Regulatory functions of the SUMO system in ribosome biogenesis and mitosis

泛素样相扑修饰物对蛋白质的可逆翻译后修饰控制着关键的细胞通路。特定底物在修饰和去修饰之间的变化可以决定其空间分布或调节蛋白质复合体的组装或拆解。SENP家族的相扑专一性蛋白酶通过催化相扑从靶蛋白上的去结合来精确地控制修饰状态。因此，对SENP家族成员的进一步描述对于获得对相扑功能的新见解具有重要意义。在这个建议中，我们集中在SENP3在核糖体生物发生中的作用(第一部分)以及SENP3和SENP6在有丝分裂过程中的调控(第二部分)。我们以前的工作发现了SENP3在核糖体生物发生的核仁步骤中的调节作用，并表明这一功能与核磷蛋白(NPM1)有关。在第一个资助期，我们发现并鉴定了一个新的SENP3相关复合体，该复合体由PELP1、TEX10和WDR18组成，并证明该复合体参与核糖体大亚基的成熟和核仁释放。我们发现，在SENP3控制的过程中，PELP1被SUMO2/3动态修饰，我们提供了证据，证明相扑系统决定了PELP1的核仁分配。我们提出了一个模型，其中平衡的求和-去甲基化控制PELP1-WDR18-TEX10复合体与60S前核糖体颗粒的动态结合，并假设SENP3的缺失导致该复合体从这些结构中提前释放。在本项目的第一部分，我们将挑战这一假设，并提出以下问题：1.求和作用如何影响核糖体颗粒的组成和动力学？2.AAA-ATPase如何参与依赖于相扑的前核糖体的重塑？3.c-myc和p14ARF如何控制SENP3和核仁相扑系统？我们和其他人以前的工作也确定了哺乳动物相扑系统在有丝分裂功能控制中的重要作用。通过siRNA耗尽SENP3可以防止细胞在添加纺锤体毒物时发生有丝分裂停滞，这表明SENP3在纺锤体组装检查点(SAC)发挥作用。我们可以将Borealin定义为SENP3的第一有丝分裂底物，它是染色体乘客复合体(CPC)的一个组成部分。在第一个资助期，我们对SENP3的有丝分裂调控有了新的认识，并揭示了SENP3与有丝分裂激酶Plk1的相互联系。此外，我们还观察到，对SUMO2/3链具有特异性的SENP6的耗尽也会影响SAC的活性。此外，我们确定SAC组分MAD1是SUMO2/3对SENP6敏感的靶标。在第二部分中，我们现在将集中讨论以下问题：1.SENP3和PLK1在功能上是如何联系的？2.MAD1是依赖于相扑的降解途径的底物吗？3.SUMO2/3链在控制MAD1活性和SENP6识别MAD1中起什么作用？

The reversible post-translational modification of proteins by the ubiquitin-like SUMO modifier controls key cellular pathways. The alteration of a given substrate between modification and demodification can determine its spatial distribution or regulate the assembly or disassembly of protein complexes. SUMO-specific proteases of the SENP family accurately govern the state of modification by catalyzing the deconjugation of SUMO from target proteins. The further characterization of SENP family members is thus of major importance to gain novel insights into SUMO function. In this proposal we concentrate on the role of SENP3 in ribosome biogenesis (Part I) and the involvement of SENP3 and SENP6 in the control of mitotic processes (Part II).Our previous work uncovered a regulatory role of SENP3 in the nucleolar steps of ribosome biogenesis and showed that this function is linked to nucleophosmin (NPM1). In the first funding period we now identified and characterized a novel SENP3-associated complex comprised of PELP1, TEX10 and WDR18 and demonstrate that this complex is involved in maturation and nucleolar release of the large ribosomal subunit. We found that PELP1 is dynamically modified by SUMO2/3 in a SENP3-controlled process and we provide evidence that the SUMO system determines the nucleolar partitioning of PELP1. We propose a model where the balanced sumoylation-desumoylation controls the dynamic association of the PELP1-WDR18-TEX10 complex with 60S pre-ribosomal particles and hypothesize that loss of SENP3 induces the premature release of the complex from these structures.In part I of the project we will challenge this hypothesis and ask the following questions:1. How does sumoylation affect composition and dynamics of ribosomal particles?2. How are AAA-ATPases involved in SUMO-dependent remodelling of pre-ribosomes?3. How do c-myc and p14ARF control SENP3 and the nucleolar SUMO system?Previous work by our group and others has also determined an important role of the mammalian SUMO system in the control of mitotic functions. Depletion of SENP3 by siRNA prevents mitotic arrest of cells upon addition of spindle poisons indicating that SENP3 functions in the spindle assembly checkpoint (SAC). We could define Borealin, a component of the chromosomal passenger complex (CPC), as a first mitotic substrate of SENP3. In the first funding period we gained new insight into mitotic regulation of SENP3 and revealed an interconnection of SENP3 with the mitotic kinase Plk1. We additionally observed that depletion of SENP6, which exerts specificity for SUMO2/3-chains, also affects SAC activity. Moreover, we identified the SAC component MAD1 as a SENP6-sensitive target of SUMO2/3.In part II the new funding period we will now concentrate on the following questions:1. How are SENP3 and PLK1 functionally connected?2. Is Mad1 a substrate for the SUMO-dependent degradation pathway?3. What is the role of SUMO2/3-chains in the control of MAD1 activity and recognitionby SENP6?