A5067772482- RNA and protein synthesis mechanisms
- RNA modifications and cancer
- RNA Research and Splicing
- RNA Interference and Gene Delivery
- Photosynthetic Processes and Mechanisms
- Plant Molecular Biology Research
- RNA regulation and disease
- Plant Reproductive Biology
- Viral Infections and Immunology Research
- Bacteriophages and microbial interactions
- Advanced biosensing and bioanalysis techniques
- Plant biochemistry and biosynthesis
- Animal Disease Management and Epidemiology
- CRISPR and Genetic Engineering
- Plant Virus Research Studies
- interferon and immune responses
- Peptidase Inhibition and Analysis
- Cancer-related molecular mechanisms research
New England Biolabs (United States)
2019-2024
University of Massachusetts Chan Medical School
2013-2020
PTC Therapeutics (United States)
2016-2017
University of Tennessee at Knoxville
2007-2014
Significance The drug ataluren restores activity to otherwise nonfunctional nonsense alleles, a capability possibly reflecting the insertion of near-cognate aminoacyl tRNAs at premature termination codons during protein synthesis. Because alleles comprise significant fraction all causing inherited disorders, drugs that promote such codon readthrough have broad therapeutic potential. However, effectiveness suppression depends on nature amino acids inserted each three codons. Here we...
Chemical modifications of RNAs have long been established as key modulators nonprotein-coding RNA structure and function in cells. There is a growing appreciation that messenger (mRNA) sequences responsible for directing protein synthesis can also be posttranscriptionally modified. The enzymatic incorporation mRNA has many potential outcomes, including changing stability, recruitment, translation. We tested how one the most common present coding regions, pseudouridine (Ψ), impacts using...
Premature termination codons (PTCs) in an mRNA ORF inactivate gene function by causing production of a truncated protein and destabilization the mRNA. Readthrough PTC allows ribosomal A-site insertion near-cognate tRNA, leading to synthesis full-length from otherwise defective To understand mechanism such nonsense suppression, we developed yeast system that purification sequence analysis readthrough products arising as consequence endogenous or compromised fidelity attributable loss Upf...
The use of synthetic RNA for therapeutics requires that the in vitro synthesis process be robust and efficient. technology used these - transcribed RNAs, predominantly using phage polymerases (RNAPs), is well established. However, transcripts synthesized with RNAPs are known to display an immune-stimulatory activity vivo often undesirable. Previous studies have identified double-stranded (dsRNA), a major by-product transcription (IVT) process, as trigger cellular immune responses. Here we...
Upstream open reading frames (uORFs) are protein coding elements in the 5′ leader of messenger RNAs. uORFs generally inhibit translation main ORF because ribosomes that perform elongation suffer either permanent or conditional loss reinitiation competence. After loss, competence may be regained by, at minimum, reacquisition a fresh methionyl-tRNA. The conserved h subunit Arabidopsis eukaryotic initiation factor 3 (eIF3) mitigates inhibitory effects certain uORFs. Here, we define more...
Abstract In vitro transcribed synthetic messenger RNAs (mRNAs) represent a novel therapeutic modality. To overcome the inherent immunogenicity, as well to increase efficacy of molecules, uridine analogs—such pseudouridine (Ψ) and N 1 -methyl-pseudouridine (m1Ψ), are incorporated in mRNA. decipher fidelity with which these modifications during transcription (IVT) process, we compared incorporation analogs different RNA polymerases. We demonstrate that m1Ψ is higher than Ψ. The nucleotide...
Abstract With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, development analytical tools for characterization long, complex RNAs has become essential. Tandem liquid chromatography–mass spectrometry (LC–MS/MS) permits direct assessment mRNA primary sequence modifications thereof without conversion to cDNA or amplification. It relies upon digestion with site-specific endoribonucleases generate pools short oligonucleotides that are then amenable MS-based...
The ribosome utilizes hydrogen bonding between mRNA codons and aminoacyl-tRNAs to ensure rapid accurate protein production. Chemical modification of nucleobases can adjust the strength pattern this alter synthesis. We investigate how N1-methylpseudouridine (m
Abstract Background Within the scanning model of translation initiation, reinitiation is a non-canonical mechanism that operates on mRNAs harboring upstream open reading frames. The h subunit eukaryotic initiation factor 3 (eIF3) boosts uORF-containing mRNA coding for Arabidopsis bZip transcription factor, AtbZip11, among others. RPL24B protein large ribosomal subunit, which encoded by SHORT VALVE1 , likewise fosters mRNAs, example auxin response factors (ARFs). Results Here we tested...
