Translational regulation in response to changes in amino acid availability in Neurospora crassa.

We examined the regulation of Neurospora crassa arg-2 and cpc-1 in response to amino acid availability.arg-2 encodes the small subunit of arginine-specific carbamoyl phosphate synthetase; it is subject to unique negative regulation by Arg and is positively regulated in response to limitation for many different amino acids through a mechanism known as cross-pathway control. cpc-1 specifies a transcriptional activator important for crosspathway control. Expression of these genes was compared with that of the cytochrome oxidase subunit V gene, cox-5. Analyses of mRNA levels, polypeptide pulse-labeling results, and the distribution of mRNA in polysomes indicated that Arg-specific negative regulation of arg-2 affected the levels of both arg-2 mRNA and arg-2 mRNA translation. Negative translational effects on arg-2 and positive translational effects on cpc-1 were apparent soon after cells were provided with exogenous Arg. In cells limited for His, increased expression of arg-2 and cpc-1, and decreased expression of cox-5, also had translational and transcriptional components. The arg-2 and cpc-1 transcripts contain upstream open reading frames (uORFs), as do their Saccharomyces cerevisiae homologs CPA1 and GCN4. We examined the regulation of arg-2-lacZ reporter genes containing or lacking the uORF start codon; the capacity for arg-2 uORF translation appeared critical for controlling gene expression.

Download Full-text

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.

Download Full-text

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2914-2924.1987 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2914-2924

Author(s):

A Hoekema ◽

R A Kastelein ◽

M Vasser ◽

H A de Boer

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Steady State ◽

Codon Usage ◽

Experimental Approach ◽

Mrna Translation ◽

Yeast Cells ◽

Expression Level ◽

Start Codon ◽

Mrna Levels

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

Download Full-text

Regulation of type I collagen mRNA in lung fibroblasts by cystine availability

Biochemical Journal ◽

10.1042/bj3310417 ◽

1998 ◽

Vol 331 (2) ◽

pp. 417-422 ◽

Cited By ~ 13

Author(s):

David C. RISHIKOF ◽

Ping-Ping KUANG ◽

Christine POLIKS ◽

Ronald H. GOLDSTEIN

Keyword(s):

Amino Acids ◽

Steady State ◽

Amino Acid ◽

Type I Collagen ◽

State Level ◽

Lung Fibroblasts ◽

Type I ◽

Mrna Levels ◽

Collagen Mrna ◽

Amino Acid Availability

The steady-state level of α1(I) collagen mRNA is regulated by amino acid availability in human lung fibroblasts. Depletion of amino acids decreases α1(I) collagen mRNA levels and repletion of amino acids induces rapid re-expression of α1(I) mRNA. In these studies, we examined the requirements for individual amino acids on the regulation of α1(I) collagen mRNA. We found that re-expression of α1(I) collagen mRNA was critically dependent on cystine but not on other amino acids. However, the addition of cystine alone did not result in re-expression of α1(I) collagen mRNA. Following amino acid depletion, the addition of cystine with selective amino acids increased α1(I) collagen mRNA levels. The combination of glutamine and cystine increased α1(I) collagen mRNA levels 6.3-fold. Methionine or a branch-chain amino acid (leucine, isoleucine or valine) also acted in combination with cystine to increase α1(I) collagen mRNA expression, whereas other amino acids were not effective. The prolonged absence of cystine lowered steady-state levels of α1(I) collagen mRNA through a mechanism involving decreases in both the rate of gene transcription as assessed by nuclear run-on experiments and mRNA stability as assessed by half-life determination in the presence of actinomycin D. The effect of cystine was not mediated via alterations in the level of glutathione, the major redox buffer in cells, as determined by the addition of buthionine sulphoximine, an inhibitor of γ-glutamylcysteine synthetase. These data suggest that cystine directly affects the regulation of α1(I) collagen mRNA.

Download Full-text

4E-BP is a target of the GCN2–ATF4 pathway during Drosophila development and aging

The Journal of Cell Biology ◽

10.1083/jcb.201511073 ◽

2016 ◽

Vol 216 (1) ◽

pp. 115-129 ◽

Cited By ~ 38

Author(s):

Min-Ji Kang ◽

Deepika Vasudevan ◽

Kwonyoon Kang ◽

Kyunggon Kim ◽

Jung-Eun Park ◽

...

