Effect of TbDRBD3 depletion on procyclic T. brucei transcriptome

In trypanosomes, the apparent lack of regulation of RNA polymerase II-dependent transcription initiation poses a challenge to understand how these eukaryotes adjust gene expression in order to adapt to the contrasting environments they find during their life cycles. Evidence so far indicates that mRNA turnover and translation are the major control points in which regulation is exerted in trypanosomes. However, very little is known about which proteins are involved, and how do they regulate the abundance and translation of different mRNAs in different life stages. In this work, an RNA-binding protein, TbDRBD3, has been identified by affinity chromatography, and its function addressed using RNA interference, microarray analysis and immunoprecipitation of mRNA-protein complexes. The results obtained indicate that TbDRBD3 binds to a subset of developmentally regulated mRNAs encoding membrane proteins and intermediate metabolism enzymes, and that this association promotes the stabilization of the target transcripts. These observations raise the possibility that TbDRBD3-mRNA complexes act as a post-transcriptional operon, and provide a framework to interpret how trypanosomes regulate gene expression in the absence of transcriptional control. Keywords: Effect of depletion of an RNA-binding protein on the transcriptome In order to analyze whether TbDRBD3 has a role in the regulation of mRNA turnover, the transcriptome of procyclic trypanosomes depleted of TbDRBD3 was compared to that of uninduced cells using hybridizations of genomic microarrays. RNA samples were isolated from cells harvested after 48 hours of induction with tetracycline. At this point, cell growth was barely affected and trypanosomes remained highly motile. 15 µg of RNA were reverse-transcribed in the presence of 100 ng of oligo(dT)12-18, 0.25 mM dATP, 0.25 mM dTTP, 0.25 mM dGTP, 0.05 mM dCTP, 0.05 mM Cy3- or Cy5-dCTP, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 5 mM DTT, 40 U of RNaseOUT (Invitrogen) and 400 U of Superscript III reverse transcriptase (Invitrogen). Reactions were incubated for 3 hours at 50ºC, stopped by heating at 70ºC for 15 min and treated with 5 U of RNase H for 20 min at 37ºC. cDNA was purified using the MinElute kit (Qiagen), ethanol precipitated and resuspended in hybridization buffer. Genomic T. brucei microarray glass slides containing ca. 24,000 independent random genomic clones (Brems et al., 2005) were pre-hybridized and hybridized as described (Diehl et al., 2002). Four hybridizations (two biological replicates with a dye swap each) were analyzed.

在锥虫（trypanosomes）中，依赖RNA聚合酶II（RNA polymerase II）的转录起始似乎缺乏调控机制，这为解析这类真核生物如何调整基因表达以适应其生命周期中遭遇的迥异环境带来了挑战。迄今已有研究证据显示，mRNA周转与翻译过程是锥虫实施基因表达调控的核心调控节点。然而，目前人们对其中涉及的蛋白质种类，以及这些蛋白质如何在不同生命周期阶段调控不同mRNA的丰度与翻译过程仍知之甚少。
本研究通过亲和色谱法（affinity chromatography）鉴定出一种RNA结合蛋白（RNA-binding protein）TbDRBD3，并借助RNA干扰（RNA interference）、微阵列分析（microarray analysis）以及mRNA-蛋白质复合物免疫沉淀技术解析了其功能。研究结果显示，TbDRBD3可结合一类受发育调控的mRNA，这类mRNA编码膜蛋白与中间代谢酶；且该结合过程可促进靶标转录本的稳定性。上述发现提示TbDRBD3与mRNA形成的复合物可作为转录后操纵子（post-transcriptional operon）发挥功能，同时为解析锥虫在缺乏转录调控的情况下如何实现基因表达调控提供了理论框架。
关键词：RNA结合蛋白敲除对转录组的影响
为探究TbDRBD3是否参与mRNA周转调控，本研究通过基因组微阵列杂交技术，将敲低TbDRBD3的循环前期锥虫（procyclic trypanosomes）的转录组与未诱导细胞的转录组进行比对。
RNA样本取自经四环素（tetracycline）诱导48小时后收获的细胞。在该时间点，细胞生长几乎未受影响，锥虫仍保持高度运动能力。
取15 μg RNA进行反转录反应，反应体系包含100 ng oligo(dT)12-18、0.25 mM dATP、0.25 mM dTTP、0.25 mM dGTP、0.05 mM dCTP、0.05 mM Cy3-或Cy5-dCTP、50 mM Tris-HCl（pH 8.3）、75 mM KCl、3 mM MgCl2、5 mM DTT、40 U RNaseOUT（Invitrogen）以及400 U Superscript III反转录酶（Invitrogen）。反应体系于50℃孵育3小时，随后通过70℃加热15分钟终止反应，并加入5 U RNase H在37℃处理20分钟。使用MinElute试剂盒（Qiagen）纯化cDNA，经乙醇沉淀后重悬于杂交缓冲液中。
包含约24000个独立随机基因组克隆的布氏锥虫（T. brucei）基因组微阵列玻片（Brems等人，2005），按照文献（Diehl等人，2002）所述方法进行预杂交与杂交。共分析4次杂交实验（包含2次生物学重复，每次重复均进行荧光染料互换）。