Translation of the coding potential a messenger RNA into protein molecule is fundamental process in all living cells and consumes large fraction metabolites energy resources growing cells. Moreover, translation has emerged as an important control point regulation gene expression. At level regulation, translational utilized to support specific life histories plants, particular their responses abiotic environment metabolites. This review summarizes diversity mechanisms plant cytoplasm,...
Abstract T7 RNA polymerase is commonly used to synthesize large quantities of for a wide variety applications, from basic science mRNA therapeutics. This in vitro system, while showing high fidelity many ways, also well known producing longer than encoded products, particularly under high-yield reaction conditions. Specifically, the resulting product pool contaminated by an often disperse collection cis-primed extension products. In addition reducing yield via conversion correctly...
Multiple factors influence translation termination efficiency, including nonsense codon identity and immediate context. To determine whether the relative position of a within an open reading frame (ORF) influences we quantitate production prematurely terminated and/or readthrough polypeptides from 26 alleles 3 genes expressed in yeast. The accumulation premature products extent for respective codons (PTCs) manifest marked dependence on PTC proximity to mRNA 3′ end. Premature increase...
The central nonsense-mediated mRNA decay (NMD) regulator, Upf1, selectively targets nonsense-containing mRNAs for rapid degradation. In yeast, Upf1 preferentially associates with that are NMD substrates, but the mechanism of its selective retention on these has yet to be elucidated. Previously, we demonstrated 40S ribosomal subunits. Here, define more precisely nature this association using conventional and affinity-based purification subunits, a two-hybrid screen identify Upf1-interacting...
Nonsense mutations resulting in a premature stop codon an open reading frame occur critical tumor suppressor genes large number of the most common forms cancers and are known to cause or contribute progression disease. Low molecular weight compounds that induce readthrough nonsense offer new means treating patients with genetic disorders from mutations. We have identified nucleoside analog clitocine as potent efficacious determined incorporation into RNA during transcription is prerequisite...
mRNA decapping commits a transcript to complete turnover in eukaryotic cells. In yeast, general requires the Dcp1/Dcp2 enzyme and set of activators, including Pat1, Dhh1, Edc3, Lsm1-7 complex. The exact function mode action each these activators largely remain elusive. Here, we analyzed role Edc3 decay yeast RPS28B mRNA, pathway triggered by negative-feedback autoregulatory mechanism. We show that Edc3-mediated either two orthologous proteins, Rps28a Rps28b, expressed from RPS28A genes,...
Summary The roles of individual eukaryotic translation initiation factor 3 (eIF3) subunits are largely unclear, although some essential, while others thought to have regulatory roles. ‘e’ subunit, also known as Int‐6/Int6, is a candidate for subunit it not essential in yeasts. eIF3e associates with the COP9 signalosome, and localizes nucleus certain tissues. To further elucidate eIF3e, we taken genetic approach using Arabidopsis model system. Overexpression results defects similar mutations...
Essentially all aboveground plant tissues develop from the stem cells in primary shoot apical meristem. Proliferation of cell population Arabidopsis meristem is tightly controlled by a feedback loop formed primarily homeodomain transcription factor WUSCHEL (WUS) and CLAVATA ligand-receptor system. In this study, it shown that mutation translation initiation factor, eIF3h, causes tendency to strikingly enlarged with elevated ectopic expression WUS CLAVATA3 (CLV3). Many mRNAs function...
Genome-wide assays performed in Arabidopsis and other organisms have revealed that the translation status of mRNAs responds dramatically to different environmental stresses genetic lesions apparatus. To identify additional features global landscape translational control, we used microarray analysis polysomal as well non-polysomal examine defects a poly(A) binding protein mutant, pab2 pab8, mutant large ribosomal subunit protein, rpl24b/shortvalve1. The mutation RPL24B stimulated ribosome...
Background Mutations in several subunits of eukaryotic translation initiation factor 3 (eIF3) cause male transmission defects Arabidopsis thaliana. To identify the stage pollen development at which eIF3 becomes essential it is desirable to examine viable and distinguish mutant from wild type. accomplish this we have developed a broadly applicable method track alleles that are not already tagged by visible marker gene through lineage Arabidopsis. Methodology/Principal Findings Fluorescence...
The ribosome relies on hydrogen bonding interactions between mRNA codons and incoming aminoacyl-tRNAs to ensure rapid accurate protein production. inclusion of chemically modified bases into mRNAs has the potential alter strength pattern synthesis. We investigated how Nl-methylpseudouridine (m 1 Ψ) modification, commonly incorporated therapeutic vaccine sequences, influences ability react with cognate near-cognate tRNAs release factors. find that presence a single m Ψ does not substantially...