UC Riverside UC Riverside Previously Published Works Title Viral RNAs are unusually compact. Permalink https://escholarship.org/uc/item/6b40r0rp Journal PloS one, 9(9) ISSN 1932-6203 Authors Gopal, Ajaykumar Egecioglu, Defne E Yoffe, Aron M et al. Publication Date 2014 DOI 10.1371/journal.pone.0105875 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Viral RNAs Are Unusually Compact Ajaykumar Gopal1, Defne E. Egecioglu1, Aron M. Yoffe1, Avinoam Ben-Shaul2, Ayala L. N. Rao3, Charles M. Knobler1, William M. Gelbart1* 1 Department of Chemistry & Biochemistry, University of California Los Angeles, Los Angeles, California, United States of America, 2 Institute of Chemistry & The Fritz Haber Research Center, The Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel, 3 Department of Plant Pathology, University of California Riverside, Riverside, California, United States of America Abstract A majority of viruses are composed of long single-stranded genomic RNA molecules encapsulated by protein shells with diameters of just a few tens of nanometers. We examine the extent to which these viral RNAs have evolved to be physically compact molecules to facilitate encapsulation. Measurements of equal-length viral, non-viral, coding and non-coding RNAs show viral RNAs to have among the smallest sizes in solution, i.e., the highest gel-electrophoretic mobilities and the smallest hydrodynamic radii. Using graph-theoretical analyses we demonstrate that their sizes correlate with the compactness of branching patterns in predicted secondary structure ensembles. The density of branching is determined by the number and relative positions of 3-helix junctions, and is highly sensitive to the presence of rare higher-order junctions with 4 or more helices. Compact branching arises from a preponderance of base pairing between nucleotides close to each other in the primary sequence. The density of branching represents a degree of freedom optimized by viral RNA genomes in response to the evolutionary pressure to be packaged reliably. Several families of viruses are analyzed to delineate the effects of capsid geometry, size and charge stabilization on the selective pressure for RNA compactness. Compact branching has important implications for RNA folding and viral assembly. Citation: Gopal A, Egecioglu DE, Yoffe AM, Ben-Shaul A, Rao ALN, et al. (2014) Viral RNAs Are Unusually Compact. PLoS ONE 9(9): e105875. doi:10.1371/journal. pone.0105875 Editor: Xi Zhou, Wuhan University, China Received May 25, 2014; Accepted July 21, 2014; Published September 4, 2014 Copyright: ß 2014 Gopal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by the U.S. National Science Foundation Division of Chemistry (http://www.nsf.gov/div/index.jsp?div = CHE) grants CHE 0714411 and 1051507 to WMG and CMK, and the Israel Science Foundation (http://www.isf.org.il/english) grant 1448/10 to ABS. The CNSI Advanced Light Microscopy/Spectroscopy Shared Resource Facility at UCLA was supported by funding from NIH-NCRR shared resources grant (CJX1-443835-WS-29646) and NSF Major Research Instrumentation grant (CHE-0722519). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] Introduction case, it is possible to predict and/or measure the radius of gyration as a function of molecular weight (number of monomeric units). Single-stranded (ss) RNA molecules are typically branched, with Very few experiments and theories [7,14–19] extend this approach physical properties that depend on the secondary and tertiary to long RNA molecules. In particular, the connection between structures determined by their primary nucleotide (nt) sequence primary sequence and branching properties, and the resulting [1–3]. High-resolution structures have been elucidated for several molecular sizes, has not been studied in long RNAs. biologically important molecules with lengths up to hundreds of nt; ssRNA viral genomes are special RNA molecules in several e.g. ribozymes, transfer RNAs, and messenger RNA sub-sequenc- significant ways. First, because they involve two or more genes, es [4–6]. For longer sequences, however, it is generally not possible they are necessarily thousands of nt long. Also, unlike other long to identify a unique secondary/tertiary structure that dominates RNAs, such as edited messenger RNA transcripts or ribosomal the ensemble of configurational states of the molecule [7,8]. [An RNA, they are constrained to have physical sizes compatible with important exception is that of ribosomal RNAs [9], but there the being packaged spontaneously by viral coat proteins into small structures of these thousands-of-nt-long RNA molecules are largely volumes corresponding to the inside of a rigid viral capsid. As determined by the many proteins with which they are bound.] proposed in earlier theoretical