RNA-Seq analysis of wild-type and ttpA-D450trx mutant D. discoideum amebae. RNA-Seq analysis of wild-type and ttpA-D450trx mutant D. discoideum amebae

In many eukaryotes, mRNAs containing specific AU-rich motifs are regulated by proteins of the tristetraprolin (TTP) family, which bind to these motifs through a tandem zinc finger (TZF) domain. This binding leads to promotion of subsequent deadenylation and decay, partly through a conserved carboxyl-terminal CNOT1 binding domain. We explored the physiological role of the single TTP family member (TtpA) expressed in Dictyostelium discoideum. This study shows the effect of a carboxyl-terminal truncation (ttpA-D450trx), which removed the predicted CNOT1 binding domain, evaluated in amebae in growth medium during surface culture. The cells exhibited external and gene expression phenotypes that were very similar to those of two distinct null mutants, raising the possibility that this domain is necessary for TtpA-promoted mRNA decay in this species. Overall design: Dictyostelium discoideum amebae (strain AX3) were grown in normal growth medium under axenic conditions in plastic flasks until they were approximately 80% confluent. Four cultures of wild-type cells and four cultures of ttpA-D450trx cells were then frozen and used for RNA extraction and RNA-Seq analysis.

在众多真核生物中，携带特定AU富集基序的mRNA会受三联脯氨酸蛋白（tristetraprolin, TTP）家族蛋白的调控，这类蛋白通过串联锌指（tandem zinc finger, TZF）结构域与上述基序结合。该结合过程可促进后续的脱腺苷酸化与mRNA降解，这一过程部分依赖于一段保守的羧基端CNOT1结合结构域。我们针对盘基网柄菌（Dictyostelium discoideum）中唯一表达的TTP家族成员TtpA，探究了其生理功能。本研究分析了羧基端截短突变体ttpA-D450trx的效应，该突变体缺失了预测的CNOT1结合结构域，实验在生长培养基中表面培养的变形虫内开展。该突变体细胞的外部表型与基因表达谱均与两种独立的TtpA敲除突变体高度相似，提示该结构域是该物种中TtpA介导的mRNA降解所必需的。实验总体设计：将盘基网柄菌变形虫（菌株AX3）于无菌条件下在塑料培养瓶中的正常生长培养基内培养，至细胞汇合度约80%。随后分别收集4瓶野生型细胞与4瓶ttpA-D450trx突变体细胞，冻存后用于RNA提取及RNA测序（RNA-Seq）分析。