DExD/H-box proteins are ubiquitously involved in RNA-mediated processes and use ATP to accelerate conformational changes in RNA. However, their mechanisms of action, and what determines which RNA species are targeted, are not well understood. Here we show that the DExD/H-box protein CYT-19, a general RNA chaperone, mediates ATP-dependent unfolding of both the native conformation and a long-lived misfolded conformation of a group I catalytic RNA with efficiencies that depend on the stabilities of the RNA species but not on specific structural features. CYT-19 then allows the RNA to refold, changing the distribution from equilibrium to kinetic control. Because misfolding is favoured kinetically, conditions that allow unfolding of the native RNA yield large increases in the population of misfolded species. Our results suggest that DExD/H-box proteins act with sufficient breadth and efficiency to allow structured RNAs to populate a wider range of conformations than would be present at equilibrium. Thus, RNAs may face selective pressure to stabilize their active conformations relative to inactive ones to avoid significant redistribution by DExD/H-box proteins. Conversely, RNAs whose functions depend on forming multiple conformations may rely on DExD/H-box proteins to increase the populations of less stable conformations, thereby increasing their overall efficiencies.

We explore the interactions of CYT-19, a DExD͞H-box protein that functions in folding of group I RNAs, with a well characterized misfolded species of the Tetrahymena ribozyme. Consistent with its function, CYT-19 accelerates refolding of the misfolded RNA to its native state. Unexpectedly, CYT-19 performs another reaction much more efficiently; it unwinds the 6-bp P1 duplex formed between the ribozyme and its oligonucleotide substrate. Furthermore, CYT-19 performs this reaction 50-fold more efficiently than it unwinds the same duplex free in solution, suggesting that it forms additional interactions with the ribozyme, most likely using a distinct RNA binding site from the one responsible for unwinding. This site can apparently bind double-stranded RNA, as attachment of a simple duplex adjacent to P1 recapitulates much of the activation provided by the ribozyme. Unwinding the native P1 duplex does not accelerate refolding of the misfolded ribozyme, implying that CYT-19 can disrupt multiple contacts on the RNA, consistent with its function in folding of multiple RNAs. Further experiments showed that the P1 duplex unwinding activity is virtually the same whether the ribozyme is misfolded or native but is abrogated by formation of tertiary contacts between the P1 duplex and the body of the ribozyme. Together these results suggest a mechanism for CYT-19 and other general DExD͞H-box RNA chaperones in which the proteins bind to structured RNAs and efficiently unwind loosely associated duplexes, which biases the proteins to disrupt nonnative base pairs and gives the liberated strands an opportunity to refold.group I RNA ͉ RNA folding ͉ RNA unwinding ͉ Tetrahymena ribozyme E ssentially all cellular processes that are mediated by structured RNAs also require one or more DExD͞H-box proteins (1). These proteins use the energy from ATP binding and hydrolysis to accelerate RNA structural transitions, which can represent folding steps toward the native state or conformational switches between functional forms. The requirement for proteins presumably arises because RNA base pairs and other local structure can be highly stable even in the absence of enforcing structure, such that folding steps or rearrangements that require significant unfolding require assistance to proceed efficiently (2-4).Despite their ubiquitous presence, key questions about the functions of DExD͞H-box proteins remain largely unanswered. First, what interactions direct different DExD͞H-box proteins to their physiological substrates? All of these proteins share a core ''helicase'' domain containing a set of conserved motifs, and most have additional domains, a few of which have been shown to recognize substrate RNAs or RNA-protein complexes (reviewed in ref. 5). On this basis, targeting has been proposed as a general role for these domains, and the specific interactions that target one DExD͞H-box protein have been delineated (6-9). Nevertheless, in general, the interactions that direct DExD͞H box proteins to their substrates remain to be identified.Second, h...

The DEAD-box protein Mss116p promotes group II intron splicing in vivo and in vitro. Here we explore two hypotheses for how Mss116p promotes group II intron splicing: by using its RNA unwinding activity to act as an RNA chaperone or by stabilizing RNA folding intermediates. We show that an Mss116p mutant in helicase motif III (SAT/AAA), which was reported to stimulate splicing without unwinding RNA, retains ATP-dependent unwinding activity and promotes unfolding of a structured RNA. Its unwinding activity increases sharply with decreasing duplex length and correlates with group II intron splicing activity in quantitative assays. Additionally, we show that Mss116p can promote ATP-independent RNA unwinding, presumably via single-strand capture, also potentially contributing to DEAD-box protein RNA chaperone activity. Our findings favor the hypothesis that DEAD-box proteins function in group II intron splicing as in other processes by using their unwinding activity to act as RNA chaperones.

The DEAD-box protein CYT-19 functions in folding of several group I introns in vivo and a diverse set of group I and group II RNAs in vitro. Recent work using the Tetrahymena group I ribozyme demonstrated that CYT-19 possesses a second RNA binding site, distinct from the unwinding active site, which enhances unwinding activity by binding non-specifically to adjacent RNA structure. Here we probe the region of CYT-19 responsible for that binding by constructing a C-terminal truncation variant that lacks 49 amino acids and terminates at a domain boundary, as defined by limited proteolysis. This truncated protein unwinds a six-base-pair duplex, formed between the oligonucleotide substrate of the Tetrahymena ribozyme and an oligonucleotide corresponding to the internal guide sequence of the ribozyme, with near-wild-type efficiency. However, the truncated protein is activated much less than the wild-type protein when the duplex is covalently linked to the ribozyme or to single-stranded or double-stranded extensions. Thus, the active site for RNA unwinding remains functional in the truncated CYT-19, but the site that binds adjacent RNA structure has been compromised. Equilibrium binding experiments confirmed that the truncated protein binds RNA less tightly than the wild-type protein. RNA binding by the compromised site is important for chaperone activity, as the truncated protein is less active in facilitating folding of a group I intron that requires CYT-19 in vivo. The deleted region contains arginine-rich sequences, as found in other RNA-binding proteins, and may function by tethering CYT-19 to structured RNAs so that it can efficiently disrupt exposed, non-native structural elements, allowing them to re-fold. Many other DExD/H-box proteins also contain arginine-rich ancillary domains, and some of them may function similarly as non-specific RNA-binding elements that enhance general RNA chaperone activity.Structured RNAs are required for a myriad of cellular processes, including mRNA processing and translation, tRNA processing, and maintenance of chromosome ends, and nearly all structured RNAs require at least one DExD/H-box protein for their functions (2,3). DExD/Hbox proteins are thought to function in large part by facilitating structural transitions of RNAs and ribonucleoprotein (RNP) complexes that would otherwise be too slow to allow the complexes to form or function. DExD/H-box proteins include a conserved motor domain, which uses energy derived from cycles of ATP binding and hydrolysis to facilitate structural rearrangements of RNAs, at least in part by 'unwinding' double-stranded segments (2,4). Many † This work was supported by grants from the NIH (R01-GM070456 to R.R. and R01-GM037951 to A.M.L.) and from the Welch Foundation (F-1563 to R.R.). M.D. was supported by a postdoctoral fellowship from the NIH (F01-GM076961).*To whom correspondence should be addressed. Tel: 512-471-1514; Fax: 512-232-3432; E-mail: rick_russell@mail.utexas.edu. 1 Abbreviations: Δ578-626, C-terminal truncation variant of CY...

A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8 + T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4 + T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 μg and 10 μg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine.