A genome-wide tethering screen reveals novel potential post-transcriptional regulators in Trypanosoma brucei.
A genome-wide tethering screen reveals novel potential post-transcriptional regulators in Trypanosoma brucei.
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DOI：
10.1371/journal.ppat.1004178
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发表时间：
2014-06
期刊：
影响因子：
6.7
通讯作者：
Clayton C
中科院分区：
文献类型：
--
作者：
Erben ED;Fadda A;Lueong S;Hoheisel JD;Clayton C
在锥虫中，基因表达主要由转录后机制调节，其影响mRNA加工、翻译和降解。目前，我们对调节mRNA稳定性或翻译的因素的理解相当有限。我们知道，通常情况下，调节因子是与3′-非翻译区结合的蛋白质;它们可能与核糖核酸酶和翻译因子相互作用。然而，很少有这样的蛋白质已被表征的任何细节。在这里，我们描述了一个全基因组的屏幕，以找到涉及在布氏锥虫的转录后调控蛋白质。我们在质粒中构建了一个随机基因组片段库，该质粒旨在表达与RNA结合结构域（lambda-N肽）融合的蛋白质。将其转染到表达编码阳性或阴性选择标记的mRNA的细胞中，并在3′-非翻译区携带“boxB”-N识别元件。筛选鉴定了约300种可能与转录后mRNA调控有关的蛋白质。这些包括已知的调节剂，降解酶和翻译因子，许多典型的RNA结合蛋白，以及通过多蛋白复合物起作用的蛋白质。然而，也有近150个潜在的调节因子没有以前注释的功能，或与mRNA代谢无关的功能。使用靶向蛋白质组阵列显示近50种新型调节剂结合RNA。筛选还提供命中候选者的功能域的精细结构映射。我们的研究结果不仅证实了RNA结合蛋白在锥虫基因表达调控中的关键作用，而且还表明了以前未知的蛋白质的新作用。锥虫的生存和适应新的环境需要激活特定的基因网络。这主要是通过转录后机制实现的，并且已知与特定mRNA结合并影响降解或翻译的蛋白质是重要的。然而，迄今为止，只有少数这样的蛋白质被表征。锥虫基因组编码超过150种具有保守RNA结合结构域的蛋白质，并且很可能不具有此类结构域的其他蛋白质也可以调节mRNA命运。在这里，我们报告了一个全基因组筛选的结果，以确定布氏锥虫的mRNA命运调节。我们使用了一种称为“拴系”的方法，将蛋白质片段人工连接到mRNA上。我们的研究结果证实了RNA结合蛋白在mRNA命运调控中的作用，也表明了许多其他蛋白质（包括一些代谢酶）的这种作用。我们的研究结果应该成为有用的资源。此外，拴系筛选方法可以很容易地适用于其他生物体。
In trypanosomatids, gene expression is regulated mainly by post-transcriptional mechanisms, which affect mRNA processing, translation and degradation. Currently, our understanding of factors that regulate either mRNA stability or translation is rather limited. We know that often, the regulators are proteins that bind to the 3′-untranslated region; they presumably interact with ribonucleases and translation factors. However, very few such proteins have been characterized in any detail. Here we describe a genome-wide screen to find proteins implicated in post-transcriptional regulation in Trypanosoma brucei. We made a library of random genomic fragments in a plasmid that was designed for expression of proteins fused to an RNA-binding domain, the lambda-N peptide. This was transfected into cells expressing mRNAs encoding a positive or negative selectable marker, and bearing the “boxB” lambda-N recognition element in the 3′-untranslated region. The screen identified about 300 proteins that could be implicated in post-transcriptional mRNA regulation. These included known regulators, degradative enzymes and translation factors, many canonical RNA-binding proteins, and proteins that act via multi-protein complexes. However there were also nearly 150 potential regulators with no previously annotated function, or functions unrelated to mRNA metabolism. Almost 50 novel regulators were shown to bind RNA using a targeted proteome array. The screen also provided fine structure mapping of the hit candidates' functional domains. Our findings not only confirm the key role that RNA-binding proteins play in the regulation of gene expression in trypanosomatids, but also suggest new roles for previously uncharacterized proteins. Survival and adaptation of trypanosomatids to new surroundings requires activation of specific gene networks. This is mainly achieved by post-transcriptional mechanisms, and proteins that bind to specific mRNAs, and influence degradation or translation, are known to be important. However, only few such proteins have been characterized to date. The trypanosome genome encodes over 150 proteins with conserved RNA-binding domains, and it is very likely that additional proteins that do not have such domains could also modulate mRNA fate. Here, we report the results of a genome-wide screen to identify mRNA-fate regulators in Trypanosoma brucei. We used a method called “tethering” to artificially attach protein fragments to an mRNA. Our findings confirmed the role of RNA-binding proteins in the regulation of mRNA fate, and also suggested such roles for many other proteins, including some metabolic enzymes. Our results should serve as a useful resource. Moreover, the tethering screen approach could readily be adapted for use in other organisms.
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