scholarly journals Smart-ORF: a single-molecule method for accessing ribosome dynamics in both upstream and main open reading frames

2020 ◽  
Author(s):  
Anthony Gaba ◽  
Hongyun Wang ◽  
Trinisia Fortune ◽  
Xiaohui Qu
Keyword(s):  
Single Molecule ◽  
Translational Regulation ◽  
Mrna Translation ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Upstream Open Reading Frame ◽  
Translation Efficiency ◽  
Reading Frame ◽  
Reading Frames

Abstract Upstream open reading frame (uORF) translation disrupts scanning 43S flux on mRNA and modulates main open reading frame (mORF) translation efficiency. Current tools, however, have limited access to ribosome dynamics in both upstream and main ORFs of an mRNA. Here, we develop a new two-color in vitro fluorescence assay, Smart-ORF, that monitors individual uORF and mORF translation events in real-time with single-molecule resolution. We demonstrate the utility of Smart-ORF by applying it to uORF-encoded arginine attenuator peptide (AAP)-mediated translational regulation. The method enabled quantification of uORF and mORF initiation efficiencies, 80S dwell time, polysome formation, and the correlation between uORF and mORF translation dynamics. Smart-ORF revealed that AAP-mediated 80S stalling in the uORF stimulates the uORF initiation efficiency and promotes clustering of slower uORF-translating ribosomes. This technology provides a new tool that can reveal previously uncharacterized dynamics of uORF-containing mRNA translation.

scholarly journals A UV-Induced Mutation in Neurospora That Affects Translational Regulation in Response to Arginine

Genetics ◽  
1996 ◽  
Vol 142 (1) ◽  
pp. 117-127 ◽  
Author(s):  
Michael Freitag ◽  
Nelima Dighde ◽  
Matthew S Sachs
Keyword(s):  
Translational Control ◽  
Translational Regulation ◽  
Fusion Gene ◽  
Mrna Translation ◽  
Small Subunit ◽  
Upstream Open Reading Frame ◽  
Control Mechanisms ◽  
Carbamoyl Phosphate ◽  
Altered Expression ◽  
Reading Frame

The Neurospora crmsu arg-2 gene encodes the small subunit of arginine-specific carbamoyl phosphate synthetase. The levels of arg-2 mRNA and mRNA translation are negatively regulated by arginine. An upstream open reading frame (uORF) in the transcript’s 5′ region has been implicated in arginine-specific control. An arg-2-hph fusion gene encoding hygromycin phosphotransferase conferred arginine-regulated resistance to hygromycin when introduced into N. crassa. We used an arg-2-hph strain to select for UV-induced mutants that grew in the presence of hygromycin and arginine, and we isolated 46 mutants that had either of two phenotypes. One phenotype indicated altered expression of both arg-2-hph and urg-2 genes; the other, altered expression of urg-2-hph but not arg-2. One of the latter mutations, which was genetically closely linked to arg-2-hph, was recovered from the 5′ region of the arg-2-hph gene using PCR. Sequence analyses and transformation experiments revealed a mutation at uORF codon 12 (Asp to Asn) that abrogated negative regulation. Examination of the distribution of ribosomes on arg-2-hph transcripts showed that loss of regulation had a translational component, indicating the uORF sequence was important for Arg-specific translational control. Comparisons with other uORFS suggest common elements in translational control mechanisms.

scholarly journals Translational regulation of Arabidopsis XIPOTL1 is modulated by phosphocholine levels via the phylogenetically conserved upstream open reading frame 30

2012 ◽  
Vol 63 (14) ◽  
pp. 5203-5221 ◽  
Author(s):  
Fulgencio Alatorre-Cobos ◽  
Alfredo Cruz-Ramírez ◽  
Celine A. Hayden ◽  
Claudia-Anahí Pérez-Torres ◽  
Anne-Laure Chauvin ◽  
...  
Keyword(s):  
Translational Regulation ◽  
Open Reading Frame ◽  
Upstream Open Reading Frame ◽  
Reading Frame

SOME HINTS ON OPEN READING FRAME STATISTICS — HOW ORF LENGTH DEPENDS ON SELECTION

1999 ◽  
Vol 10 (04) ◽  
pp. 635-643 ◽  
Author(s):  
AGNIESZKA GIERLIK ◽  
PAWEŁ MACKIEWICZ ◽  
MARIA KOWALCZUK ◽  
STANISŁAW CEBRAT ◽  
MIROSŁAW R. DUDEK
Keyword(s):  
Genetic Code ◽  
Dna Sequences ◽  
Nucleotide Composition ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Antisense Strand ◽  
Coding Sequences ◽  
Reading Frame ◽  
Intergenic Sequences ◽  
Reading Frames

Coding sequences of DNA generate Open Reading Frames (ORFs) inside them with much higher frequency than random DNA sequences do, especially in the antisense strand. This is a specific feature of the genetic code. Since coding sequences are selected for their length, the generated ORFs are indirect results of this selection and their length is also influenced by selection. That is why ORFs found in any genome, even much longer ones than those spontaneously generated in random DNA sequences, should be considered as two different sets of ORFs: The first one coding for proteins, the second one generated by the coding ORFs. Even intergenic sequences possess greater capacity for generating ORFs than random DNA sequences of the same nucleotide composition, which seems to be a premise that intergenic sequences were generated from coding sequences by recombinational mechanisms.

scholarly journals Translation Termination Reinitiation between Open Reading Frame 1 (ORF1) and ORF2 Enables Capsid Expression in a Bovine Norovirus without the Need for Production of Viral Subgenomic RNA

