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至关重要。锥虫是研究mRNA降解的优秀模型，因为在个体开放阅读框水平上没有RNA聚合酶II活性的控制。这使得寄生虫非常依赖于转录后机制，具有广泛的mRNA衰变率和翻译调节。我们已经确定了锥虫mRNA降解所需的大多数酶和复合物，并测量了转录组范围内mRNA的衰减率。迄今为止的研究结果表明，通常mrna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是未知的。我们进行了全基因组范围的“捆绑”筛选，发现了当与报告基因mRNA结合时，近300种蛋白质增加或减少基因表达。那些基因表达降低的细胞富含rna结合域蛋白。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。我们将从几个候选对象开始，并根据初始结果专注于一个子集。我们将选择在栓系筛选中表现出强烈下调的蛋白质，并且至少在一个容易培养的锥虫生命周期阶段对生长至关重要。将确定蛋白质的亚细胞位置，并选择细胞质蛋白。利用多聚体分析和Northern blot，我们将发现人工附着（栓系）蛋白的影响主要是对报告mRNA的翻译还是稳定性。对于选定的蛋白质，将确定（通过RNAi）耗尽蛋白质对转录组和翻译谱的影响：特别受影响的mrna是潜在目标。与该蛋白结合的RNA序列将通过对受RNAi影响的mrna进行Motif搜索来确定，并使用具有或不具有候选基序的mrna进行报告基因分析。在抑制mRNA衰变后，我们将尝试使用uv交联，亲和纯化，然后RNA测序来寻找体内结合的RNA。生产可溶性蛋白和体外rna结合试验是另一种选择。为了找出作用机制，将通过酵母-2杂交试验和下拉试验来研究蛋白质-蛋白质相互作用。相互作用在RNA调控中的作用将通过结构-功能分析来确定。该项目是一个长期计划的一部分，该计划包括对增加表达的蛋白质的表征，以及在捆绑筛选中呈阳性但没有任何与RNA代谢相关的结构域的蛋白质。最终目的是获得对锥虫转录后调控网络的定量理解。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.