Poster abstracts
Poster number 156 submitted by Margaret Bohmer
Identification and Characterization of ATP-Binding Noncoding RNAs in Viruses and Humans
Margaret Bohmer (Center for RNA Nanobiotechnology and Nanomedicine, Center for RNA Biology, College of Pharmacy, James Comprehensive Cancer Center, College of Medicine, The Ohio State University, USA ), Kai Jin (Center for RNA Nanobiotechnology and Nanomedicine, Center for RNA Biology, College of Pharmacy, James Comprehensive Cancer Center, College of Medicine, The Ohio State University, USA), Daniel W. Binzel (Center for RNA Nanobiotechnology and Nanomedicine, Center for RNA Biology, College of Pharmacy, James Comprehensive Cancer Center, College of Medicine, The Ohio State University, USA), Wen Zhang (Department of Biochemistry and Molecular Biology, Melvin and Bren Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana 46202, United States), Peixuan Guo (Center for RNA Nanobiotechnology and Nanomedicine, Center for RNA Biology, College of Pharmacy, James Comprehensive Cancer Center, College of Medicine, The Ohio State University, USA)
Abstract:
During the last stage of replication of double-stranded RNA or DNA viruses, their genome is packaged into a preassembled protein capsid. The bacterial virus phi29 dsDNA packaging motor uses a noncoding packaging RNA (pRNA) molecule to gear its genomic DNA translocation. In this study, we constructed chimeric pRNAs by fusing the pRNA of bacterial virus M2 and that of phi29. The chimeric pRNAs can form dimers or trimers. The dimeric or trimeric pRNAs were active in the packaging of the phi29 dsDNA genome into the purified procapsid, which was subsequently converted into infectious viruses, as proven by counting plaque-forming units (PFU). These data show that the stoichiometry of the chimeric pRNAs on the motor is 6 subunits, a multiple of 2 and 3. Furthermore, AFM studies on pRNA fused to an RNA-triangle revealed hexamer formation. But how do the six identical RNA anchor on the 12-subunit connector with the double stoichiometry? Structural analysis in combination with enzymatic and chemical probing data revealed that each native pRNA contributes 2 domains to bind to the 12-subunit DNA-packaging channel at 3 positively changed residues RKR, proving the formation of the hexameric ring. Resolving the hexamer versus pentamer debate clarifies the mechanism of dsDNA translocation in living organisms.
Additionally, the phi29 pRNA has previously been shown to bind ATP due to its similarity to a synthetic ATP-binding RNA aptamer. The existence of a naturally occurring viral ATP-binding raised the possibility of an endogenous ATP-binding RNA in humans. Due to the abundance of noncoding RNA (ncRNA) discovered in the human body, it is expected that many short or long noncoding RNAs will be involved in ATP binding and hydrolysis. Here, we isolated human RNA and identified an ATP-binding RNA via iterative ATP binding and amplification of the eluate. This ATP-binding ncRNA, which was found to be G-quadruplex-forming, was characterized by ATP column binding and circular dichroism.
Keywords: DNA-packaging motor, ATP-binding RNA, G-quadruplex