Robin White

Characterization of oligodendroglial sncRNA715 synthesis, its functional role in myelination and identification of novel RNA-transport granule-associated ncRNAs



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1st. Financiation

Oligodendrocytes myelinate neuronal axons to enable fast and efficient information propagation in the central nervous system. Myelin Basic Protein (MBP) is an essential protein for the process of myelination and its synthesis is tightly controlled to allow localized translation at the plasma membrane in response to neuronal stimuli. We recently identified the small non-coding RNA 715 (sncRNA715) as an inhibitor of MBP translation during oligodendrocyte maturation and intracellular mRNA transport. Importantly, we found that sncRNA715 levels are abnormally high in demyelinated lesions of multiple sclerosis patients which contain MBP mRNA but no protein. We therefore want to understand the synthesis and functional impact of sncRNA715 on myelination in the proposed project. Interestingly sncRNA715 appears to be part of the 47S pre-ribosomal RNA and we plan to characterize its processing in more detail. We further intend to analyze if overexpression and inhibition of sncRNA715 modulate myelin synthesis in myelinating cortical slice cultures. This can be particularly important for the development of novel remyelination therapies in demyelinating diseases. We expect additional mRNAs to be localized in oligodendrocytes which makes it very likely that their translation is inhibited by sncRNAs during intracellular transport and we intend to identify these RNA molecules by RNA sequencing.

Some key technologies for internal use :

- Molecular Biology/ Biochemistry :
Generation and optimization of viral-, pro-and eukaryotic expression vectors (all standard cloning techniques), DNA manipulation (site-directed mutagenesis); DNA/ RNA/ protein extraction from cells and tissue; protein purification (immunoprecipitation, affinity-chromatography, gelfiltration), conjugation/ chemical crosslinking, SDS-PAGE, Western Blot and quantitative densitometric analysis, ELISA; Reverse-transcription, PCR, qPCR (realtime); miRNA quantification (qPCR) and manipulation;

- Cell Biology :
RNAi (siRNA, shRNA), Transfection of plasmids and siRNA/ miRNA (electroporation, nucleofection, lipofection), adeno-associated virus (AAV)-transduction in primary neural cells, Immunocyto-/ histochemistry, expression of GFP-tagged proteins and localisation by fluorescence- and confocal laser scanning microsopy; expression of recombinant proteins for subsequent purification in pro- and eukaryotic systems, cell-ELISA, luciferase assays for translational regulational studies (DualGlo),

- Cell culture :
Preparation, culture and differentiation of primary oligodendrocytes and neurons from rodents including quality controls in respect to purity and differentiation status; MACS sorting; culture and maintenance of several cell lines (Oli-neu, HT22, Neuro2A, Cos7, HEK293); neuron-oligodendrocyte co-cultures and transient co-cultures of neurons and T-Cells; myelinating murine cortical slice cultures and semi-quantitative quantification of myelination;

Last Publications

White, R; Gonsior, C; Krämer-Albers, E-M, Stoehr, N; Hüttelmaier, S & Trotter, J.: ‘Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in HnRNP A2-dependent RNA granules’ (Journal of Cell Biology, 2008, 181(4):579-586)

Krämer-Albers, E-M & White, R: ‘From axon-glial signalling to myelination: The integrating role of oligodendroglial Fyn Kinase’. (Cellular and Molecular Life Sciences, 2011 68(12):2003-12)

White, R; Gonsior, C, Bauer, NM; Krämer-Albers, E-M; Luhmann, HJ; Trotter, J: ‘HnRNP F is a novel component of oligodendroglial RNA transport granules contributing to the regulation of MBP protein synthesis’ (Journal of Biological Chemistry, 2012;287(3):1742-54)

Bauer, NM, Moos, C; van Horssen, J; Witte, M; van der Valk, P; Altenhein, B; Luhmann, HJ; White, R: ‘Myelin Basic Protein synthesis is regulated by small non-coding RNA 715.’ (EMBO Reports, 2012;13(9):827-34 )

Müller, C; Bauer, NM; Schäfer, I; White, R: ‘Making Myelin Basic Protein -from mRNA transport to localized translation’ (Frontiers in Cellular Neuroscience, 2013 7:169. doi:10.3389/fncel.2013.00169

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