A wide range of RNA viruses use programmed −1 ribosomal frameshifting for the production of viral fusion proteins. Inspection of the overlap regions between ORF1a and ORF1b of the SARS-CoV genome revealed that, similar to all coronaviruses, a programmed −1 ribosomal frameshift could be used by the virus to produce a fusion protein. Computational analyses of the frameshift signal predicted the presence of an mRNA pseudoknot containing three double-stranded RNA stem structures rather than two. Phylogenetic analyses showed the conservation of potential three-stemmed pseudoknots in the frameshift signals of all other coronaviruses in the GenBank database. Though the presence of the three-stemmed structure is supported by nuclease mapping and two-dimensional nuclear magnetic resonance studies, our findings suggest that interactions between the stem structures may result in local distortions in the A-form RNA. These distortions are particularly evident in the vicinity of predicted A-bulges in stems 2 and 3. In vitro and in vivo frameshifting assays showed that the SARS-CoV frameshift signal is functionally similar to other viral frameshift signals: it promotes efficient frameshifting in all of the standard assay systems, and it is sensitive to a drug and a genetic mutation that are known to affect frameshifting efficiency of a yeast virus. Mutagenesis studies reveal that both the specific sequences and structures of stems 2 and 3 are important for efficient frameshifting. We have identified a new RNA structural motif that is capable of promoting efficient programmed ribosomal frameshifting. The high degree of conservation of three-stemmed mRNA pseudoknot structures among the coronaviruses suggests that this presents a novel target for antiviral therapeutics.

There is something special about mRNA pseudoknots that allows them to elicit efficient levels of programmed −1 ribosomal frameshifting. Here, we present a synthesis of recent crystallographic, molecular, biochemical, and genetic studies to explain this property. Movement of 9 Å by the anticodon loop of the aminoacyl-tRNA at the accommodation step normally pulls the downstream mRNA a similar distance along with it. We suggest that the downstream mRNA pseudoknot provides resistance to this movement by becoming wedged into the entrance of the ribosomal mRNA tunnel. These two opposing forces result in the creation of a local region of tension in the mRNA between the A-site codon and the mRNA pseudoknot. This can be relieved by one of two mechanisms; unwinding the pseudoknot, allowing the downstream region to move forward, or by slippage of the proximal region of the mRNA backwards by one base. The observed result of the latter mechanism is a net shift of reading frame by one base in the 5 direction, that is, a −1 ribosomal frameshift.Keywords: Virus; ribosome; translation; genetic code; recoding; structure/function After a generation spent in the shadows, the ribosome is enjoying a renaissance. Recent breakthroughs in X-ray crystallography and cryoelectron microscopy have given us atomic-level views of this complex molecular machine Wimberly et al. 2000;Harms et al. 2001;Spahn et al. 2001;) that are bringing into focus the relationship between ribosome structure and function (Gabashvili et al. 1999;Agrawal et al. 2000;Carter et al. 2000;Frank and Agrawal 2000;Mueller et al. 2000;Nissen et al. 2000;Schluenzen et al. 2000;Beckmann et al. 2001;Nissen et al. 2001; Pioletti et al. 2001;Polacek et al. 2001;Thompson et al. 2001;Yusupova et al. 2001;Noller et al. 2002;Schmeing et al. 2002;Simonson and Lake 2002). One of the major requirements of the ribosome is to maintain translational reading frame, and an increasing number of cis-acting mRNA signals that alter this have been used to probe this essential function of the translational machinery. These translational "recoding" events (Gesteland and Atkins 1996) can take many forms, for example, "slips" of one or more bases, "hops" spanning as many as 50 nucleotides, and "shunts" around large mRNA secondary structures (for review, see Jacks 1990;Brierley 1995;Farabaugh 1996;Giedroc et al. 2000). Programmed −1 ribosomal frameshifting (−1 PRF) is the most widely used translational recoding mechanism of RNA viruses. The −1 PRF signal can be broken down into three discrete parts: the "slippery site", a linker region, and a downstream region of secondary mRNA structure, typically an mRNA pseudoknot. Mutagenesis studies from many different laboratories have demonstrated that the primary sequence of the slippery site and its placement in relation to the incoming translational reading frame is critical: It must be X XXY YYZ, where X must be a stretch of three identical nucleotides, Y is either AAA or UUU, and Z is A, C, or U. Although less is known about the linker region, ...

Bicistronic reporter assay systems have become a mainstay of molecular biology. While the assays themselves encompass a broad range of diverse and unrelated experimental protocols, the numerical data garnered from these experiments often have similar statistical properties. In general, a primary dataset measures the paired expression of two internally controlled reporter genes. The expression ratio of these two genes is then normalized to an external control reporter. The end result is a 'ratio of ratios' that is inherently sensitive to propagation of the error contributed by each of the respective numerical components. The statistical analysis of this data therefore requires careful handling in order to control for the propagation of error and its potentially misleading effects. A careful survey of the literature found no consistent method for the statistical analysis of data generated from these important and informative assay systems. In this report, we present a detailed statistical framework for the systematic analysis of data obtained from bicistronic reporter assay systems. Specifically, a dual luciferase reporter assay was employed to measure the efficiency of four programmed À1 frameshift signals. These frameshift signals originate from the L-A virus, the SARSassociated Coronavirus and computationally identified frameshift signals from two Saccharomyces cerevisiae genes. Furthermore, these statistical methods were applied to prove that the effects of anisomycin on programmed À1 frameshifting are statistically significant. A set of Microsoft Excel spreadsheets, which can be used as templates for data generated by dual reporter assay systems, and an online tutorial are available at our website (http://dinmanlab. umd.edu/statistics). These spreadsheets could be easily adapted to any bicistronic reporter assay system.

The subcellular localization and translation of mRNA supports functional differentiation between cellular compartments. In neuronal dendrites, local translation of mRNA provides a rapid and specific mechanism for synaptic plasticity and memory formation, and might be involved in the pathophysiology of certain brain disorders. Despite the importance of dendritic mRNA translation, little is known about which mRNAs can be translated in dendrites in vivo and when their translation occurs. Here we collect ribosome-bound mRNA from the dendrites of CA1 pyramidal neurons in the adult mouse hippocampus. We find that dendritic mRNA rapidly associates with ribosomes following a novel experience consisting of a contextual fear conditioning trial. High throughput RNA sequencing followed by machine learning classification reveals an unexpected breadth of ribosome-bound dendritic mRNAs, including mRNAs expected to be entirely somatic. Our findings are in agreement with a mechanism of synaptic plasticity that engages the acute local translation of functionally diverse dendritic mRNAs.