Nuclear envelope spectrin repeat proteins, or Nesprins, are a novel family of nuclear and cytoskeletal proteins with rapidly expanding roles as intracellular scaffolds and linkers. Nesprins are characterized by a central spectrin repeat (SR) rod domain and a C-terminal KASH domain, which acts as a nuclear envelope (NE) targeting motif. At the NE, via interactions with the Sun domain family of proteins and the nuclear lamina, nesprins on both the inner and outer nuclear membranes form the linker of the nucleoskeleton and cytoskeleton (LING) complex. This complex requires the giant nesprin-1 and nesprin-2 isoforms, which possess a pair of N-terminal calponin homology domains that bind directly to Filamentous-actin. However, via alternative promoter usage and alternative 3’ end processing, the nesprins are able to generate multiple mRNA transcripts, leading to the production of diverse tissue specific isoforms with potential roles beyond the NE. To explore further the capacity of nesprin-1 to generate alternative transcripts, 5’ and 3’ RACE was performed to identify cDNA ends which represent novel 5’ and 3’ untranslated regions (UTRs) respectively. By alternatively combining the differential 5’ and 3’ UTRs, multiple tissue specific nesprin-1 variants could be generated. Transfection of tagged constructs showed localizations to multiple sub-cellular compartments such as the nucleolus, focal adhesions, actin stress fibres and cytoplasmic particles, supporting the notion that nesprins are more than NE-cytoskeletal couplers. One of these novel nesprin-1 variants, p50Nesp1, was found to localize to cytoplasmic RNA granules called mRNA processing bodies (P-bodies). Using GST pull-downs and co-immunoprecipitations (co-IPs), p50Nesp1 was found to complex with mRNA decapping factors and translational repressers. More importantly, p50Nesp1 associated strongly with microtubules, both in vitro and in vivo, and was required for scaffolding P-body complexes to microtubules. By disrupting P-body-microtubule association with a dominant negative p50Nesp1 construct, time-lapse microscopy demonstrated impairment of fluorescently-labelled P-body proteins. Furthermore, this disruption resulted in P-bodies failing to associate with RNA stress granules and transferring p-globin mRNA reporter transcripts between compartments during the stress response. Further co-IP experiments identified a host of mRNA processing proteins that also associated with nesprin-1, including Matrin-3; an abundant nuclear matrix protein involved in a number of key nuclear processes. A novel isoform of matrin-3 localized to P-bodies and was required for miRNA-mediated translational repression. By identifying and tethering matrin-3 P-body localizing domains to a luciferase reporter construct, it was also found that matrin-3 could induce translational repression, which was significantly hampered when a single point mutation (S85C), previously described in a form of autosomal dominant distal myopathy, was introduced.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:628433 |
| Date | January 2013 |
| Creators | Rajgor, Dipen |
| Publisher | King's College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://kclpure.kcl.ac.uk/portal/en/theses/characterisation-of-the-multiisomeric-protein-nesprin1-in-pbody-and-mrna-dynamics(aa49c0e2-15fb-4a02-aa88-8fca5575b892).html |
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