Quantitative Mass Spectrometric Analysis of RNA-Protein Cross-Links
2019
In recent times much emphasis has been laid on revealing the composition and regulation of various RNP complexes. The structural studies of the RNP complexes provide a valuable insight into the binding modes and functional implications of interactions between the RNAs and RNA binding proteins within the complexes. To investigate the interactions of the RNA-binding proteins within RNP complexes, UV-induced cross-linking followed by mass spectrometry (MS) has proved to be a promising and straightforward technique. But the limitations of most of the purification methods as well as the intricate mass spectrometric data analysis have hampered the study of these RNP complexes. During the course of this study, the protocol was modified and optimized for the interaction analysis of large RNP complex assemblies like RNP complexes isolated from the HeLa nuclear extract which led to the identification of predicted as well as unknown RBMs. Moreover, the qualitative analysis of the protein-RNA cross-links derived from in vitro assembled Brat-NHL-hb RNA complex and CWC2-U6/U4 snRNAs complexes was also carried out. Based on the qualitative analysis of CWC2-U6/U4 cross-links, the quantitative analysis of protein-RNA cross-links has been established. The studies conducted during the research work have contributed in the identification and characterization of protein-RNA interactions within the aforementioned complexes and also provided the quantitative insight into the protein-RNA interactions.
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