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...

RNA chaperones are ubiquitous, heterogeneous proteins essential for RNA structural biogenesis and function. We investigated the mechanism of chaperone-mediated RNA folding by following the time-resolved dimerization of the packaging domain of a retroviral RNA at nucleotide resolution. In the absence of the nucleocapsid (NC) chaperone, dimerization proceeded via multiple, slow-folding intermediates. In the presence of NC, dimerization occurred rapidly via a single structural intermediate. The RNA binding domain of hnRNP A1 protein (UP1), a structurally unrelated chaperone, also accelerated dimerization. Both chaperones interacted primarily with guanosine residues. Replacing guanosine with more weakly pairing inosine yielded an RNA that folded rapidly without a facilitating chaperone. These results show RNA chaperones can simplify RNA folding landscapes by weakening intramolecular interactions involving guanosine and explain many RNA chaperone activities.

Higher-order structure influences critical functions in nearly all non-coding and coding RNAs. Most single-nucleotide resolution RNA structure determination technologies cannot be used to analyze RNA from scarce biological samples, like viral genomes. To make quantitative RNA structure analysis applicable to a much wider array of RNA structure-function problems, we developed and applied high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to structural analysis of authentic genomic RNA of the xenotropic murine leukemia virus-related virus (XMRV). For analysis of fluorescently labeled cDNAs generated in high-sensitivity SHAPE experiments, we developed a two-color capillary electrophoresis approach with zeptomole molecular detection limits and sub-femtomole sensitivity for complete SHAPE experiments involving hundreds of individual RNA structure measurements. High-sensitivity SHAPE data correlated closely (R = 0.89) with data obtained by conventional capillary electrophoresis. Using high-sensitivity SHAPE, we determined the dimeric structure of the XMRV packaging domain, examined dynamic interactions between a packaging domain RNA and viral nucleocapsid protein inside virion particles, and identified the packaging signal for this virus. Despite extensive sequence differences between XMRV and the intensively studied Moloney murine leukemia virus, architectures of the regulatory domains are similar and reveal common principles of gammaretrovirus RNA genome packaging.

The thermostable Geobacillus stearothermophilus GsI-IIC intron is among the few bacterial group II introns found to proliferate to high copy number in its host genome. Here, we developed a bacterial genetic assay for retrohoming and biochemical assays for protein-dependent and self-splicing of GsI-IIC. We found that GsI-IIC, like other group IIC introns, retrohomes into sites having a 5'-exon DNA hairpin, typically from a bacterial transcription terminator, followed by short intron-binding sequences (IBSs) recognized by base pairing of exon-binding sequences (EBSs) in the intron RNA. Intron RNA insertion occurs preferentially but not exclusively into the parental lagging strand at DNA replication forks, using a nascent lagging strand DNA as a primer for reverse transcription. In vivo mobility assays, selections, and mutagenesis indicated that a variety of GC-rich DNA hairpins of 7-19 bp with continuous base pairs or internal elbow regions support efficient intron mobility and identified a critically recognized nucleotide (T-5) between the hairpin and IBS1, a feature not reported previously for group IIC introns. Neither the hairpin nor T-5 is required for intron excision or lariat formation during RNA splicing, but the 5'-exon sequence can affect the efficiency of exon ligation. Structural modeling suggests that the 5'-exon DNA hairpin and T-5 bind to the thumb and DNA-binding domains of GsI-IIC reverse transcriptase. This mode of DNA target site recognition enables the intron to proliferate to high copy number by recognizing numerous transcription terminators and then finding the best match for the EBS/IBS interactions within a short distance downstream.

Retroviral virions initially assemble in an immature form that differs from that of the mature infectious particle. The RNA genomes in both immature and infectious particles are dimers, and interactions between the RNA dimer and the viral Gag protein ensure selective packaging into nascent immature virions. We used high-sensitivity selective 2=-hydroxyl acylation analyzed by primer extension (SHAPE) to obtain nucleotide-resolution structural information from scarce, femtomole quantities of Moloney murine leukemia virus (MuLV) RNA inside authentic virions and from viral RNA extracted from immature (protease-minus) virions. Our secondary structure model of the dimerization and packaging domain indicated that a stable intermolecular duplex known as PAL2, previously shown to be present in mature infectious MuLV particles, was sequestered in an alternate stem-loop structure inside immature virions. The intermediate state corresponded closely to a late-folding intermediate that we detected in time-resolved studies of the free MuLV RNA, suggesting that the immature RNA structure reflects trapping of the intermediate folding state by interactions in the immature virion. We propose models for the RNA-protein interactions that trap the RNA in the immature state and for the conformational rearrangement that occurs during maturation of virion particles. IMPORTANCEThe structure of the RNA genome in mature retroviruses has been studied extensively, whereas very little was known about the RNA structure in immature virions. The immature RNA structure is important because it is the form initially selected for packaging in new virions and may have other roles. This lack of information was due to the difficulty of isolating sufficient viral RNA for study. In this work, we apply a high-sensitivity and nucleotide-resolution approach to examine the structure of the dimerization and packaging domain of Moloney murine leukemia virus. We find that the genomic RNA is packaged in a high-energy state, suggesting that interactions within the virion hold or capture the RNA before it reaches its most stable state. This new structural information makes it possible to propose models for the conformational changes in the RNA genome that accompany retroviral maturation.A n early step in the formation of an infectious retroviral particle involves the association of retroviral RNA with the cell membrane via interactions with the viral Gag protein. Budding from the cell yields a particle in which Gag protein domains are arrayed roughly in concentric spheres and the RNA is bound in the interior of the particle. Subsequent cleavage of the Gag structural protein results in large-scale rearrangements to yield a mature particle with a complex structure (1, 2). The genomic RNA also undergoes conformational rearrangements during particle maturation (3-5). In both immature and mature particles, two copies of the genomic RNA are present, and it is known that the structure adopted by the immature form of the viral RNA is less thermodynamically stable ...