Characterisation and regulation of the alternative cap binding complex consisting of NCBP1 and -3

基因组信息从DNA到蛋白质的流动需要聚合酶-II转录物，其特征在于存在5个引物帽结构。由核帽结合蛋白（NCBP）2及其接头NCBP 1组成的帽结合复合物（CBC）被认为结合所有加帽的RNA，并且是加工和细胞内定位所必需的。然而，NCBP 1而不是NCBP 2是细胞活力和mRNA输出所必需的，这表明存在参与mRNA生物学的其他因子。我们最近发现了一种替代的帽结合复合物，由高度保守的蛋白质C17 orf 85（现在正式命名为核帽结合蛋白3的NCBP 3）和NCBP 1组成。只有同时消耗NCBP 2和NCBP 3才能减少NCBP 1与cap-RNA的结合，损害细胞生长和核mRNA输出。mRNA结合NCBP 2或NCBP 3，而小RNA种类如snRNA优先与NCBP 2结合。因此，除了由NCBP 1/2组成的典型CBC之外，由NCBP 1/3组成的替代CBC也在大多数真核细胞中发挥作用。目前还不清楚为什么我们的生物体进化出两种CBCs，在稳态条件下共享它们大部分的活动。有趣的是，在细胞应激条件下，如病毒感染，NCBP 3成为调节适当的抗病毒免疫应答的关键。这表明，mRNA的成熟和易位可能受到不同的调节，这取决于环境刺激。在这里，我们的目标是功能特性的替代CBC在稳态和刺激条件下。因此，NCBP 1和-3之间的相互作用以及RNA加工因子与NCBP 1/3复合物的关联应进行详细研究。另一个目的是研究为什么mRNA而不是其他类型的加帽RNA（例如snRNA，lincRNA，.）与另一种CBC有关。在功能上，在抗病毒免疫应答期间似乎需要替代CBC，这表明先天性病原体感知对mRNA输出途径的影响。在初步实验中，我们可以确定NCBP 1和NCBP 3上独特的磷酸化位点，这些位点在病毒感染期间动态变化。将在抗病毒免疫应答的背景下评价这些磷酸化位点的功能。最近开发的NCBP 3缺陷小鼠将对在生物体水平上获得功能性见解提供关键帮助。总而言之，该建议旨在进一步了解基本的细胞过程，即从DNA到蛋白质的信息传递的调节。初步数据让我们假设，这一过程是通过整合环境刺激引起的信号控制的。

The flow of genomic information from DNA to protein requires polymerase-II transcripts, which are characterized by the presence of a 5 primes cap structure. The cap-binding complex (CBC), consisting of the nuclear cap binding protein (NCBP) 2 and its adaptor NCBP1, is believed to bind all capped RNA and to be necessary for processing and intracellular localization. However, NCBP1 but not NCBP2 is required for cell viability and mRNA export suggesting the existence of additional factors involved in mRNA biology. We recently identified an alternative cap-binding complex consisting of the highly conserved protein C17orf85 (now officially named NCBP3 for Nuclear cap-binding protein 3) and NCBP1. Only simultaneous depletion of NCBP2 and -3 diminishes association of NCBP1 to cap-RNA, impairs cell growth and nuclear mRNA export. mRNA binds either NCBP2 or -3 whereas small RNA species such as snRNA preferentially associates to NCBP2. Thus, besides the canonical CBC consisting of NCBP1/2, an alternative CBC consisting of NCBP1/3 is operating in most eukaryotic cells. It is currently unclear why our organism evolved two CBCs that under steady state conditions share large parts of their activity. Intriguingly, under cellular stress conditions such as virus infection NCBP3 becomes pivotal to regulate a proper antiviral immune response. This suggests that mRNA maturation and translocation may be differentially regulated depending on environmental stimuli. Here, we aim to functionally characterize the alternative CBC under steady state and stimulated conditions. Therefore, the interactions between NCBP1 and -3 as well as association of RNA processing factors to the NCBP1/3 complex should be studied in detail. A further aim is to study why mRNA but not other types of capped RNAs (e.g. snRNA, lincRNA,...) are associated with the alternative CBC. Functionally, the alternative CBC appears to be required during the antiviral immune response suggesting influence of innate pathogen sensing on the mRNA export pathway. In preliminary experiments we could identify unique phosphorylation sites on NCBP1 and -3 that are dynamically changing during virus infection. The function of these phosphorylation sites will be evaluated in the context of the antiviral immune response. A recently developed NCBP3 deficient mouse will be of critical help to gain functional insights on an organismal level. In sum this proposal aims to gain further insights in a fundamental cellular process, namely the regulation of information transfer from DNA to protein. Preliminary data let us hypothesize that this process is controlled through integrating signals elicited by environmental stimuli.