Research

Background: our research program aims to reveal the role of viral RNA structures in events critical to the viral life cycle. While our current research interests extend to RNA structures in many different viruses, our active projects are united by a common theme: we want to understand how RNA structures within a protein-coding transcript change the way they are decoded. The RNA structures we study are found in locations known as “programmed -1 ribosomal frameshift (-1 PRF) sites”. These sites get their name because they cause ribosomes, the macromolecular machines that decode RNA to produce proteins, to slip backwards by a single nucleotide (-1) during translation (Figure 1). This repositioning of a ribosome on the transcript changes the way the RNA is subsequently decoded. While a -1 frameshift would normally be a deleterious mistake by a ribosome, many viruses intentionally trigger these “mistakes” to permit the synthesis of proteins encoded in alternate reading frames.

Figure 1. A cartoon depiction of a -1 PRF. On the left, a ribosome is paused on a frameshift site in the original (0) reading frame. The RNA transcript is shown 5’ to 3’ with select nucleotides included to emphasize the change in reading frame before (left) and after (right) a -1 PRF.

-1 PRFs are used by many viruses with RNA genomes to regulate the synthesis of viral enzymes critical to viral replication. Viral -1 PRF sites include three components: a “slippery sequence”, a spacer, and an RNA structure (Figure 2). When a ribosome encounters a -1 PRF site during translation, it becomes temporarily stalled with the slippery sequence positioned in its A- and P-sites (See the example in Figure 1, which includes a UUUAAAC slippery sequence). The RNA structure in the frameshift site, typically a very stable stem-loop or pseudoknot (Figure 2), acts as a steric block to translation because it cannot fit inside the Ribosome’s mRNA entry channel. The structure’s resistance to unwinding promotes ribosomal stalling over the slippery sequence and modifies a step within translation in a way that promotes -1 ribosomal frameshifting.

Figure 2. A viral -1 PRF site includes three elements. The slippery sequence (XXXYYYZ) is indispensable and alone can increase the basal rate of ribosomal frameshifting from less than 0.005% per codon to as high as ~1%. Here, “X” is any nucleotide (A, U, C, or G), “Y” is A or U, and “Z” is any nucleotide except for G. The 5-8 nucleotide spacer separates the slippery sequence from the downstream structure. This structure, typically a very stable stem-loop or pseudoknot, acts as a steric block to translation. Stem-loop structures are formed through the base-pairing of complementary intra-strand nucleotides. The “stem” includes the base-pairs, which are depicted with a –. Pseudoknot’s include additional nucleotides downstream of the base-paired region that form base-pairs with nucleotides in the loop.

How often a ribosome slips when a -1 PRF site is encountered is described by its frameshift efficiency. Frameshift efficiencies can range from ~5-70%, and depend on the unique composition of RNA elements in the frameshift site. Despite this variance, -1 PRF efficiencies appear to be optimized for each viruses’ particular replicative needs. Decreasing or increasing the frameshift efficiency can have severe impacts on viral infectivity, as is the case for HIV-1, and viral replication, as was demonstrated for the SARS coronavirus. How RNA structures participate in -1 PRF events is a fundamental question of relevance to human health, due to their prevalence in viruses that infect and cause human disease.