Survival Motor Neuron protein interaction partners in Drosophila melanogster Public Deposited

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  • March 21, 2019
Creator
  • Gray, Kelsey
    • Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology
Abstract
  • Spinal Muscular Atrophy (SMA) is a neuromuscular disorder that results from biallelic loss-of-function mutations in the human survival motor neuron 1 (SMN1) gene. Tissue-specific and housekeeping functions have been ascribed to SMN; however, their relevance to SMA pathology is not well understood. We generated transgenic Drosophila melanogaster that express only flag-tagged wild-type SMN. Our objective is to characterize novel protein-protein interactions of SMN. We collected embryos and analyzed Flag-purified lysates by mass spectrometry. We identified Flag-SMN along with other known interactors such as the Sm proteins and the Gemins. We also identified Slmb, SkpA, and Cullin 1 as being highly enriched in Flag-SMN samples as compared to the control sample. Together, these proteins comprise the SCFSlmb E3 ubiquitin ligase. These interactions were verified in Drosophila S2 cells and human cells. In vitro experiments revealed Slmb and SMN can directly interact. Identification of a putative Slmb degron in the self-oligomerization domain of SMN led us to generate a serine to alanine mutation that stabilizes full length and truncated SMN, with strongest effects on SMN with poor self-oligomerization capability. This same point mutation decreases SMN’s interaction with Slmb, demonstrating the putative Slmb degron is indeed mediating degradation of SMN. Finally, expression of truncated SMN stabilized by the mutation modifies viability of a mild SMA mouse model. We identified additional protein interactions of SMN with CG2941, nucleosome assembly protein 1 (Nap1), and Bendless (Ben). Each of these interactions was verified in cell culture or using antibodies generated specifically for the protein of interest. Preliminary investigation of CG2941 has revealed it is an essential gene that produces protein that localizes to both the nucleus and the cytoplasm. We have examined SMN protein interactions in the context of developing Drosophila melanogaster embryos, with follow-up studies in mouse, and human systems. When SMN is unable to self-oligomerize, the Slmb degron is highly accessible, and thus SMN is degraded. SMN also interacts with previously unknown partners that may be relevant to SMA pathology. This work elucidates a disease-relevant mechanism where SMN levels are regulated by self-multimerization and identifies candidate proteins for further study of the molecular mechanisms underlying SMA.
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Advisor
  • Conlon, Frank
  • Matera, Arnold
  • Damania, Blossom
  • Sekelsky, Jeff
  • Peifer, Mark
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill
Graduation year
  • 2018
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