uORFs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs

Ivanov, Ivaylo P.; Loughran, Gary; Atkins, John F.

doi:10.1073/pnas.0801590105

Cited by 93 publications

(102 citation statements)

References 41 publications

(38 reference statements)

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“…These are the same uORFs that we found do suppress translation, but spermine had no effect. Polyamine-responsive uORF elements have been found for other polyamine metabolic enzymes (24,28), and our data do not exclude the possibility of responses to other polyamines or polyamine-related compounds. Alternatively, these uORFs could act as constitutive repressors.…”

Section: Discussionmentioning

confidence: 67%

Polyamine-Regulated Translation of Spermidine/Spermine-N¹-Acetyltransferase

Pérez-Leal

Barrero

Clarkson

et al. 2012

Molecular and Cellular Biology

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Rapid synthesis of the polyamine catabolic enzyme spermidine/spermine-N 1 -acetyltransferase (SSAT) in response to increased polyamines is an important polyamine homeostatic mechanism. Indirect evidence has suggested that there is an important control mechanism involving the release of a translational repressor protein that allows the immediate initiation of SSAT protein synthesis without RNA transcription, maturation, or translocation. To identify a repressor protein, we used a mass spectroscopy-based RNA-protein interaction system and found six proteins that bind to the coding region of SSAT mRNA. Individual small interfering RNA (siRNA) experiments showed that nucleolin knockdown enhances SSAT translation. Nucleolin exists in several isoforms, and we report that the isoform that binds to SSAT mRNA undergoes autocatalysis in the presence of polyamines, a result suggesting that there is a negative feedback system that helps control the cellular content of polyamines. Preliminary molecular interaction data show that a nucleolin isoform binds to a 5= stem-loop of the coding region of SSAT mRNA. The glycine/arginine-rich C terminus of nucleolin is required for binding, and the four RNA recognition motif domains are included in the isoform that blocks SSAT translation. Understanding SSAT translational control mechanisms has the potential for the development of therapeutic strategies against cancer and obesity. P olyamines are small positively charged molecules present in all cells. The common polyamines putrescine, spermidine, and spermine are essential for cell growth. The rate of synthesis and content both increase with increased cell proliferation (47). Polyamine functions include stabilization of polynucleotides, regulation of transcription and translation, control of enzyme activities, modulation of ion channels, and response to oxidative stress (42, 61). Because so many processes are affected, levels are maintained within a relatively narrow range by shifts in anabolism/catabolism and import/export (1, 46). Manipulation of polyamine metabolism has been an anticancer strategy, with pool depletion in tumor cells used as a surrogate marker of efficacy (21,34,38,48).Many mechanisms contribute to control of eukaryotic polyamine metabolic enzymes (15,41,43,50). Ornithine decarboxylase (ODC), the rate-limiting anabolic enzyme, is regulated allosterically, at transcription, at translation, and by "ODC antizyme," a protein that binds to ODC monomers, thereby blocking homodimerization required for activity and accelerating monomer degradation (41). Antizyme itself is regulated by a translation control mechanism involving polyamine-induced ribosomal frameshifting (15, 35). Spermidine/spermine-N 1 -acetyltransferase (SSAT) is the principal catabolic regulator. SSAT acetylates spermidine and spermine using acetyl-coenzyme A (CoA), thereby altering their charge and facilitating excretion. SSAT basal activity is very low but increases quickly when polyamines are in excess (11). There is evidence for transcription, translati...

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Section: Discussionmentioning

confidence: 67%

Polyamine-Regulated Translation of Spermidine/Spermine-N¹-Acetyltransferase

Pérez-Leal

Barrero

Clarkson

et al. 2012

Molecular and Cellular Biology

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“…Intriguingly, there have been hints that uORFs may be more common than initially believed. Translation can initiate (mostlikely using tRNA i Met ) at near cognate codons (NCCs)-sequences that differ from the canonical AUG start codon by one nucleotide (Zitomer et al 1984;Peabody 1989;Ivanov et al 2008Ivanov et al , 2011Kolitz et al 2009;Starck et al 2012;Kochetov et al 2013;Gao et al 2014). Thus, many NCC uORFs may be lying hidden in eukaryotic genomes.…”

