys-derived opal, ochre, and amber suppressor tRNAs

In vitro incorporation of nonnatural amino acids into protein using tRNA^Cys-derived opal, ochre, and amber suppressor tRNAs