The RNA-binding protein RBP42 regulates cellular energy metabolism in mammalian-infective Trypanosoma brucei. Trypanosoma brucei brucei strain:427

RNA-binding proteins are key players in coordinated post-transcriptional regulation of functionally related genes, defined as RNA regulons. RNA regulons play particularly critical roles in parasitic trypanosomes, which exhibit unregulated co-transcription of long unrelated gene arrays. In this report, we present a systematic analysis of an essential RNA-binding protein, RBP42, in the mammalian-infective bloodstream form of African trypanosome, and show that RBP42 is a key regulator of parasite’s central carbon and energy metabolism. Using individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to identify genome-wide RBP42-RNA interactions, we show that RBP42 preferentially binds within the coding region of mRNAs encoding core metabolic enzymes. Global quantitative transcriptomic and proteomic analyses reveal that loss of RBP42 reduces the abundance of target mRNA-encoded proteins, but not target mRNA, suggesting a positive translational regulatory role of RBP42. To identify direct targets of RBP42, iCLIP was performed using two independent antibodies (anti-RBP42 and BB2), and from extracts prepared from cells following two increasing UV doses, at low (150mJ/cm2) and at high (300mJ/cm2) constant energy. For transcriptomic change mRNA-seq was performed on three biological replicate samples of before (day 0) and three consecutive days after (day 1 -3) RBP42 knockdown.

RNA结合蛋白（RNA-binding proteins）是协调调控功能相关基因转录后表达的核心参与者，这类基因调控单元被定义为RNA调控子（RNA regulons）。RNA调控子在寄生性锥虫中发挥尤为关键的作用，此类生物会对长片段且功能无关的基因阵列进行非调控性共转录。本研究针对感染哺乳动物的非洲锥虫（African trypanosome）血流形阶段中一种必需的RNA结合蛋白RBP42开展了系统性分析，证实RBP42是该寄生虫中枢碳代谢与能量代谢的核心调控因子。通过单核苷酸分辨率紫外交联免疫沉淀（individual-nucleotide resolution UV cross-linking and immunoprecipitation, iCLIP）技术在全基因组范围内鉴定RBP42与RNA的互作位点，我们发现RBP42优先结合编码核心代谢酶的mRNA的编码区。全局定量转录组与蛋白质组分析结果显示，敲低RBP42会降低靶mRNA编码的蛋白质丰度，但不影响靶mRNA自身的水平，这提示RBP42发挥正向翻译调控作用。为鉴定RBP42的直接靶标，我们使用两种独立抗体（抗RBP42与BB2），分别以低剂量（150mJ/cm²）与高剂量（300mJ/cm²）的恒定紫外能量照射细胞后制备提取物，开展iCLIP实验。针对转录组变化分析，我们对RBP42敲低前（第0天）以及敲低后连续3天（第1至3天）的三份生物学重复样本开展了mRNA测序（mRNA-seq）。