Instituto de Parasitología y Biomedicina "López-Neyra" (IPBLN), CSIC, Parque Tecnológico de Ciencias de la Salud, Avenida del Conocimiento, 17, 18016, Armilla, Granada, Spain.
Estación Experimental del Zaidín (EEZ), CSIC, Prof. Albareda 1, 18008, Granada, Spain.
Parasit Vectors. 2023 Mar 31;16(1):118. doi: 10.1186/s13071-023-05728-x.
Trypanosomes are single-celled eukaryotes that rely heavily on post-transcriptional mechanisms to regulate gene expression. RNA-binding proteins play essential roles in regulating the fate, abundance and translation of messenger RNAs (mRNAs). Among these, zinc finger proteins of the cysteine3histidine (CCCH) class have been shown to be key players in cellular processes as diverse as differentiation, regulation of the cell cycle and translation. ZC3H41 is an essential zinc finger protein that has been described as a component of spliced leader RNA granules and nutritional stress granules, but its role in RNA metabolism is unknown.
Cell cycle analysis in ZC3H41- and Z41AP-depleted cells was carried out using 4',6-diamidino-2-phenylindole staining, microscopic examination and flow cytometry. The identification of ZC3H41 protein partners was done using tandem affinity purification and mass spectrometry. Next-generation sequencing was used to evaluate the effect of ZC3H41 depletion on the transcriptome of procyclic Trypanosoma brucei cells, and also to identify the cohort of mRNAs associated with the ZC3H41/Z41AP complex. Levels of 5S ribosomal RNA (rRNA) species in ZC3H41- and Z41AP-depleted cells were assessed by quantitative reverse transcription-polymerase chain reaction. Surface sensing of translation assays were used to monitor global translation.
We showed that depletion of the zinc finger protein ZC3H41 resulted in marked cell cycle defects and abnormal cell morphologies. ZC3H41 was found associated with an essential protein, which we named Z41AP, forming a stable heterodimer, and also with proteins of the poly(A)-binding protein 1 complex. The identification of mRNAs associated with the ZC3H41/Z41AP complex revealed that it is primarily composed of ribosomal protein mRNAs, and that binding to target transcripts is diminished upon nutritional stress. In addition, we observed that mRNAs encoding several proteins involved in the maturation of 5S rRNA are also associated with the ZC3H41/Z41AP complex. Finally, we showed that depletion of either ZC3H41 or Z41AP led to the accumulation of 5S rRNA precursors and a decrease of protein translation.
We propose that ZC3H41 and Z41AP play important roles in controlling the fate of ribosomal components in response to environmental cues.
锥虫是依赖于转录后机制来调节基因表达的单细胞真核生物。RNA 结合蛋白在调节信使 RNA（mRNA）的命运、丰度和翻译方面发挥着重要作用。在这些蛋白中，半胱氨酸 3 组氨酸（CCCH）类锌指蛋白已被证明是分化、细胞周期调控和翻译等多种细胞过程的关键参与者。ZC3H41 是一种必需的锌指蛋白，已被描述为拼接 leader RNA 颗粒和营养应激颗粒的组成部分，但它在 RNA 代谢中的作用尚不清楚。
使用 4'，6-二脒基-2-苯基吲哚染色、显微镜检查和流式细胞术对 ZC3H41 和 Z41AP 耗尽细胞的细胞周期进行分析。使用串联亲和纯化和质谱鉴定 ZC3H41 蛋白伴侣。使用下一代测序评估 ZC3H41 耗尽对 Procyclic Trypanosoma brucei 细胞转录组的影响，并鉴定与 ZC3H41/Z41AP 复合物相关的 mRNA 群体。通过定量逆转录聚合酶链反应评估 ZC3H41 和 Z41AP 耗尽细胞中 5S 核糖体 RNA（rRNA）物种的水平。使用表面翻译感应测定法监测全局翻译。
我们表明，锌指蛋白 ZC3H41 的耗竭导致明显的细胞周期缺陷和异常的细胞形态。发现 ZC3H41 与一种必需的蛋白质 Z41AP 相关联，形成稳定的异二聚体，并且还与聚（A）结合蛋白 1 复合物的蛋白质相关联。与 ZC3H41/Z41AP 复合物相关联的 mRNA 的鉴定表明，它主要由核糖体蛋白 mRNAs 组成，并且在营养胁迫下与靶转录本的结合减少。此外，我们观察到，编码参与 5S rRNA 成熟的几种蛋白质的 mRNAs 也与 ZC3H41/Z41AP 复合物相关联。最后，我们表明，耗尽 ZC3H41 或 Z41AP 都会导致 5S rRNA 前体的积累和蛋白质翻译的减少。
我们提出 ZC3H41 和 Z41AP 在响应环境线索控制核糖体成分命运方面发挥着重要作用。
