Biochemical dissection, functional characterization and regulatory cues of the assembly machinery for U snRNPs

由蛋白质或蛋白质和核酸组成的大分子复合体执行各种不同的细胞任务。这些大分子复合体的组装通常依赖于体内的反式作用因子，因为分离的亚基可能聚集或参与错误的相互作用。一种辅助组装的范例是形成共同的Sm/LSM核心，该核心由剪接体和组蛋白-mRNA加工U SnRNPs组成。这一过程的关键组装因子统一在PRMT5-和SMN-复合体中，并促进7个Sm/LSM蛋白与U-SnRNA结合。组装是由PRMT5复合体亚基pICln启动的，它将Sm/LSM蛋白预先安排到成熟的U SnRNP中的空间位置。然后，SMN复合体接管这些Sm/LSM单位，取代pICln，并将它们与U SnRNA结合。我们在过去的资助期间已经证明，新合成的Sm/LSM蛋白即使在翻译终止后仍然与核糖体结合，并且Sm/LSM对组装途径的引导依赖于pICln。我们的研究揭示了一条精心设计的U SnRNPs装配线，其中核糖体作为质量控制中心和伴侣介导的组装过程的起点起着至关重要的作用。在这些发现的基础上，我们现在计划测试这一假设，即Sm/LSM蛋白从核糖体到组装机械的协调移交防止了它们的错误组装和/或聚集，并决定了细胞U SnRNP的生物发生。该提案的第二部分建立在我们的发现基础上，即SMN复合体的几个成分被磷酸化，并且这种翻译后修饰对SMN复合体的功能和亚细胞分布产生影响。我们的目标是识别和进一步表征修改SMN复合体的信号线索，并提出实验，揭示这些修改对U SnRNP生物发生和动态平衡的功能相关性。

Macromolecular complexes composed of proteins or proteins and nucleic acids perform a wide spectrum of different cellular tasks. The assembly of these macromolecular complexes often depends on trans-acting factors in vivo, as isolated subunits may aggregate or engage in wrong interactions. A paradigm for assisted assembly is the formation of the common Sm/LSm core of spliceosomal and histone-mRNA processing U snRNPs. The key assembly factors of this process are united in PRMT5- and SMN-complexes and facilitate binding of seven Sm/LSm proteins onto U snRNA. Assembly is initiated by the PRMT5-complex subunit pICln, which pre-arranges Sm/LSm proteins into spatial positions occupied in the mature U snRNP. The SMN complex then takes over these Sm/LSm units, displaces pICln and unites them with U snRNA. We have shown in the past funding period that newly synthesized Sm/LSm proteins remain bound to the ribosome even upon translation termination and that Sm/LSm guidance into the assembly pathway is dependent on pICln. Our studies uncovered an elaborate assembly line for U snRNPs in which the ribosome plays a crucial part as a quality control hub and the starting point for the chaperone-mediated assembly process. Building on these findings we are now planning to test the hypothesis that the coordinated hand-over of Sm/LSm proteins from the ribosome to the assembly machinery prevents their mis-assembly and/or aggregation and determines cellular U snRNP biogenesis. The second part of the proposal builds on our finding that several components of the SMN complex are phosphorylated and that this post-translational modification impacts on the function and sub-cellular distribution of the SMN complex. We aim to identify and further characterize signalling cues that modify the SMN complex and propose experiments that will shed light on the functional relevance of these modifications for U snRNP biogenesis and homeostasis.