Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate

Hall, Mary P.; Unch, James; Binkowski, Brock F.; Valley, Michael P.; Butler, Braeden L.; Wood, Monika G.; Otto, Paul; Zimmerman, Kristopher; Vidugiris, Gediminas; Machleidt, Thomas; Robers, Matthew B.; Benink, Hélène A; Eggers, Christopher T.; Slater, Michael; Meisenheimer, Poncho; Klaubert, Dieter H.; Fan, Frank; Encell, Lance P.; Wood, Keith V.

doi:10.1021/cb3002478

Cited by 1,378 publications

(1,592 citation statements)

References 35 publications

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“…S2). Ataluren treatment of mammalian 293H cells stably transfected with wild-type (WT) or PTC(W12X)-containing NanoLuc (19) reporters promoted a bell-shaped dose-response, with a peak ninefold increase in luciferase activity derived from the PTC reporter at 10 μM (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

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Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

Roy

Friesen

Tomizawa

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

189

218

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A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren’s likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren’s retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.

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Section: Resultsmentioning

confidence: 99%

“…Procedures for RNA analysis, protein purification, and luciferase assays were as described previously (3,19,21,38) or in SI Appendix. Western analyses and protein purification largely used antibodies or affinity resins targeted to the HA, FLAG, StrepII, T-GFP, and 6× His epitope tags expressed from the respective reporter constructs.…”

Section: Methodsmentioning

confidence: 99%

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

Roy

Friesen

Tomizawa

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

189

218

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“…Our study used NanoLuc as a reporter for the evaluation, as it is the most highly luminescent luciferase and provides 150-fold brighter bioluminescence than firefly luciferase. 17) In other words, the detection limit of our NanoLuc assay is sub pM, which is at least 1000-fold more sensitive than the ELISA used in the published reports.…”

Section: Discussionmentioning

confidence: 83%

“…17) In addition, to locate L. lactis in the tissues, we performed an ex vivo analysis using fluorescence-labeled L. lactis, and found that L. lactis accumulated in the Peyer's patches (PPs) irrespective of mInlA expression. Our observations suggested that DNA transfer from L. lactis occurred predominantly in the PPs not in the intestinal epithelial cells.…”

mentioning

confidence: 99%

Peyer’s Patches as a Portal for DNA Delivery by <i>Lactococcus lactis</i> <i>in Vivo</i>

Yano

Takahashi

Mori

et al. 2018

Biological & Pharmaceutical Bulletin

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Application of food-grade Lactococcus lactis (L. lactis) as a safe delivery tool for DNA vaccines and therapeutic proteins has been well investigated. Although some studies showed that eukaryotic expression plasmids were transferred from L. lactis to enterocytes, the precise mechanism of the DNA transfer remains unknown. In this study, we generated an invasive L. lactis strain that expresses "murinized" Internalin A, an invasin of intracellular bacteria Listeria monocytogenes with two amino acid alterations for invasion into murine cells, and confirmed that this L. lactis strain delivered DNA in an invasin-dependent manner into a monolayer of epithelial cells polarized to mimic the gastrointestinal tract environment. Although invasive L. lactis inoculated orally can deliver DNA into enterocytes in the gastrointestinal tract of mice, the efficiency of DNA transfer was similar to that of non-invasive L. lactis strain, suggesting that the in vivo DNA transfer from L. lactis occurs invasin-independently. A ligated-intestinal loop assay, a method for a short-term culturing of the whole intestine filled with materials to evaluate the interaction of the materials with intestinal cells, demonstrated that both non-invasive and invasive L. lactis strains were present in the Peyer's patches of the small intestine. On the other hand, few L. lactis was detected in the non-Peyer's patch epithelial region. Thus, our observations lead us to speculate that DNA transfer from L. lactis occurs predominantly in the Peyer's patches in an invasin-independent manner. Key words Lactococcus lactis;Peyer's patch; DNA delivery; Internalin A Vaccination with a plasmid containing the DNA sequence encoding an antigen has been of great interest of late as it can induce both humoral and cell-mediated immune responses. 1)Despite numerous studies, no DNA vaccines have been licensed for human use to date. The administration of naked DNA is less immunogenic in large animals and humans, probably due to the low level of protein expression.2) To improve the potency of DNA vaccination, various DNA delivery platforms, including electroporation, needle-free jet injection and liposomes, have been developed. 3,4) The use of bacteria as a delivery vehicle for DNA vaccines has emerged recently as a promising approach to enhance potency, 5) and one of the attractive features of the use of bacteria is the potential for oral administration to induce not only systemic but also mucosal immune responses. 5) Salmonella typhimurium and Listeria monocytogenes (L. monocytogenes) have been studied as experimental delivery vehicles as they are known to invade intestinal epithelial cells. 6) Even though the strains employed are extensively attenuated for virulence, the possibility of reversion to virulence as well as preexisting immunity limits their clinical use, especially for infants, the elderly and immunocompromised hosts. 7,8) The mechanisms involving DNA transfer from bacteria to mammalian cells and tissues remain to be clarified.The use of food-grade Lactococcus ...

