Arginine-specific Regulation Mediated by the Neurospora crassa arg-2 Upstream Open Reading Frame in a Homologous, Cell-free in Vitro Translation System

Zhong, Wei; Sachs, Matthew S.

doi:10.1074/jbc.272.1.255

Cited by 63 publications

(90 citation statements)

References 32 publications

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“…Primer extension inhibition (toeprint) assays used cell-free translation extracts derived from Neurospora crassa that were prepared as described (18) and treated with cycloheximide upon addition of the ␣-globin-CHOP-Luc mRNA to measure initiating ribosomes (time 0), 15 min after addition of the transcript to measure ribosomal localization during steady-state translation (time 15), or left untreated to measure prominent ribosomal stalls. Toeprint assays were conducted using primer ZW4 (5Ј-TCCAGGAACCAGGGCGTA-3Ј) as previously described (19).…”

Section: Methodsmentioning

confidence: 99%

Ribosome Elongation Stall Directs Gene-specific Translation in the Integrated Stress Response

Young

Palam

et al. 2016

Journal of Biological Chemistry

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Upon exposure to environmental stress, phosphorylation of the ␣ subunit of eIF2 (eIF2␣-P) represses global protein synthesis, coincident with preferential translation of gene transcripts that mitigate stress damage or alternatively trigger apoptosis. Because there are multiple mammalian eIF2 kinases, each responding to different stress arrangements, this translational control scheme is referred to as the integrated stress response (ISR). Included among the preferentially translated mRNAs induced by eIF2␣-P is that encoding the transcription factor CHOP (DDIT3/GADD153). Enhanced levels of CHOP promote cell death when ISR signaling is insufficient to restore cell homeostasis. Preferential translation of CHOP mRNA occurs by a mechanism involving ribosome bypass of an inhibitory upstream ORF (uORF) situated in the 5-leader of the CHOP mRNA. In this study, we used biochemical and genetic approaches to define the inhibitory features of the CHOP uORF and the biological consequences of loss of the CHOP uORF on CHOP expression during stress. We discovered that specific sequences within the CHOP uORF serve to stall elongating ribosomes and prevent ribosome reinitiation at the downstream CHOP coding sequence. As a consequence, deletion of the CHOP uORF substantially increases the levels and modifies the pattern of induction of CHOP expression in the ISR. Enhanced CHOP expression leads to increased expression of key CHOP target genes, culminating in increased cell death in response to stress.

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Section: Methodsmentioning

confidence: 99%

Ribosome Elongation Stall Directs Gene-specific Translation in the Integrated Stress Response

Young

Palam

et al. 2016

Journal of Biological Chemistry

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“…Plasmids pPQ-LUC-pA30 (Wang and Sachs 1997) and pT3-LUC-pA98 (Bergamini et al 2000) plasmids were kindly provided by Matthew Sachs (Oregon Health and Science University, Portland, OR) and Matthias Hentze (EMBL, Heidelberg, Germany), respectively. pET-19b-hnRNPL, pET19b-PTB, and pTandem1-hnNASAP plasmids were kindly provided by Tom Cooper (Baylor College of Medicine, Houston, TX) and Paul Fox (Cleveland Clinic, Cleveland, OH).…”

Section: Methodsmentioning

confidence: 99%

An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

Zeenko

Wang

Majumder

et al. 2008

RNA

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In vitro translation systems are used to investigate translational mechanisms and to synthesize proteins for characterization. Most available mammalian cell-free systems have reduced efficiency due to decreased translation initiation caused by phosphorylation of the initiation factor eIF2a on Ser51. We describe here a novel cell-free protein synthesis system using extracts from cultured mouse embryonic fibroblasts that are homozygous for the Ser51 to-Ala mutation in eIF2a (A/A cells). The translation efficiency of a capped and polyadenylated firefly luciferase mRNA in A/A cell extracts was 30-fold higher than in wild-type extracts. Protein synthesis in extracts from A/A cells was active for at least 2 h and generated up to 20 mg/mL of luciferase protein. Additionally, the A/A cell-free system faithfully recapitulated the selectivity of in vivo translation for mRNA features; translation was stimulated by a 59-end cap (m 7 GpppN) and a 39-end poly(A) tail in a synergistic manner. The system also showed similar efficiencies of cap-dependent and IRES-mediated translation (EMCV IRES). Significantly, the A/A cell-free system supported the post-translational modification of proteins, as shown by glycosylation of the HIV type-1 gp120 and cleavage of the signal peptide from b-lactamase. We propose that cell-free systems from A/A cells can be a useful tool for investigating mechanisms of mammalian mRNA translation and for the production of recombinant proteins for molecular studies. In addition, cell-free systems from differentiated cells with the Ser51Ala mutation should provide a means for investigating cell type-specific features of protein synthesis.

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“…For in vitro studies, capped, polyadenylated RNA was synthesized with T7 RNA polymerase from EcoRIlinearized plasmid templates and yields of RNA were quantified as described [12]. For in vivo studies, capped and polyadenylated mRNAs were prepared as previously described [13,14]; the yield of RNA transcripts were quantified by measuring UV-absorbance at 260nm and the quality of each RNA preparation assessed by SYBR gold (Molecular Probes; Eugene, OR) staining following fractionation on formaldehyde/1% agarose gels.…”

Section: Preparation Of Synthetic Rnamentioning

confidence: 99%

her-2 upstream open reading frame effects on the use of downstream initiation codons

Spevak¹,

Park²,

Geballe³

et al. 2006

Biochemical and Biophysical Research Communications

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The her-2 (neu, erbB-2) oncogene encodes a 185-kDa transmembrane receptor tyrosine kinase. HER2 overexpression occurs in numerous primary human tumors and contributes to 25-30% of breast and ovarian carcinomas. Synthesis of HER2 is controlled in part by an upstream open reading frame (uORF) present in the transcript. We used synthetic capped and polyadenylated mRNAs containing sequences derived from the 5′ region of the her-2 transcript fused to firefly luciferase (LUC) reporter to examine this ORF's effect on translation in cell-free systems derived from reticulocytes, wheat germ and Neurospora crassa, and in RNA-transfected HeLa cells. The uORF reduced translation of the downstream cistron in all systems. [ 35 S]Met-labeling of in vitro translation products obtained indicated that the uORF also affected downstream start-site selection. Primer extension inhibition (toeprint) assays of ribosomes loaded at initiation codons in reticulocyte lysates indicated that the uORF affected the interaction of ribosomes with the primary her-2 AUG codon.

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Arginine-specific Regulation Mediated by the Neurospora crassa arg-2 Upstream Open Reading Frame in a Homologous, Cell-free in Vitro Translation System

Cited by 63 publications

References 32 publications

Ribosome Elongation Stall Directs Gene-specific Translation in the Integrated Stress Response

Ribosome Elongation Stall Directs Gene-specific Translation in the Integrated Stress Response

An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

her-2 upstream open reading frame effects on the use of downstream initiation codons

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