New strategies for RNA crystallization and applications to the pri-miRNA processing machinery
- Author(s): Shoffner, Grant
- Advisor(s): Guo, Feng
- et al.
Although the regulatory, signaling, and catalytic functions of RNA are comparable to their protein counterparts, the fact that RNA represent less than 1% of the atomic structures in the Protein Data Bank highlights the gap in our understanding of the RNA structure-function relationship. Overcoming the challenge of RNA structure determination requires innovation in crystallization techniques and further insight into the principles of RNA crystal packing. We propose a scaffold-directed crystallization method for short RNA fragments and employ this technique to determine the three-dimensional conformation of loop sequences from hairpin-structured microRNA (miRNA) precursors. These data reveal common structured elements of the primary miRNA transcripts that may designate the RNA for processing by the Drosha-DGCR8 complex. We further show that mutation of DGCR8, particularly in cancer, can disrupt this process. To study RNA crystal packing, we devise an in crystal selection scheme and show that this selection experiment can generate new lattice contacts in RNA crystals. From these contacts we infer strategies for the rational engineering of RNA lattice contacts. Lastly, we explore the RNA-protein interactions involved in the process of X-chromosome inactivation and uncover a unique Xist RNA-binding region in the regulatory factor SHARP.