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“…This time the reporter was generated with Nano-luciferase (nLUC) that is smaller in size and thus more sensitive in cellular responses compared to firefly-luciferase (Hall et al, 2012). To substantiate this splicing assay, evolutionarily conserved splicing sites of the intron in gHSFA3 were mutated to be unspliced at splice donor (Do) or acceptor (Ac) site (Fig.…”

Section: Sta1 Function In Pre-mrna Splicingmentioning

confidence: 99%

STABILIZED1 Modulates Pre-mRNA Splicing for Thermotolerance

Kim

Cho

Lee³

et al. 2017

Plant Physiol.

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High-temperature stress often leads to differential RNA splicing, thus accumulating different types and/or amounts of mature mRNAs in eukaryotic cells. However, regulatory mechanisms underlying plant precursor mRNA (pre-mRNA) splicing in the environmental stress conditions remain elusive. Herein, we describe that a U5-snRNP-interacting protein homolog STABILIZED1 (STA1) has pre-mRNA splicing activity for heat-inducible transcripts including HEAT STRESS TRANSCRIPTION FACTORs and various HEAT SHOCK PROTEINs for the establishment of heat stress tolerance in Arabidopsis (Arabidopsis thaliana). Our cell-based splicing reporter assay demonstrated STA1 acts on pre-mRNA splicing for specific subsets of stressrelated genes. Cellular reconstitution of heat-inducible transcription cascades supported the view that STA1-dependent premRNA splicing plays a role in DREB2A-dependent HSFA3 expression for heat-responsive gene expression. Further genetic analysis with a loss-of-function mutant sta1-1, STA1-expressing transgenic plants in Col background, and STA1-expressing transgenic plants in the sta1-1 background verified that STA1 is essential in expression of necessary genes including HSFA3 for two-step heat stress tolerance in plants. However, constitutive overexpression of the cDNA version of HSFA3 in the sta1-1 background is unable to execute plant heat stress tolerance in sta1-1. Consistently our global target analysis of STA1 showed that its splicing activity modulates a rather broad range of gene expression in response to heat treatment. The findings of this study reveal that heat-inducible STA1 activity for pre-mRNA splicing serves as a molecular regulatory mechanism underlying the plant stress tolerance to high-temperature stress.The splicing of precursor mRNA (pre-mRNA) is a necessary step for intron-containing gene expression in eukaryotic cells to produce mature transcripts for protein translation (Wahl et al., 2009). This process is highly ordered and tightly controlled by multisubunit spliceosome activity to mix and match introns and exons of pre-mRNAs. The high molecular weight spliceosome complex comprises small nuclear ribonucleoprotein particles (snRNPs) called U1, U2, U4/U6, and U5 snRNPs. For splicing of pre-mRNA introns, U1 snRNP recognizes the 59-splicing site, and U2 snRNP binds to the adenosine at the branch point of introns with the assistance of U2 auxiliary factors. U4/U6 and U5 trimeric snRNPs associate with each other and undergo a stepwise 39-splicing site cleavage process. Eventually, U5 snRNP dissociates from the complex along with a lariat form of the intron. In this process, U5 snRNP accurately and dynamically swaps interacting partners with other snRNP subunits (Wahl et al., 2009).

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Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate

Cited by 1,378 publications

References 35 publications

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

Peyer’s Patches as a Portal for DNA Delivery by <i>Lactococcus lactis</i> <i>in Vivo</i>

STABILIZED1 Modulates Pre-mRNA Splicing for Thermotolerance

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