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Functional analysis of importins and paraspeckle proteins in spermatogenesis
thesisposted on 22.05.2017, 04:15 by Major, Andrew Thomas
All eukaryotic developmental processes require progressive, tightly controlled changes in gene expression and nuclear activities. Transport into the nucleus is mediated by importins (IMPs) which display preferential cargo binding specificities and are now known to be dynamically synthesized throughout development and differentiation processes, including spermatogenesis. These observations give rise to the core theme of this thesis: regulated expression and/or activity of indervidual nuclear transport machinery components can act as a gatekeeper of development and differentiation by transporting key cargoes to influence cell fate outcomes. A comprehensive overview of when and where the major nuclear transport machinery components are synthesised within the testis and as sperm develop was first constructed using existing transcriptome data. This provides a platform for understanding regulation of nucleocytoplasmic transport in the testis in the context of distinct shifts in synthesis of specific nuclear transport components. IMPα2 transcript levels were found to peak at E12.5, just after sex determination in the embryonic mouse testis and within spermatocytes and spermatids in the adult mouse testis. This was interpreted to suggest that cargoes of IMPα2 could be performing important nuclear functions at these particular stages of development. Yeast two hybrid (Y2H) screens were performed and identified amongst others, paraspeckle protein 1 (PSPC1) as a putative IMPα2 cargo. The interaction between PSPC1 and IMPα2 was validated using several techniques, with co-immunoprecipitations and pulldowns demonstrating that an IMPα2-PSPC1 complex is present in testis lysates. ELISA-based importin binding assays using purified recombinant proteins measured the relative affinity of IMPα2, IMPα4 and IMPα6 binding for PSPC1. PSPC1 is one of three core Drosophila behaviour, human splicing (DBHS) proteins first identified as components of the subnuclear domain termed paraspeckles, and observed as nuclear speckles or foci. The three core DBHS proteins, PSPC1, PSF and NONO, are multifunctional and execute diverse nuclear roles. Paraspeckles are built upon the long non-coding RNA (lncRNA) NEAT1. They can contain many other proteins (>40) and are also rich in RNA. Development of an automated, high throughput, image analysis pipeline allowed non-subjective identification of nuclear foci, nuclei and cells. The nuclear accumulation and localization of PSPC1 and PSF to paraspeckles was assessed in a HeLa cell model in which the functional levels of multiple IMPα subtypes were modulated. This large-scale, unbiased analysis demonstrated that the relative abundance both of individual IMPαs and of the paraspeckle-bound cargo protein(s) contribute to the detectable numbers and size of these subnuclear structures within the population of cells. The expression of DBHS paraspeckle proteins and NEAT1 were investigated in the mouse testis and during its development. Remarkably, these are not all present within the same testicular cell types or at the same spermatogenic developmental stages. NEAT1 was detected throughout the adult mouse testis, with punctate NEAT1 nuclear foci, delineating paraspeckles, observed only within Leydig cells, while an anomalous cytoplasmic NEAT1 signal was observed within spermatogonia and spermatocytes. The significance of these findings is discussed within the context of spermatogenesis, but more broadly, these studies demonstrate a mechanism by which regulated importin synthesis or factors which alter nucleocytoplasmic transport capacity can influence nuclear activities of a cell.