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Transgenic zebrafish model to study translational control mediated by upstream open reading frame of human chop gene.

ABSTRACT: Upstream open reading frame (uORF)-mediated translational inhibition is important in controlling key regulatory genes expression. However, understanding the underlying molecular mechanism of such uORF-mediated control system in vivo is challenging in the absence of an animal model. Therefore, we generated a zebrafish transgenic line, termed huORFZ, harboring a construct in which the uORF sequence from human CCAAT/enhancer-binding protein homologous protein gene (huORF(chop)) is added to the leader of GFP and is driven by a cytomegalovirus promoter. The translation of transgenic huORF(chop)-gfp mRNA was absolutely inhibited by the huORF(chop) cassette in huORFZ embryos during normal conditions, but the downstream GFP was only apparent when the huORFZ embryos were treated with endoplasmic reticulum (ER) stresses. Interestingly, the number and location of GFP-responsive embryonic cells were dependent on the developmental stage and type of ER stresses encountered. These results indicate that the translation of the huORF(chop)-tag downstream reporter gene is controlled in the huORFZ line. Moreover, using cell sorting and microarray analysis of huORFZ embryos, we identified such putative factors as Nrg/ErbB, PI3K and hsp90, which are involved in huORF(chop)-mediated translational control under heat-shock stress. Therefore, using the huORFZ embryos allows us to study the regulatory network involved in human uORF(chop)-mediated translational inhibition.

SUBMITTER: Lee HC

PROVIDER: S-EPMC3203588 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Transgenic zebrafish model to study translational control mediated by upstream open reading frame of human chop gene.

Lee Hung-Chieh HC Chen Yi-Jiun YJ Liu Yu-Wei YW Lin Kai-Yen KY Chen Shaio-Wen SW Lin Cheng-Yung CY Lu Yi-Chin YC Hsu Pei-Chun PC Lee Sheng-Chung SC Tsai Huai-Jen HJ

Nucleic acids research 20110825 20

Upstream open reading frame (uORF)-mediated translational inhibition is important in controlling key regulatory genes expression. However, understanding the underlying molecular mechanism of such uORF-mediated control system in vivo is challenging in the absence of an animal model. Therefore, we generated a zebrafish transgenic line, termed huORFZ, harboring a construct in which the uORF sequence from human CCAAT/enhancer-binding protein homologous protein gene (huORF(chop)) is added to the lead ...[more]

PMID: 21873270

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Project description:Translation of an mRNA in eukaryotes starts at AUG in most cases. Near-cognate codons (NCCs) such as UUG, ACG and AUU are also used as start sites at low levels in S. cerevisiae. Initiation from NCCs or AUGs in the 5’-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature. Using reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs in S. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 ˚C relative to 30 ˚C and decreased efficiency at 20 ˚C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5’-leader relative to the 5’-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation. Overall, our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.

Dataset Information

Transgenic zebrafish model to study translational control mediated by upstream open reading frame of human chop gene.

Publications

Transgenic zebrafish model to study translational control mediated by upstream open reading frame of human chop gene.

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