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Oct 15, 2013 (Vol. 33, No. 18)

Deciphering RNA Secondary Structure

  • 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.”

  • Novel RNA Modeling

    Delineating the secondary structure of large, multidomain RNA can be complicated when more than one structural conformer is present or when molecular multimerization occurs. “Most RNAs in vivo likely adopt alternate functional conformations involving intermolecular or intramolecular interactions,” said Julia C. Kenyon Ph.D., a post-doctoral fellow in the laboratory of Andrew M. L. Lever, Ph.D., who heads the division of infectious diseases at the University of Cambridge’s department of medicine.

    Traditionally, a variety of biochemical techniques are used to analyze RNA secondary structure, including nuclease mapping and chemical modifications. With these techniques, reactive sites are examined by primer extension or 3′ labeling with radioactive isotopes. With the SHAPE method, however, investigators take advantage of recently found compounds that acylate the ribose 2′-hydroxyl such as occurs in single-stranded regions.

    Dr. Kenyon developed a novel method called in-gel SHAPE in which a mixed structural population of RNA molecules is first separated by native polyacrylamide gel electrophoresis and then conformers are subsequently individually isolated and analyzed. “By probing the individual RNAs in a population within the gel matrix, we can determine the structure of each conformer without the need for mutagenesis or variation of refolding conditions,” said Dr. Kenyon. “It’s a straightforward approach, and sometimes when we present this at meetings people comment, ‘Why hasn’t this been done before?’”

    To validate her in-gel technique, Dr. Kenyon used the well-characterized, structurally known HIV-1 trans-activation response element (TAR). “Our method provided an authentic and accurate structural analysis that was also highly reproducible. We then further examined the utility of in-gel SHAPE analysis for separating monomeric and dimeric species of HIV-1 packaging signal RNA,” said Dr. Kenyon. “We found extensive differences between monomer and dimer that supported a recently proposed structural switch model of the RNA genomic dimerization and packaging.”

    Although current analyses are limited to RNAs of 1,000 or fewer nucleotides, the group is examining different gel matrixes to solve that challenge. “We plan on utilizing this approach in the future to study RNA/RNA interactions as well as RNA/protein interactions. It’s a great way to increase throughput over traditional methods,” said Dr. Kenyon.

  • RNA Mapping

    Chemical probing is a valuable tool for inferring RNA functional secondary structures and tertiary RNA interactions. The gold standards of chemical probing are comparative sequence analysis, X-ray crystallography, and nuclear magnetic resonance (NMR). These techniques, however, require significant investigator insight and effort. Even worse, they are not always tractable for RNAs.

    “We have created a whole new platform utilizing Illumina sequencing to probe thousands of RNA molecules at once,” reported Rhiju Das, Ph.D., assistant professor of biochemistry at Stanford University. “It takes about a day and only requires a tabletop Illumina machine followed by analysis with our software package, which is called MAPseeker.”

    Initially, Julius B. Lucks, Ph.D., and his group at Cornell University’s department of chemical and biomolecular engineering developed an experimental protocol and bioinformatics pipeline to enable highly parallel chemical mapping. “We decided to extend this to RNAs of many distinct sequences,” said Dr. Das. “We also decided to advance this protocol into a MAP-seq (multiplexed accessibility probing) method in order to increase the rate at which large numbers of RNAs could be probed and to reduce bias.”

    The strategy allows multiple chemical reactions, such as carbodiimide base modification and acylation of the 2′-hydroxyl to be carried out in parallel. Conditions that give near-quantitative, single-stranded DNA ligation are used, and an Illumina adapter sequence is added to the reverse-transcribed DNA. “Unique sequence identifiers, placed at the 3′ ends of RNAs, are then sequestered into hairpins,” said Dr. Das. “This helps prevent interference with the fold of the RNA segments.” Finally, analysis is carried out with MAPseeker, an automated tool that can, according to Dr. Das, “quantify all the reactivities of the nucleotides in each RNA of every condition.”

    MAP-seq continues to evolve rapidly to enhance multiplexing, to probe oligonucleotides pools, and to tackle RNAs with lengths reaching into the kilobases. “Right now one limitation of the technology is size. The limit is about 100 nucleotides. That will likely be increased in the coming years,” said Dr. Das.

    Dr. Das also has constructed a fun, Web-based site where anyone can type in an RNA sequence and then get MAP-seq structural data through his lab’s experimental pipeline. “It’s really becoming quite popular,” said Dr Das. “You can check it out at http://eterna.stanford.edu/web/.”



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