Inosine-specific cleavage of RNA for microarray analysis of RNA A-to-I editing targets.

Dysregulations of RNA A-to-I editing are associated with developmental defects in mouse and human diseases. Although several methods of identifying RNA A-to-I editing sites are currently available, most of the critical editing targets responsible for the important biological functions of adenosine deaminases that act on RNA (ADARs) remain unknown. Here we report a modified I-specific cleavage method that improves the quality of the RNA product. Preliminary microarray comparison of RNAs subjected to I-specific cleavage or mock digestion reported 165 genes that showed more than 0.2-fold reductions due to the cleavage. Six of the 165 genes were randomly selected for further verification, and three were verified to be targets of I-specific cleavage. This method may provide an alternative method of identifying novel RNA A-to-I editing sites using a microarray and facilitate the inquiry into the roles of RNA A-to-I editing in various biological processes.

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

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.