Post-transcriptional repression of gene expression in trypanosomes: roles of RNA-binding proteins in translation and mRNA decay

转录后机制对所有生物体的基因表达调控都是至关重要的。锥体是研究mRNA降解的优秀模型，因为在单个开放阅读框的水平上不能控制RNA聚合酶II的活性。这使得寄生虫精致地依赖转录后机制，广泛地调节信使核糖核酸的衰减率和翻译。我们已经确定了锥虫mRNA降解所需的大多数酶和复合体，并测量了整个转录组的mRNA衰减率。到目前为止的研究结果表明，通常情况下，mRNAs的3‘非翻译区通过与RNA结合蛋白的相互作用来控制降解和翻译。然而，RNA结合蛋白的作用机制大多尚不清楚。我们进行了全基因组的“拴系”筛选，鉴定出了近300种蛋白质，当连接到报告基因时，基因表达会增加或减少。那些降低基因表达的基因富含带有RNA结合域的蛋白质。在目前的项目中，我们的目标是确定其中一些蛋白质的功能。我们将从几个候选者开始，并根据初步结果专注于子集。我们将选择在系留筛选中产生强烈下调的蛋白质，这些蛋白质对至少一个容易培养的锥虫生命周期阶段的生长是必不可少的。蛋白质的亚细胞位置将被确定，胞浆蛋白将被选择。利用多聚体图谱和Northern杂交，我们将发现人工附着(拴)蛋白的作用主要是对报告基因的翻译或稳定性的影响。对于选定的蛋白质，耗尽蛋白质(通过RNAi)对转录组和翻译谱的影响将被确定：特定受影响的mRNAs是潜在的靶标。与蛋白质结合的RNA序列将通过对受RNAi影响的mRNAs进行基序搜索和报告程序分析来确定，其中包含和不包含候选基序的mRNAs。在抑制了mRNA的衰变后，我们将尝试通过紫外光交联、亲和纯化、然后进行RNA测序来寻找体内结合的RNA。可溶性蛋白质的生产和体外RNA结合试验是一种替代方法。为了找出作用机制，蛋白质之间的相互作用将通过酵母-2-杂交法和下拉试验进行研究。相互作用在RNA调控中的作用将通过结构-功能分析来确定。这个项目是一个长期计划的一部分，该计划包括表征增加表达的蛋白质，以及在系留筛选中呈阳性但没有任何将它们与RNA新陈代谢联系起来的蛋白质。最终目的是对锥虫体内转录后调控网络有一个定量的了解。我们还希望发现新的调节机制，其中一些可能也存在于其他生物体中。

Post-transcriptional mechanisms are vital for regulation of gene expression in all organisms. Trypanosomes are excellent models for the study of mRNA degradation because there is no control of RNA polymerase II activity at the level of individual open reading frames. This makes the parasites exquisitely reliant on post-transcriptional mechanisms, with extensive regulation of mRNA decay rates and translation. We have identified most of the enzymes and complexes that are required for trypanosome mRNA degradation, and measured mRNA decay rates transcriptome-wide. Results so far suggest that usually, the 3'-untranslated regions of mRNAs control degradation and translation, through interactions with RNA-binding proteins. However, the mechanisms by which the RNA-binding proteins act are mostly unknown. We conducted a genome wide "tethering" screen and identified nearly 300 proteins that increase or decrease gene expression when attached to a reporter mRNA. Those that decreased gene expression were enriched for proteins with RNA-binding domains. In the current project, we aim to determine the functions of some of these proteins. We will start with several candidates and focus on a sub-set depending on initial results. We will choose proteins that gave strong down-regulation in the tethering screen and are essential for growth in at least one of the easily cultivated trypanosome life-cycle stages. The subcellular location of the protein will be determined, and cytosolic proteins will be selected. Using polysome profiling and Northern blot, we will find out whether the effect of the artificially attached (tethered) protein is primarily on translation or stability of the reporter mRNA. For selected proteins, the effects of depleting the protein (by RNAi) on the transcriptome and translation profiles will be determined: specifically affected mRNAs are potential targets. The RNA sequences bound by the protein will be determined by Motif searches on mRNAs affected by the RNAi, and reporter assays using mRNAs with and without candidate motifs. After inhibition of mRNA decay, we will attempt to find in vivo bound RNAs using UV-cross linking, affinity purification, then RNA sequencing. Production of soluble protein and in vitro RNA-binding assays are an alternative. To find the mechanism of action, protein-protein interactions will be investigated by yeast-2-hybrid assays and pull-downs. The roles of interactions in RNA regulation will be determined by structure-function analyses.This project is part of a longer-term plan which includes the characterization of proteins that increase expression, and of proteins that were positive in the tethering screen but do not have any domains that link them to RNA metabolism. The ultimate aim is to obtain a quantitative understanding of the network of post-transcriptional regulation in trypanosomes. We hope also to discover novel regulatory mechanisms, some which may also be present in other organisms.