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

转录后机制对于所有生物体中基因表达的调节至关重要。锥虫是研究mRNA降解的极好模型，因为在单个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，广泛调节mRNA的衰变率和翻译。我们已经确定了锥虫mRNA降解所需的大多数酶和复合物，并测量了全转录组的mRNA衰减率。迄今为止的结果表明，通常情况下，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未知。我们进行了一个全基因组的“拴系”筛选，并确定了近300个蛋白质，增加或减少基因表达时，连接到一个报告mRNA。那些降低基因表达的细胞富含具有RNA结合结构域的蛋白质。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。我们将从几个候选人开始，并根据初始结果集中在一个子集上。我们将选择在系链筛选中具有强烈下调作用的蛋白质，并且这些蛋白质在至少一个容易培养的锥虫生命周期阶段中对生长至关重要。将确定蛋白质的亚细胞位置，并选择胞质蛋白质。使用多核糖体分析和北方印迹，我们将发现人工连接的（栓系的）蛋白质的作用是否主要是对报告mRNA的翻译或稳定性。对于选定的蛋白质，将确定耗尽蛋白质（通过RNAi）对转录组和翻译谱的影响：特异性受影响的mRNA是潜在的靶标。通过对受RNAi影响的mRNA进行基序搜索，并使用具有和不具有候选基序的mRNA进行报告基因测定，确定蛋白质结合的RNA序列。在抑制mRNA衰变后，我们将尝试使用UV交联、亲和纯化、然后RNA测序来发现体内结合的RNA。可溶性蛋白质的生产和体外RNA结合测定是一种替代方法。为了找到作用机制，蛋白质-蛋白质相互作用将通过酵母双杂交测定和下拉法进行研究。RNA调控中相互作用的作用将通过结构-功能分析来确定，该项目是一个长期计划的一部分，该计划包括表征增加表达的蛋白质，以及在系链筛选中呈阳性但没有任何结构域将其与RNA代谢联系起来的蛋白质。最终目的是获得一个定量的了解锥虫的转录后调控网络。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.