2008 ◽  
Vol 82 (17) ◽  
pp. 8917-8921 ◽  
Author(s):  
Christopher J. McCormick ◽  
Omar Salim ◽  
Paul R. Lambden ◽  
Ian N. Clarke
Keyword(s):  
Hepg2 Cells ◽  
Nonstructural Protein ◽  
Translation Termination ◽  
Surrogate Marker ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Subgenomic Rna ◽  
Reading Frame ◽  
Reading Frames ◽  
Open Reading Frame 1

ABSTRACT A generally accepted view of norovirus replication is that capsid expression requires production of a subgenomic transcript, the presence of capsid often being used as a surrogate marker to indicate the occurrence of viral replication. Using a polymerase II-based baculovirus delivery system, we observed capsid expression following introduction of a full-length genogroup 3 norovirus genome into HepG2 cells. However, capsid expression occurred as a result of a novel translation termination/reinitiation event between the nonstructural-protein and capsid open reading frames, a feature that may be unique to genogroup 3 noroviruses.

scholarly journals The Nitric Oxide Cycle: Translational Regulation of Argininosuccinate Synthase by an Upstream Open Reading Frame

2006 ◽  
Vol 20 (4) ◽  
Author(s):  
Laura Carol Pendleton ◽  
Lindsey R. Jackson ◽  
Sarah L. Yong ◽  
Larry P. Solomonson ◽  
Duane C. Eichler
Keyword(s):  
Nitric Oxide ◽  
Translational Regulation ◽  
Open Reading Frame ◽  
Upstream Open Reading Frame ◽  
Reading Frame ◽  
Argininosuccinate Synthase ◽  
Nitric Oxide Cycle

scholarly journals Autonomous functionality of an upstream open reading frame in polycistronic mammalian mRNAs

2018 ◽  
Author(s):  
Shohei Kitano ◽  
Gabriel Pratt ◽  
Keizo Takao ◽  
Yasunori Aizawa
Keyword(s):  
Brain Regions ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Upstream Open Reading Frame ◽  
Translation Regulation ◽  
Reading Frame ◽  
Eukaryotic Translation ◽  
Upstream Open Reading Frames ◽  
Human And Mouse ◽  
Reading Frames

SUMMARYUpstream open reading frames (uORFs) are established as cis-acting elements for eukaryotic translation of annotated ORFs (anORFs) located on the same mRNAs. Here, we identified a mammalian uORF with functions that are independent from anORF translation regulation. Bioinformatics screening using ribosome profiling data of human and mouse brains yielded 308 neurologically vital genes from which anORF and uORFs are polycistronically translated in both species. Among them, Arhgef9 contains a uORF named SPICA, which is highly conserved among vertebrates and stably translated only in specific brain regions of mice. Disruption of SPICA translation by ATG-to-TAG substitutions did not perturb translation or function of its anORF product, collybistin. SPICA-null mice displayed abnormal maternal reproductive performance and enhanced anxiety-like behavior, characteristic of ARHGEF9-associated neurological disorders. This study demonstrates that mammalian uORFs can be independent genetic units, revising the prevailing dogma of the monocistronic gene in mammals, and even eukaryotes.

scholarly journals Saccharomyces cerevisiae positive regulatory gene PET111 encodes a mitochondrial protein that is translated from an mRNA with a long 5' leader.

1987 ◽  
Vol 7 (8) ◽  
pp. 2728-2734 ◽  
Author(s):  
C A Strick ◽  
T D Fox
Keyword(s):  
Amino Acids ◽  
Regulatory Gene ◽  
Mitochondrial Protein ◽  
Nuclear Gene ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Amino Terminal ◽  
Interesting Pattern ◽  
Reading Frames

The yeast nuclear gene PET111 is required specifically for translation of the mitochondrion-coded mRNA for cytochrome c oxidase subunit II. We have determined the nucleotide sequence of a 3-kilobase segment of DNA that carries PET111. The sequence contains a single long open reading frame that predicts a basic protein of 718 amino acids. The PET111 gene product is a mitochondrial protein, since a hybrid protein which includes the amino-terminal 154 amino acids of PET111 fused to beta-galactosidase is specifically associated with mitochondria. PET111 is translated from a 2.9-kilobase mRNA which, interestingly, has an extended 5'-leader sequence containing four short open reading frames upstream of the long open reading frame. These open reading frames exhibit an interesting pattern of overlap with each other and with the PET111 reading frame.

Saccharomyces cerevisiae positive regulatory gene PET111 encodes a mitochondrial protein that is translated from an mRNA with a long 5' leader

1987 ◽  
Vol 7 (8) ◽  
pp. 2728-2734
Author(s):  
C A Strick ◽  
T D Fox
Keyword(s):  
Amino Acids ◽  
Regulatory Gene ◽  
Mitochondrial Protein ◽  
Nuclear Gene ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Amino Terminal ◽  
Interesting Pattern ◽  
Reading Frames

The yeast nuclear gene PET111 is required specifically for translation of the mitochondrion-coded mRNA for cytochrome c oxidase subunit II. We have determined the nucleotide sequence of a 3-kilobase segment of DNA that carries PET111. The sequence contains a single long open reading frame that predicts a basic protein of 718 amino acids. The PET111 gene product is a mitochondrial protein, since a hybrid protein which includes the amino-terminal 154 amino acids of PET111 fused to beta-galactosidase is specifically associated with mitochondria. PET111 is translated from a 2.9-kilobase mRNA which, interestingly, has an extended 5'-leader sequence containing four short open reading frames upstream of the long open reading frame. These open reading frames exhibit an interesting pattern of overlap with each other and with the PET111 reading frame.

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