A profusion of upstream open reading frame mechanisms in polyamine-responsive translational regulation

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.

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Ribosome stalling is responsible for arginine-specific translational attenuation in Neurospora crassa.

Molecular and Cellular Biology ◽

10.1128/mcb.17.9.4904 ◽

1997 ◽

Vol 17 (9) ◽

pp. 4904-4913 ◽

Cited By ~ 86

Author(s):

Z Wang ◽

M S Sachs

Keyword(s):

Neurospora Crassa ◽

Translational Regulation ◽

Open Reading Frame ◽

Upstream Open Reading Frame ◽

Initiation Codon ◽

Free System ◽

Reading Frame ◽

Ribosome Stalling ◽

A Cell ◽

Specific Regulation

The Neurospora crassa arg-2 upstream open reading frame (uORF) plays a role in negative arginine-specific translational regulation. Primer extension inhibition analyses of arg-2 uORF-containing RNA translated in a cell-free system in which arginine-specific regulation was retained revealed "toeprints" corresponding to ribosomes positioned at the uORF initiation and termination codons and at the downstream initiation codon. At high arginine concentrations, the toeprint signal corresponding to ribosomes at the uORF termination codon rapidly increased; a new, broad toeprint that represents additional ribosomes stalled on the uORF appeared 21 to 30 nucleotides upstream of this site; and the toeprint signal corresponding to ribosomes at the downstream initiation codon decreased. These data suggest that arginine increases ribosomal stalling and thereby decreases translation from the downstream initiation codon.

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Faculty Opinions recommendation of Mechanism of translational regulation by miR-2 from sites in the 5' untranslated region or the open reading frame.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.7491957.7786055 ◽

2011 ◽

Author(s):

Miles Wilkinson

Keyword(s):

Untranslated Region ◽

Translational Regulation ◽

Open Reading Frame ◽

Reading Frame

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

Genetics ◽

10.1093/genetics/142.1.117 ◽

1996 ◽

Vol 142 (1) ◽

pp. 117-127 ◽

Cited By ~ 3

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.

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