In vitro / in vivo investigation of association between protein and long non-coding RNA
A large part of the mammalian transcriptome comprises long RNAs that have little or no protein-coding capacity. Only a few of such transcripts have been analyzed in depth but their role in the cell is still unclear. Non-coding RNAs (ncRNAs) can be involved in the facilitation of nucleic acid hybridization, regulation of cellular signals and formation of protein assemblies.
My research focus on the functional investigation of macromolecular associations between long ncRNAs and protein. For this purpose I'll combine a variety of coordinated in vitro and in vivo experiments with in silico prediction methods (catRAPID). In particular, I plan to isolate non-coding transcripts that exhibit multiple protein binding domains, study their activity as cell regulators and elucidate their role in pathology (neurological diseases). More in details, I will proceed with: i) identification of protein partners for specific long ncRNAs; ii) detection of common RNA structures for protein recognition and cellular localization; iii) mapping of RNA-protein interaction networks to pinpoint disease-related targets; iv) structure-function elucidation of selected ncRNAs-protein complexes.
Experimental techniques that I plan to use are summarized here below:
In vitro transcription using T7 RNA polymerase-based methods; RNA Electrophoretic Mobility Shift (REMSA); RNA Filter Binding Assays; In vitro affinity-tagged isolation methods for protein-RNA interactions; RNA ChIP and RIP-ChIP; In vitro Cross-linking and RNase footprinting assays; Targeted Purification of RNA-Specific RNPs (Ribotrap); In vivo Cross-Linking and ImmunoPrecipitation of RNA-binding proteins (CLIP and PAR-CLIP); Fluorescence-based localization of RNAs and Proteins; Surface Plasmon Resonance (SPR) using Biacore device; Isothermal Titration Calorimetry (ITC); NMR Spectroscopy; X-ray Crystallography.
Past Research activity: In vitro characterization of Protein-Protein Interactions
ISBN-10: 3639311604, ISBN-13: 978-3639311600
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