Browsing by Subject "Heterogeneous-Nuclear Ribonucleoprotein Group A-B"
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Item Characterization of the Roles of Intrinsically Disordered Regions from RNA-Binding Proteins in Phase Separation(2016-05-27) Lin, Yuan; Ross, Elliott M.; Liu, Qinghua; Tu, Benjamin; Rosen, Michael K.Eukaryotic cells organize complex biochemical reactions through compartmentalization. While many intracellular compartments are enclosed by membranes, others are not. Messenger ribonucleoprotein (mRNP) granules are membrane-less organelles that enrich RNA and RNA-binding proteins containing intrinsically disordered regions (IDRs). I demonstrate that IDRs, coupled with RNA binding domain and RNA, can phase separate in vitro, producing dynamic liquid droplets. Over time, these droplets mature into more stable states, as assessed by slowed fluorescence recovery after photobleaching and resistance to salt. Maturation often coincides with the formation of fibrous structures. Pathological mutation within IDRs leads to the acceleration of maturation. Different disordered domains can co-assemble into phase-separated droplets. In the case of the IDR from FUS (fused in sarcoma), I show that tyrosine residues are important in promoting phase separation. Either mutation of these aromatic residues or phosphorylation of the IDR disassembles liquid droplets. I further discover that the disassembly is due to the disruption of aromatic interactions mediated by critical tyrosine residues and therefore an increase in the overall solubility of proteins. Taken together, these studies demonstrate a plausible mechanism by which interactions between IDRs, coupled with RNA binding, could contribute to mRNP granule assembly in vivo by promoting phase separation. Progression from dynamic liquids to stable fibers may be regulated to produce cellular structures with diverse physiochemical properties and functions. Misregulation of maturation could contribute to diseases that are associated with aberrant mRNP granules. Posttranslational modifications of IDRs could modulate the assembly and disassembly of mRNP granules by altering the solubility of IDRs.Item Chemical Footprinting of Polymeric Structure of hnRNPA2 Low Complexity Domain(2016-05-24) Xiang, Siheng; Nijhawan, Deepak; Rosen, Michael K.; Yu, Hongtao; Kliewer, Steven A.; McKnight, Steven L.Many DNA and RNA regulatory proteins contain polypeptide domains that are unstructured when analyzed in cell lysates. These domains are typified by an over-representation of a limited number of amino acids and have been termed prion-like, intrinsically disordered or low complexity domains. These low complexity sequences have been shown to induce phase transition in low salt buffer. When incubated at high concentration, certain of these low complexity domains polymerize into labile, amyloid-like fibers. I developed a chemical footprinting method to probe solvent accessible residues in the low complexity domain polymers. By acetylating protein side chains with N-acetylimidazole, and comparing the acetylation in native and denatured conformation by use of SILAC mass spectrometry, I generated an NAI footprint for hnRNPA2 polymers. I deployed this footprinting technique to probe the structure of the native hnRNPA2 protein present in isolated nuclei, and offered evidence that its low complexity domain exists in a similar conformation as that described for recombinant polymers of the protein. To study the structure of the low complexity sequence in liquid-like droplets, I systematically mutated individual tyrosine or phenylalanine residues to serine, assayed the ratio of these mutants that partitioned into the droplet phase, and compared the results with their abilities to grow polymeric fibers from wild-type seeds. The same region which contained mutations impeding fiber growth were found to display decreased partitioning into liquid-like droplets. Additionally, the NAI footprint of hnRNPA2 in these liquid-like droplets appeared to be similar to the footprint found in fibers. These observations suggest that the hnRNPA2 low complexity domain adopts a similar structure in amyloid-like fibers and liquid-like droplets. Combining these results, my studies favor the perspective that cross-beta polymerization commonly drives the formation of hydrogels, the retention of low complexity domains trapped by hydrogels, the formation of liquid-like droplets, the partitioning of low complexity domains into existing liquid-like droplets, and the formation and maturation of RNA granules. In other words, my results provide evidence that the involvement of low complexity domains in the formation of RNA granules, liquid-like droplets and hydrogels all rely on one in the same phenomenon - cross-beta polymerization.