Regulated transcript stability

既然许多生物的基因蓝图已经被破译，生物学的下一个重大挑战是理解指令是如何用来创造生物的。这突出了基因调控的主题，即研究哪些基因在任何时候都在使用中。最不被理解的部分之一是，一旦不再需要基因(信使RNA、mRNA、转录本)，细胞如何迅速处理它们的工作副本。已知有几种途径可以降解信使核糖核酸，但人们对保护信使核糖核酸的控制措施知之甚少，这些信使核糖核酸仍然是必需的，同时又能迅速去除不需要的信使核糖核酸。基因控制的这一基本水平很可能在所有生物体中都有类似的特征，信号和控制系统根据它们所参与的过程精细地适应一组基因，甚至是单个重要基因。可控的mRNA降解是丝状真菌nidulans氮代谢的主要调节机制。我们最近发现了一种分子(谷氨酰胺)，当有足够的优质氮源可供真菌使用时，它可以加速一些但不是所有参与氮代谢的mRNAs的衰退。更令人兴奋的是，发现另一种分子(硝酸盐)与特定mRNA的这种信号相反。因此，它提供了一种很好的方法来识别和表征调节mRNA稳定性的成分。这个经过充分研究的系统拥有所有的遗传、信息和技术资源，可以解决这样的问题：当真菌对不同的氮源做出反应时，一个mRNA如何保持稳定，而另一个如何在几分钟内被破坏。信使核糖核酸降解的关键第一步是从分子中去除聚(A)尾巴。酶复合体在所有形式的生命中高度保守，含有执行这一功能的蛋白质，尽管细节仍未被发现。基于我们的初步实验，我们认为一种蛋白质Ccr4p是一般mRNA衰变的主要死烯基酶，而另一种蛋白质Pop2p在特定的调控途径中是必需的。我们将在这个项目中确认并进一步表征这些蛋白质，包括它们对特定mRNAs的活性如何被特定的信号硝酸盐抑制。必须需要更多的蛋白质来调节降解，我们已经从初步实验和生物信息学分析中确定了三个有希望的候选者。我们将描述它们的功能，并进行实验以识别更多的调节蛋白。监测单个mRNAs的半衰期是我们的基本技术之一。DNA微阵列将让我们看到这些调控蛋白的全球影响。然而，我们也将使用经典遗传分析、定点突变、蛋白质标记、双杂交和三杂交分析以及免疫共沉淀等方法来实现我们的目标。我们将使用的另一种方法是实时成像，与在细胞成像和在弧菌中使用荧光标记蛋白质方面非常有经验的实验室合作，以获得对mRNA周转的空间方面的新见解。作为基因调控和信号传递的一部分，对mRNA降解协调的研究还处于初级阶段，这个在一个易受影响的有机体内的优雅系统将使我们能够做出重大贡献。

Now that the genetic blueprint of many organisms has been deciphered, the next big challenge in Biology is to understand how the instructions are used to make a living creature. This highlights the subject of gene regulation, the study of which genes are in use at any time. One of the least understood parts is how the cell quickly gets rid of working copies of genes (the messenger RNA, mRNA, transcripts) once they are no longer needed. Several pathways are known that degrade mRNA, but the controls to protect mRNA that is still required while swiftly removing unwanted mRNA are little understood. It is likely that this fundamental level of gene control will have similar features in all organisms, with signalling and control systems exquisitely adapted for group of genes or even individual important genes according to the processes in which they are involved. Controlled mRNA degradation is a major regulatory mechanism in nitrogen metabolism in the filamentous fungus Aspergillus nidulans. We have recently identified the molecule (glutamine) that signals accelerated decay of some, but not all, mRNAs involved in nitrogen metabolism when there is sufficient good-quality nitrogen source available to the fungus. An even more exciting aspect is the discovery that a second molecule (nitrate) opposes this signal for specific mRNAs. It therefore provides an excellent way to identify and characterise the components that regulate mRNA stability. This well-studied system has all the genetic, information and technical resources to tackle the problem of how one mRNA remains stable while another is destroyed within minutes as the fungus responds to different nitrogen sources. The essential first step in mRNA degradation is to remove the poly(A) tail from the molecule. Enzyme complexes, highly conserved among all forms of life, contain proteins that carry out this function although the details remain to be uncovered. Based on our initial experiments, we propose that one protein, Ccr4p, is the main deadenylase for general mRNA decay, while another, Pop2p, is needed in specific regulated pathways. We will confirm and further characterise these proteins within this project, including how their activity against specific mRNAs is inhibited by the specific signal nitrate. Additional proteins must be required to regulate degradation and we have identified three promising candidates from initial experiments and bioinformatics analysis. We will characterise their functions and also carry out experiments to identify further regulatory proteins. Monitoring the half-lives of individual mRNAs is one of our basic techniques. DNA microarrays will let us see the global effects of these regulatory proteins. However, we will also use methodologies from classical genetic analysis, site-specific mutagenesis, protein tagging, two and three hybrid analysis and co-immunoprecipitation to achieve our goals. One further approach that we will use is real-time imaging to gain new insights into the spatial aspects of mRNA turnover, in collaboration with laboratories that are very experienced with cell imaging and the use of fluorescently tagged proteins in A. nidulans. Research into the co-ordination of mRNA degradation as part of gene regulation and signalling is in its infancy and this elegant system within an amenable organism will allow us to make a significant contribution.