Sizes of Long RNA Molecules Are Determined by the Branching Patterns of Their Secondary Structures

Long RNA molecules are at the core of gene regulation across all kingdoms of life, whilst also serving as genomes in RNA viruses. Few studies have addressed the basic physical properties of long single-stranded RNAs. Long RNAs with non-repeating sequences usually adopt highly ramified secondary structures and are better described as branched polymers. In order to test whether a branched polymer model can estimate the overall sizes of large RNAs we employed fluorescence correlation spectroscopy to examine the hydrodynamic radii of a broad spectrum of biologically important RNAs, ranging from viral genomes to long non-coding regulatory RNAs. The relative sizes of long RNAs measured at low ionic strength correspond well to those predicted by two theoretical approaches that treat the effective branching associated with secondary structure formation – one employing the Kramers theorem for calculating radii of gyration, and the other featuring the metric of “maximum ladder distance”. Upon addition of multivalent cations, most RNAs are found to be compacted as compared with their original, low-ionic-strength sizes. These results suggest that sizes of long RNAmolecules are determined by the branching pattern of their secondary structures. They also experimentally validate the proposed computational approaches for estimating hydrodynamic radii of single-stranded RNAs, which use generic RNA structure prediction tools and thus can be universally applied to a wide range of long RNAs

Role of RNA Branchedness in the Competition for Viral Capsid Proteins

To optimize bindingand packagingby their capsid proteins (CP), single-stranded (ss) RNA viral genomes often have local secondary/tertiary structures with high CP affinity, with these “packaging signals” serving as heterogeneous nucleation sites for the formation of capsids. Under typical <i>in vitro</i> self-assembly conditions, however, and in particular for the case of many ssRNA viruses whose CP have cationic N-termini, the adsorption of CP by RNA is nonspecific because the CP concentration exceeds the largest dissociation constant for CP–RNA binding. Consequently, the RNA is saturated by bound protein before lateral interactions between CP drive the homogeneous nucleation of capsids. But, before capsids are formed, the binding of protein remains reversible and introduction of another RNA specieswith a different length and/or sequenceis found experimentally to result in significant redistribution of protein. Here we argue that, for a given RNA mass, the sequence with the highest affinity for protein is the one with the most compact secondary structure arising from self-complementarity; similarly, a long RNA steals protein from an equal mass of shorter ones. In both cases, it is the lateral attractions between bound proteins that determines the relative CP affinities of the RNA templates, even though the individual binding sites are identical. We demonstrate this with Monte Carlo simulations, generalizing the Rosenbluth method for excluded-volume polymers to include branching of the polymers and their reversible binding by protein

Characterization of Viral Capsid Protein Self-Assembly around Short Single-Stranded RNA

For many viruses, the packaging of a single-stranded RNA (ss-RNA) genome is spontaneous, driven by capsid protein–capsid protein (CP) and CP–RNA interactions. Furthermore, for some multipartite ss-RNA viruses, copackaging of two or more RNA molecules is a common strategy. Here we focus on RNA copackaging <i>in vitro</i> by using cowpea chlorotic mottle virus (CCMV) CP and an RNA molecule that is short (500 nucleotides (nts)) compared to the lengths (≈3000 nts) packaged in wild-type virions. We show that the degree of cooperativity of virus assembly depends not only on the relative strength of the CP–CP and CP–RNA interactions but also on the RNA being short: a 500-nt RNA molecule cannot form a capsid by itself, so its packaging requires the aggregation of multiple CP–RNA complexes. By using fluorescence correlation spectroscopy (FCS), we show that at neutral pH and sufficiently low concentrations RNA and CP form complexes that are smaller than the wild-type capsid and that four 500-nt RNAs are packaged into virus-like particles (VLPs) only upon lowering the pH. Further, a variety of bulk-solution techniques confirm that fully ordered VLPs are formed only upon acidification. On the basis of these results, we argue that the observed high degree of cooperativity involves equilibrium between multiple CP/RNA complexes