RNAs play an integral part in all kingdoms of life and mediate critical processes from gene regulation to genomic maintenance and protein synthesis.
RNAs also have an amazing diversity in structure and function. The importance of RNA was highlighted at the “International Conference of RNA Nanotechnology and Therapeutics.” At the conference, several cutting-edge RNA technologies and emerging trends were discussed.
Scientists described how to decipher RNA secondary structure via novel mathematic approaches or via in-gel analysis following electrophoresis. Others are modifying technologies such as Illumina sequencing to probe thousands of RNAs simultaneously, or employing tandem mass spectrometry for tRNA analysis. Finally, in the arena of therapeutics, self-assembling RNA nanoparticles are being designed for diagnostics and therapeutics.
RNA processing is a critical facet driving infection by HIV. In the course of infection, HIV RNA is multiply spliced, exported from the nucleus, and translated into viral accessory proteins such as Rev (regulator of expression of viral proteins). To further assist HIV replication, Rev shuttles back into the nucleus and binds the viral Rev response element (RRE), a ~350 nucleotide RNA segment necessary for export of full-length and singly spliced mRNAs.
HIV can be divided into two subgroups. While HIV-1 is responsible for the AIDS pandemic, its cousin, HIV-2, also can cause AIDS.
“Much is known about HIV-1 and Rev1, yet comparatively little is known about Rev2 and its interactions with the homologous RRE2,” said Stuart F.J. Le Grice, Ph.D., a researcher who holds senior positions at both the HIV Drug Resistance Program and the Center for Cancer Research.
“Similar to its HIV-1 counterpart, RRE2 transports HIV-2 viral RNA from the nucleus to the cytoplasm,” Dr. Le Grice continued.
“Our goal was to determine the structure of RRE2 RNA to better understand the nucleoprotein complexes involved in this process. To do this we utilized complementary chemical and biochemical probing techniques.”
Dr. Le Grice’s group employed an RNA probing method that had been pioneered by Kevin Weeks, Ph.D., Kenan Distinguished Professor, department of chemistry, University of North Carolina at Chapel Hill. The method, called SHAPE (selective 2′-hyroxyl acylation analyzed by primer extension), takes data from capillary electrophoresis experiments and automates the interpretation.
“SHAPE works exceeding well as long as there is a single conformation of the RNA. When we evaluated RRE2, we found three bands in our polyacrylamide gels,” said Dr. Le Grice. “One of our very talented post-docs, Sabrina Lusvarghi, Ph.D., came up with the idea to add a novel mathematical approach for determining the secondary structure of each RNA conformer.”
Coupling site-directed hydroxyl radical footprinting along with SHAPE profiling did the trick. The breakthrough, according to Dr. Le Grice, occurred when the method was used to identify transitional states of the RNA. The group found that RRE2 contains five peripheral stem loops linked by adjacent four-way and three-way junctions.
“We don’t know whether RNA structure changes in the context of a virus-infected cell, but do know there is a structural transition in vitro that provides a basis for the first 3D glimpses of this critical viral RNA element,” said Dr. Le Grice. “Approaches such as this will contribute toward developing RNA-based therapeutics against HIV.”