Keyword(s):

Amino Acid ◽

Binding Sites ◽

Normal Development ◽

Mrna Translation ◽

Model Organisms ◽

Metabolic Labeling ◽

Amino Acid Availability ◽

Development And Aging ◽

Development Analysis ◽

Translational Inhibitor

Reduced amino acid availability attenuates mRNA translation in cells and helps to extend lifespan in model organisms. The amino acid deprivation–activated kinase GCN2 mediates this response in part by phosphorylating eIF2α. In addition, the cap-dependent translational inhibitor 4E-BP is transcriptionally induced to extend lifespan in Drosophila melanogaster, but through an unclear mechanism. Here, we show that GCN2 and its downstream transcription factor, ATF4, mediate 4E-BP induction, and GCN2 is required for lifespan extension in response to dietary restriction of amino acids. The 4E-BP intron contains ATF4-binding sites that not only respond to stress but also show inherent ATF4 activity during normal development. Analysis of the newly synthesized proteome through metabolic labeling combined with click chemistry shows that certain stress-responsive proteins are resistant to inhibition by 4E-BP, and gcn2 mutant flies have reduced levels of stress-responsive protein synthesis. These results indicate that GCN2 and ATF4 are important regulators of 4E-BP transcription during normal development and aging.

Download Full-text

Regulation of RAR beta 2 mRNA expression: evidence for an inhibitory peptide encoded in the 5'-untranslated region.

The Journal of Cell Biology ◽

10.1083/jcb.134.4.827 ◽

1996 ◽

Vol 134 (4) ◽

pp. 827-835 ◽

Cited By ~ 45

Author(s):

K Reynolds ◽

A M Zimmer ◽

A Zimmer

Keyword(s):

Amino Acid ◽

Untranslated Region ◽

Mutational Analysis ◽

Point Mutations ◽

Mrna Translation ◽

Start Codon ◽

Inhibitory Peptide ◽

Specific Expression ◽

Control Of Gene Expression ◽

Beta 2

Regulation of mRNA translation and stability plays an important role in the control of gene expression during embryonic development. We have recently shown that the tissue-specific expression of the RAR beta 2 gene in mouse embryos is regulated at the translational level by short upstream open reading frames (uORFs) In the 5'-untranslated region (Zimmer, A., A.M. Zimmer, and K. Reynolds. 1994. J. Cell Biol. 127:1111-1119). To gain insight into the molecular mechanism, we have performed a systematic mutational analysis of the uORFs. Two series of constructs were tested: in one series, each uORF was individually inactivated by introducing a point mutation in its start codon; in the second series, all but one ORF were inactivated. Our results indicate that individual uORFs may have different functions. uORF4 seems to inhibit translation of the major ORF in heart and brain, while uORFs 2 and 5 appear to be important for efficient translation in all tissues. To determine whether the polypeptide encoded by uORF4 or the act of translating it, is the significant event, we introduced point mutations to create silent mutations or amino acid substitutions in uORF4. Our results indicate that the uORF4 amino acid coding sequence is important for the inhibitory effect on translation of the downstream major ORF.

Download Full-text

Identification of GCD14 and GCD15, Novel Genes Required for Translational Repression of GCN4 mRNA in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/148.3.1007 ◽

1998 ◽

Vol 148 (3) ◽

pp. 1007-1020 ◽

Cited By ~ 2

Author(s):

Rafael Cuesta ◽

Alan G Hinnebusch ◽

Mercedes Tamame

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Translation Initiation ◽

Translational Control ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Mrna Levels ◽

New Genes ◽

General Translation

Abstract In Saccharomyces cerevisiae, expression of the transcriptional activator GCN4 increases at the translational level in response to starvation for an amino acid. The products of multiple GCD genes are required for efficient repression of GCN4 mRNA translation under nonstarvation conditions. The majority of the known GCD genes encode subunits of the general translation initiation factor eIF-2 or eIF-2B. To identify additional initiation factors in yeast, we characterized 65 spontaneously arising Gcd− mutants. In addition to the mutations that were complemented by known GCD genes or by GCN3, we isolated mutant alleles of two new genes named GCD14 and GCD15. Recessive mutations in these two genes led to highly unregulated GCN4 expression and to derepressed transcription of genes in the histidine biosynthetic pathway under GCN4 control. The derepression of GCN4 expression in gcd14 and gcd15 mutants occurred with little or no increase in GCN4 mRNA levels, and it was dependent on upstream open reading frames (uORFs) in GCN4 mRNA that regulate its translation. We conclude that GCD14 and GCD15 are required for repression of GCN4 mRNA translation by the uORFs under conditions of amino acid sufficiency. The gcd14 and gcd15 mutations confer a slow-growth phenotype on nutrient-rich medium, and gcd15 mutations are lethal when combined with a mutation in gcd13. Like other known GCD genes, GCD14 and GCD15 are therefore probably required for general translation initiation in addition to their roles in GCN4-specific translational control.

