Characterization of GW/P Body Components in Astrocyte and Astrocytoma Cells

Date
2013-09-09
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Abstract
GW/P bodies (GWB) are cytoplasmic foci that contain GW182 and Ago2 that bind micro RNA (miRNA) within the RNA-induced silencing complex (RISC) to silence and degrade messenger RNA (mRNA). Clinical studies indicate that 33% of patients with GWB autoantibodies have a motor/sensory neuropathy and/or ataxia. Preliminary data show that GWB are highly expressed in the central nervous system, particularly in astrocytes. Characterizing the protein and RNA components of GWB is key to elucidating their function in astrocyte and astrocytoma cells. First, a detailed immunoprecipiation (IP)-Western blot (WB) protocol was developed to addresses challenges in isolating low abundance protein GW182 and GWB-associated proteins. This protocol provides a biochemical approach for detecting GW182 and associated proteins in biological samples facilitating the elucidation of the diverse functions of GWB. Second, astrocytoma GWB exhibit complex heterogeneity with combinations of LSm4 and XRN1 as well as Ago2 and Dicer, key proteins involved in mRNA degradation and RNA interference respectively. GWB subsets contained the mRNA transport and stabilization proteins SYNCRIP, hnRNPA1 and FMRP, not previously described as part of the GWB complex. Astrocytoma GWB-IP suggested that Dicer, hDcp, LSm4, XRN1, SYNCRIP and FMRP form a multiprotein complex and are involved in a number of regulatory mRNA pathways. Third, GWB/RISC mRNA and miRNA components were profiled by microarray analysis of astrocytoma cells and astrocytes. MiRNAs were highly enriched in astrocyte RISC compared to astrocytoma RISC, astrocytoma and astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in astrocytoma RISC as compared to astrocyte RISC, and the RISC contained decreased levels of mRNAs in astrocyte and astrocytoma cells. Biological pathway analysis of RISC mRNA suggests that ultimately GWB play a pivotal role in malignancy. In future, it is important to move beyond characterizing the GWB components of resting cells, but determine the functional response of GWB when subjected to physiologically relevant stimuli. Clinically, there is much to learn about other GWB autoantigens, other diseases associated with GWB autoantibodies and the genetic basis for GWB autoimmunity.
Description
Keywords
Cell, Molecular, Neuroscience, Molecular
Citation
Moser, J. J. (2013). Characterization of GW/P Body Components in Astrocyte and Astrocytoma Cells (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/25275