Translation Toeprinting Assays Using Fluorescently Labeled Primers and Capillary Electrophoresis

Gould, Phillip S.; Bird, Helen; Easton, Andrew J.

doi:10.2144/05383st02

Cited by 28 publications

(30 citation statements)

References 19 publications

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“…Toeprinting Assay-The general procedure followed was gleaned from Gould et al (40). Two micrograms of mRNA produced from a linearized dual luciferase construct using the RiboMAX Large Scale RNA Production system-T7 (Promega) was added to rabbit reticulocyte lysates from TNT Quick-coupled Transcription/Translation System (Promega), with and without the plasmid encoding mutant human eRF1 (see Fig.…”

Section: Methodsmentioning

confidence: 99%

Identification of a Cellular Factor That Modulates HIV-1 Programmed Ribosomal Frameshifting

Kobayashi¹,

Zhuang²,

Peltz

et al. 2010

Journal of Biological Chemistry

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Programmed ؊1 ribosomal frameshifting (PRF) is a distinctive mode of gene expression utilized by some viruses, including human immunodeficiency virus type 1 (HIV-1), to produce multiple proteins from a single mRNA. ؊1 PRF induces a subset of elongating ribosomes to shift their translational reading frame by 1 base in the 5 direction. The appropriate ratio of Gag to Gag-Pol synthesis is tightly regulated by the PRF signal which promotes ribosomes to shift frame, and even small changes in PRF efficiency, either up or down, have significant inhibitory effects upon virus production, making PRF essential for HIV-1 replication. Although little has been reported about the cellular factors that modulate HIV-1 PRF, the cis-acting elements regulating PRF have been extensively investigated, and the PRF signal of HIV-1 was shown to include a slippery site and frameshift stimulatory signal. Recently, a genome-wide screen performed to identify cellular factors that affect HIV-1 replication demonstrated that down-regulation of eukaryotic release factor 1 (eRF1) inhibited HIV-1 replication. Because of the eRF1 role in translation, we hypothesized that eRF1 is important for HIV-1 PRF. Using a dual luciferase reporter system harboring a HIV-1 PRF signal, results showed that depletion or inhibition of eRF1 enhanced PRF in yeast, rabbit reticulocyte lysates, and mammalian cells. Consistent with the eRF1 role in modulating HIV PRF, depleting eRF1 increased the Gag-Pol to Gag ratio in cells infected with replication-competent virus. The increase in PRF was independent of a proximal termination codon and did not result from increased ribosomal pausing at the slippery site. This is the first time that a cellular factor has been identified which can promote HIV-1 PRF and highlights HIV-1 PRF as essential for replication and an important but under exploited antiviral drug target.The ability of ribosomes to maintain the correct translational open reading frame (ORF) 2 is fundamental to the integrity and fidelity of protein synthesis. However, there are a number of examples in which elongating ribosomes are programmed to shift their translational ORF 1 base in either the 5Ј or 3Ј direction (Ϫ1 or ϩ1 ribosomal programmed ribosomal frameshift (PRF), respectively) to translate multiple proteins from a single mRNA (1-5). HIV-1 is one example of a virus that uses this process as an integral part of its replication cycle.The HIV-1 Gag and Pol proteins are synthesized as p55 Gag or p160Gag-Pol polyprotein precursors using the same translation start codon but different ORFs in the full-length viral mRNA (6, 7). The gag ORF is located at the 5Ј end of the viral mRNA, whereas the pol ORF is 3Ј of the HIV-1 PRF element and outof-frame with the gag ORF (Fig. 1). Therefore, the enzymatic protein products of the pol gene are only translated through a Ϫ1 PRF event (8 -10). Importantly, the frequency of the PRF is controlled precisely via the interaction between viral RNA cisacting elements and host translation factors. The frequency of the PRF in HIV-1...

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Section: Methodsmentioning

confidence: 99%

Identification of a Cellular Factor That Modulates HIV-1 Programmed Ribosomal Frameshifting

Kobayashi¹,

Zhuang²,

Peltz

et al. 2010

Journal of Biological Chemistry

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“…Primer extensions were conducted by eliminating the in vitro translation step of the toeprinting assay (19). Thus, the mRNA was synthesized in vitro and then the FAM-labeled primer was annealed to the template, which was immediately added to the reverse transcription reaction carried out at 25, 37, 42, or 45°C and analyzed using the GeneScan software as described previously (19).…”

Section: Methodsmentioning

confidence: 99%

“…The primer M2toe1 (Table I) was annealed to the mRNA and added to in vitro translation reaction mixture with RRL. Areas where the ribosomes paused on the mRNA transcript would be represented as peaks on the resulting electropherogram, because further progression of the RT is inhibited (19). As a control, the same mRNA/primer mixture was added to the translation reaction mixture but with no RRL present.…”

Section: Coupled Translation Of Non-rsv Proteins-the Construct Pwildcmentioning

confidence: 99%

Coupled Translation of the Respiratory Syncytial Virus M2 Open Reading Frames Requires Upstream Sequences

Gould

Easton

2005

Journal of Biological Chemistry

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We have investigated the mechanism of the translation of the second open reading frame (ORF) of the respiratory syncytial virus M2 transcript that uses a novel coupled translation process requiring prior translation of the upstream ORF. The second M2-2 ORF sequences play no role in the coupling process and can be replaced with other gene sequences. Surprisingly, the overlap region of the two ORFs alone was not sufficient for coupled translation to occur. An analysis of the sequences required for the coupling process showed that portions of the transcript located along the length of the first ORF M2-1, upstream of the ORF overlap region, were essential for coupled translation to occur. A critically important region for this process was centered ϳ150 nucleotides upstream of the ORF2 initiation codons. This region was shown to contain a significant degree of secondary structure, and mutation of this sequence to remove predicted areas of base pairing significantly reduced coupled translation, confirming that the secondary structure was important for the coupling process. Additional sequences further upstream increased the efficiency of the coupled translation process. These data indicate that upstream sequences act in conjunction with the M2-1/M2-2 overlap region to promote coupled translation.

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“…To address this, we mapped the site of ribosome binding on the 5=-UTR using a toeprint assay with a fluorescent primer as described previously (23). The assay is based on the fact that ribosomes bound to the RNA inhibit the extension of a downstream antisense primer by reverse transcriptase as depicted in Fig.…”

Section: L65mentioning

confidence: 99%

“…1B) (62,90). Although it has been noted previously that in vitro translation by RRL may lead to artifacts due to ectopic translation initiation (23), this element could attract and bind ribosomes either at its CTG or GTG sites. This site has been found to play a cis regulatory role in previous work of our laboratory on SP-A2 (62,90) and has the unique characteristic of including alternative start codons in all three reading frames.…”

Section: L65mentioning

confidence: 99%

Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

Tsotakos

Silveyra

Lin

et al. 2015

American Journal of Physiology-Lung Cellular and Molecular Phys

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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.

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Translation Toeprinting Assays Using Fluorescently Labeled Primers and Capillary Electrophoresis

Cited by 28 publications

References 19 publications

Identification of a Cellular Factor That Modulates HIV-1 Programmed Ribosomal Frameshifting

Identification of a Cellular Factor That Modulates HIV-1 Programmed Ribosomal Frameshifting

Coupled Translation of the Respiratory Syncytial Virus M2 Open Reading Frames Requires Upstream Sequences

Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

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