Transcriptome profile of Dictyostelium cell during ER-stress

The Unfolded Protein Response (UPR) is an adaptive pathway that restores cellular homeostasis after endoplasmic reticulum (ER) stress caused by an impairment of its protein folding capacity. The ER-resident kinase/ribonuclease Ire1 is the only UPR sensor that has been conserved during evolution from yeast to mammals; in these organisms, Ire1 transmits information from the ER to the nucleus trough the non-conventional splicing of Hac1 (yeast)/Xbp1 (metazoans) mRNA. We described the Dictyostelium discoideum ER-stress response and characterized its single bonafide Ire1 orthologue, IreA. We found that tunicamycin (TN) triggers a gene-expression program that increases the protein folding capacity of the ER and that alleviates ER protein load. Further, IreA resulted essential not only for cell-survival after TN-induced ER-stress, but also to accomplish about nearly 40% of the transcriptional changes induced upon a TN treatment. In addition, we described that autophagy is activated in Dictyostelium cells after a TN treatment and that autophagy-defective mutants exhibited increased sensitivity to this drug. The response of Dictyostelium cells to ER-stress involves the combined activation of an IreA-dependent gene expression program and the autophagy pathway. Overall design: Poly A RNA profile of wild-type (WT) and ireA- mutant Dictyostelium cells treated and non-treated with tunicamycin (TN) were generated by deep sequencing, in duplicate, using illumina MiSeq. The assembly of the data was performed using TopHat and Cufflinks software. Onthology enrichment analysis were performed with PANTHER and DAVID web tools

未折叠蛋白反应（Unfolded Protein Response, UPR）是一种适应性通路，可在蛋白质折叠能力受损导致内质网（endoplasmic reticulum, ER）应激后恢复细胞稳态。定位于内质网的激酶/核糖核酸酶Ire1是从酵母到哺乳动物进化过程中唯一保守的UPR传感器；在该类生物中，Ire1通过对Hac1（酵母）/Xbp1（后生动物）mRNA进行非经典剪接，将信号从内质网传递至细胞核。本研究阐述了盘基网柄菌（Dictyostelium discoideum）的内质网应激响应过程，并对其唯一的功能性Ire1同源物IreA进行了表征。研究发现，衣霉素（tunicamycin, TN）可触发一种基因表达程序，该程序能够增强内质网的蛋白质折叠能力并减轻内质网的蛋白质负载。进一步实验表明，IreA不仅是衣霉素诱导内质网应激后细胞存活的必需因子，同时也介导了近40%的衣霉素处理所诱导的转录变化。此外，本研究证实，衣霉素处理后盘基网柄菌细胞会激活自噬（autophagy）通路，且自噬缺陷突变体对该药物的敏感性显著升高。盘基网柄菌细胞对内质网应激的响应依赖于IreA介导的基因表达程序与自噬通路的协同激活。

总体实验设计：采用Illumina MiSeq测序平台，对经衣霉素（TN）处理与未处理的野生型（wild-type, WT）及ireA敲除突变体盘基网柄菌细胞进行双份重复深度测序，以获取聚腺苷酸RNA表达谱。数据组装工作通过TopHat与Cufflinks软件完成。基因本体富集分析借助PANTHER和DAVID在线工具进行。