Section: [Supplemental Materials Is Available For This Article]mentioning

confidence: 99%

Conserved non-AUG uORFs revealed by a novel regression analysis of ribosome profiling data

et al. 2017

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Upstream open reading frames (uORFs), located in transcript leaders (5′ UTRs), are potent cis-acting regulators of translation and mRNA turnover. Recent genome-wide ribosome profiling studies suggest that thousands of uORFs initiate with non-AUG start codons. Although intriguing, these non-AUG uORF predictions have been made without statistical control or validation; thus, the importance of these elements remains to be demonstrated. To address this, we took a comparative genomics approach to study AUG and non-AUG uORFs. We mapped transcription leaders in multiple Saccharomyces yeast species and applied a novel machine learning algorithm (uORF-seqr) to ribosome profiling data to identify statistically significant uORFs. We found that AUG and non-AUG uORFs are both frequently found in Saccharomyces yeasts. Although most non-AUG uORFs are found in only one species, hundreds have either conserved sequence or position within Saccharomyces. uORFs initiating with UUG are particularly common and are shared between species at rates similar to that of AUG uORFs. However, non-AUG uORFs are translated less efficiently than AUG-uORFs and are less subject to removal via alternative transcription initiation under normal growth conditions. These results suggest that a subset of non-AUG uORFs may play important roles in regulating gene expression.

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“…These sequences and 44 known AAP sequences (8) were compared as described (30). Specific details are indicated in the legends of Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Bioinformatic Analysis of AAP Conservation-For comparative sequence assembly and analysis, 76 new AAP sequences were compiled using methodology described previously (30). These sequences and 44 known AAP sequences (8) were compared as described (30).…”

Section: Methodsmentioning

confidence: 99%

Sequence Requirements for Ribosome Stalling by the Arginine Attenuator Peptide

Spevak

Ivanov

Sachs

2010

Journal of Biological Chemistry

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The 5 regions of eukaryotic mRNAs often contain upstream open reading frames (uORFs). The Neurospora crassa arg-2 uORF encodes the 24-residue arginine attenuator peptide (AAP). This regulatory uORF-encoded peptide, which is evolutionarily conserved in fungal transcripts specifying an arginine biosynthetic enzyme, functions as a nascent peptide within the ribosomal tunnel and negatively regulates gene expression. The nascent AAP causes ribosomes to stall at the uORF stop codon in response to arginine, thus, blocking ribosomes from reaching the ARG-2 initiation codon. Here scanning mutagenesis with alanine and proline was performed to systematically determine which AAP residues were important for conferring regulation. Changing many of the most highly conserved residues (Asp-12, Tyr-13, Lys-14, and Trp-19) abolished regulatory function. The minimal functional domain of the AAP was determined by positioning AAP sequences internally within a large polypeptide. Pulse-chase analyses revealed that residues 9 -20 of the AAP composed the minimal domain that was sufficient to confer regulatory function. An extensive analysis of predicted fungal AAPs revealed that the minimal functional domain of the N. crassa AAP corresponded closely to the region that was most highly conserved among the fungi. We also observed that the tripeptide RGD could function similarly to arginine in triggering AAP-mediated ribosome stalling. These studies provide a better understanding of the elements required for a nascent peptide and a small regulatory molecule to control translational processes.

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uORFs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs

Cited by 93 publications

References 41 publications

Polyamine-Regulated Translation of Spermidine/Spermine-N¹-Acetyltransferase

Polyamine-Regulated Translation of Spermidine/Spermine-N¹-Acetyltransferase

Conserved non-AUG uORFs revealed by a novel regression analysis of ribosome profiling data

Sequence Requirements for Ribosome Stalling by the Arginine Attenuator Peptide

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uORFs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs

Cited by 93 publications

References 41 publications

Polyamine-Regulated Translation of Spermidine/Spermine-N1-Acetyltransferase

Polyamine-Regulated Translation of Spermidine/Spermine-N1-Acetyltransferase

Conserved non-AUG uORFs revealed by a novel regression analysis of ribosome profiling data

Sequence Requirements for Ribosome Stalling by the Arginine Attenuator Peptide

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Polyamine-Regulated Translation of Spermidine/Spermine-N¹-Acetyltransferase

Polyamine-Regulated Translation of Spermidine/Spermine-N¹-Acetyltransferase