Regulatory functions of the SUMO system in ribosome biogenesis and mitosis

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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?