work [7,14], the above factors Coarse-grained statistical properties – such as radius of gyration suggest that viral RNA molecules might be more compact than and shape anisotropy – have been measured for viral RNAs other sequences of identical length, in order to enhance their several thousand nt long [8,10], but how these relate to the encapsulation efficiency and hence the survivability of the virus. primary sequence or even the underlying secondary structures has In this paper, we study the correlation between the nt sequence not been studied systematically. and the physical size of large RNA molecules – in particular, On the other hand, the statistics of model branched molecules whether the sequence of a viral RNA codes not just for required and aggregates are well studied [11–13], e.g. ‘‘star’’ polymers, protein products but also for its own physical size. We compare the dendrimers, diffusion-limited-aggregation clusters, mathematical experimentally determined size of a 2117-nt viral RNA with those tree structures and ideal randomly-branched polymers. In each of non-viral sequences of identical length and find the viral PLOS ONE | www.plosone.org 1 September 2014 | Volume 9 | Issue 9 | e105875 Viral RNAs Are Unusually Compact sequence to be the most compact. The relative sizes of these conclusions relate exclusively to differences in these properties sequences are explained by analyzing the nature of branching in between viral and non-viral sequences. predicted ensembles of secondary structures. We show that compactness originates from an increased density of branching Relative Gel Electrophoretic Mobilities defined by the number, degree and organization of multi-helix Sizes are first investigated by electrophoresis (Fig. 1) through a junctions in secondary structures. We compare several families of 1% agarose gel prepared and run in pH 7.4 TAE buffer (see ssRNA viruses and find that genomes with propensity for denser Methods). Prior to loading, the RNAs were equilibrated in TE branching typically belong to families where other means of RNA buffer for 24 hours to obtain reliable hydrodynamic properties compaction (e.g., polyvalent cations) may not exist. Finally, we [21]. Each lane contains 1 mg RNA (*1011 molecules) and 1 ng of outline how compactness improves the robustness of RNA folding a 2141-bp linear dsDNA added as an internal marker. and enhances the ability of a viral genome to package in a capsid. The RNA band positions in Fig. 1 indicate that B3 (lane 1) has the highest mobility; B3A, B3R and B3RA migrate a slightly Results shorter distance, whereas the yeast-based sequences Y1–Y5 are most retarded by the gel. The viral and viral-based RNAs are RNA Sequence and Buffer Choice therefore effectively smaller in size compared to Y1–Y5, with B3 To test the relationship between the primary nt sequence of an being the most compact. To confirm these trends, B3 and Y2 were RNA and its physical size, we study nine RNAs of identical lengths mixed prior to loading in lane 6. Electrophoresis clearly separates (2117 nt), but different nt compositions and biological functions. the two bands, demonstrating that the physical properties of their The first is genomic RNA3 of Brome Mosaic Virus (BMV) [20], a molecular ensembles are distinct. In other words, although each plant pathogen. BMV RNA3 (B3) is a two-gene plus-strand RNA band represents *1011 molecules with various secondary and coding for a movement protein (MP) and a capsid protein (CP). tertiary configurations, the molecular sizes and shapes in a given The second molecule is the anti-sense (i.e. reverse-complement or band (sequence) are closer to each other than to those in other minus-strand) RNA of B3, hereafter denoted as B3A (BMV RNA3 bands. Differences in mobilities have similarly been observed Anti-sense). An anti-sense strand can differ in composition and between evolved and random sequences of short (, 100 nt) RNAs pattern only in the unpaired regions of the sense strand, therefore [22]. representing a sequence with most of the original nt patterns and The mobility of an RNA can be quantified as its distance from about 20% change in composition. The third molecule is a B3 the DNA marker band (see Methods). Relative mobilities (mr) are mutant, hereafter called B3R (BMV RNA3-Reverse), with the calculated with respect to the fastest migrating RNA, in this case positions of the MP and CP genes swapped. This alteration B3 in lane 1 (see Table S1 in File S1). Because the RNAs all have changes the overall sequence, but not the nt composition. It also the same formal charge, differences in their mobilities are expected hampers the ability of B3 to package into virions both in vivo and to arise from differences in their ability to diffuse through the gel in vitro [20].