Download Full-text

Translational regulation of light-induced ribulose 1,5-bisphosphate carboxylase gene expression in amaranth

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2347-2353.1986 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2347-2353

Author(s):

J O Berry ◽

B J Nikolau ◽

J P Carr ◽

D F Klessig

Keyword(s):

Translational Regulation ◽

Small Subunit ◽

In Vitro Translation ◽

Mrna Levels ◽

Mrna Accumulation ◽

Bisphosphate Carboxylase ◽

Genes Encoding ◽

Translational Level

The regulation of the genes encoding the large and small subunits of ribulose 1,5-bisphosphate carboxylase was examined in amaranth cotyledons in response to changes in illumination. When dark-grown cotyledons were transferred into light, synthesis of the large- and small-subunit polypeptides was initiated very rapidly, before any increase in the levels of their corresponding mRNAs. Similarly, when light-grown cotyledons were transferred to total darkness, synthesis of the large- and small-subunit proteins was rapidly depressed without changes in mRNA levels for either subunit. In vitro translation or in vivo pulse-chase experiments indicated that these apparent changes in protein synthesis were not due to alterations in the functionality of the mRNAs or to protein turnover, respectively. These results, in combination with our previous studies, suggest that the expression of ribulose 1,5-bisphosphate carboxylase genes can be adjusted rapidly at the translational level and over a longer period through changes in mRNA accumulation.

Download Full-text

Amino acids inhibit Agrp gene expression via an mTOR-dependent mechanism

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00675.2006 ◽

2007 ◽

Vol 293 (1) ◽

pp. E165-E171 ◽

Cited By ~ 117

Author(s):

Christopher D. Morrison ◽

Xiaochun Xi ◽

Christy L. White ◽

Jianping Ye ◽

Roy J. Martin

Keyword(s):

Gene Expression ◽

Amino Acids ◽

Amino Acid ◽

Food Intake ◽

Mtor Signaling ◽

Acid Mixture ◽

Mrna Levels ◽

Amino Acid Availability ◽

The Brain ◽

Amino Acid Concentrations

Metabolic fuels act on hypothalamic neurons to regulate feeding behavior and energy homeostasis, but the signaling mechanisms mediating these effects are not fully clear. Rats placed on a low-protein diet (10% of calories) exhibited increased food intake ( P < 0.05) and hypothalamic Agouti-related protein ( Agrp) gene expression ( P = 0.002). Direct intracerebroventricular injection of either an amino acid mixture (RPMI 1640) or leucine alone (1 μg) suppressed 24-h food intake ( P < 0.05), indicating that increasing amino acid concentrations within the brain is sufficient to suppress food intake. To define a cellular mechanism for these direct effects, GT1–7 hypothalamic cells were exposed to low amino acids for 16 h. Decreasing amino acid availability increased Agrp mRNA levels in GT1–7 cells ( P < 0.01), and this effect was attenuated by replacement of the amino acid leucine ( P < 0.05). Acute exposure to elevated amino acid concentrations increased ribosomal protein S6 kinase phosphorylation via a rapamycin-sensitive mechanism, suggesting that amino acids directly stimulated mammalian target of rapamycin (mTOR) signaling. To test whether mTOR signaling contributes to amino acid inhibition of Agrp gene expression, GT1–7 cells cultured in either low or high amino acids for 16 h and were also treated with rapamcyin (50 nM). Rapamycin treatment increased Agrp mRNA levels in cells exposed to high amino acids ( P = 0.01). Taken together, these observations indicate that amino acids can act within the brain to inhibit food intake and that a direct, mTOR-dependent inhibition of Agrp gene expression may contribute to this effect.

Download Full-text