Effect of the depletion of PuREBP1 (Tb927.4.4550) on the transcriptome of Trypanosoma brucei [RNAi]

We have previously characterized a short RNA stem-loop cis-element within the 3'-UTR of the NT8 nucleobase transporter mRNA (PuRE, Purine Responsive Element) that is necessary and sufficient to confer a strong repression of gene expression in response to purines in the human parasite Trypanosoma brucei (PMID: 24813448). In this study, we have identified a protein complex composed of two RNA-binding proteins (Tb927.4.4550 and Tb927.3.3060, named here as PuREBP1 and PuREBP2 respectively) that binds to the PuRE in vitro and to NT8 mRNA in vivo. Depletion of PuREBP1 by RNAi results in the upregulation of just NT8 and the mRNAs encoding the amino acid transporter AATP6 paralogues. Moreover, we found that the PuREBP1/2 complex is associated with only a handful of mRNAs, and that it is responsible for the observed purine-dependent regulation of NT8 expression. Depletion of PuRBP1 results in the upregulation of just two mRNAs (encoding membrane transporters NT8 and AATP6) and downregulation of four other mRNAs (including the one encoding PuREBP1, as expected) Overall design: Total RNA was obtained from non-induced (control) vs PuREBP1-depleted trypanosomes after 48h of RNAi induction. Two biological replicates = 4 samples in total

我们此前已在人类寄生虫布氏锥虫（*Trypanosoma brucei*）NT8核苷碱基转运蛋白mRNA的3'非翻译区（3'-UTR）中鉴定出一段RNA茎环顺式作用元件——嘌呤响应元件（Purine Responsive Element，缩写PuRE），该元件是响应嘌呤信号强效抑制基因表达的必要且充分条件（相关研究发表于PubMed编号24813448的文献）。本研究中，我们鉴定出一种由两种RNA结合蛋白组成的蛋白复合物（分别为Tb927.4.4550与Tb927.3.3060，本文中将其命名为PuREBP1与PuREBP2），该复合物可在体外结合PuRE，在体内结合NT8 mRNA。通过RNA干扰（RNAi）敲低PuREBP1，仅会导致NT8以及编码氨基酸转运蛋白AATP6旁系同源物的mRNA表达上调。此外，我们发现PuREBP1/2复合物仅与少量mRNA结合，并负责调控已观测到的、依赖嘌呤信号的NT8基因表达。敲低PuREBP1仅会使两种mRNA（编码膜转运蛋白NT8与AATP6）表达上调，同时使另外四种mRNA表达下调（包括编码PuREBP1的mRNA，符合预期）。实验整体设计：在RNA干扰诱导48小时后，分别提取未诱导组（对照组）与PuREBP1敲低组锥虫的总RNA；设置2次生物学重复，共计4个样本。