RNA binding activates RIG-I by releasing an autorepressed signaling domain.

The retinoic acid-inducible gene I (RIG-I) innate immune receptor is an important immunotherapeutic target, but we lack approaches for monitoring the physical basis for its activation in vitro. This gap in our understanding has led to confusion about mechanisms of RIG-I activation and difficulty discovering agonists and antagonists. We therefore created a novel fluorescence resonance energy transfer-based method for measuring RIG-I activation in vitro using dual site-specific fluorescent labeling of the protein. This approach enables us to measure the conformational change that releases the signaling domain during the first step of RIG-I activation, making it possible to understand the role of stimulatory ligands. We have found that RNA alone is sufficient to eject the signaling domain, ejection is reversible, and adenosine triphosphate plays but a minor role in this process. These findings help explain RIG-I dysfunction in autoimmune disease, and they inform the design of therapeutics targeting RIG-I.

Transcriptome analysis of human cumulus cells reveals hypoxia as the main determinant of follicular senescence.

STUDY QUESTION: Can RNA sequencing of human cumulus cells (CC) reveal molecular pathways involved in the physiology of reproductive aging? STUDY FINDING: Senescent but not young CC activate gene pathways associated with hypoxia and oxidative stress. WHAT IS KNOWN ALREADY: Shifts in socioeconomic norms are resulting in larger numbers of women postponing childbearing. The reproductive potential is sharply decreased with aging, and the reasons are poorly understood. Since CCs play an integral role in oocyte maturation and direct access to human oocytes is limited, we used whole transcriptome analysis of these somatic cells to gain insights into the molecular mechanisms playing a role in follicular senescence. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Twenty CC samples (from a total of 15 patients) were obtained from oocytes of either male factor or egg donor patients. RNA sequencing and bioinformatic tools were used to identify differentially expressed genes between CCs from seven aged and eight young patients (<35 (years old) y.o. vs >40 y.o.). Quantitative-PCR and immunoflourescent staining were used for validation. MAIN RESULTS AND THE ROLE OF CHANCE: RNA sequencing identified 11 572 genes expressed in CC of both age cohorts, 45 of which were differentially expressed. In CC collected from patients >40 y.o., genes involved in the hypoxia stress response (NOS2, RORA and NR4A3), vasculature development (NR2F2, PTHLH), glycolysis (RALGAPA2 and TBC1D4) and cAMP turnover (PDE4D) were significantly overexpressed when compared with CC of patients younger than 35 y.o. LIMITATIONS, REASONS FOR CAUTION: This study focused almost exclusively on assessing the genetic differences in CC transcriptome between young and older women. These genetic findings were not fully correlated with embryonic development and clinical outcome. WIDER IMPLICATIONS OF THE FINDINGS: Our data provide a new hypothesis-follicular hypoxia-as the main mechanism leading to ovarian follicular senescence and suggest a link between cumulus cell aging and oocyte quality decay. If specific molecular findings of hypoxia would be confirmed also in oocytes, genetic platforms could screen CC for hypoxic damage and identify healthier oocytes. Protocols of ovarian stimulation in older patients could also be adjusted to diminish oocyte exposure time to hypoxic follicles. LARGE SCALE DATA: GEO accession number: GSE81579 STUDY FUNDING AND COMPETING INTERESTS: Funded in part by EMD Serono Grant for Fertility Innovation (GFI).

Control of branch-site choice by a group II intron.

The branch site of group II introns is typically a bulged adenosine near the 3'-end of intron domain 6. The branch site is chosen with extraordinarily high fidelity, even when the adenosine is mutated to other bases or if the typically bulged adenosine is paired. Given these facts, it has been difficult to discern the mechanism by which the proper branch site is chosen. In order to dissect the determinants for branch-point recognition, new mutations were introduced in the vicinity of the branch site and surrounding domains. Single mutations did not alter the high fidelity for proper branch-site selection. However, several combinations of mutations moved the branch site systematically to new positions along the domain 6 stem. Analysis of those mutants, together with a new alignment of domain 5 and domain 6 sequences, reveals a set of structural determinants that appear to govern branch-site selection by group II introns.

Visualizing the solvent-inaccessible core of a group II intron ribozyme.

Group II introns are well recognized for their remarkable catalytic capabilities, but little is known about their three-dimensional structures. In order to obtain a global view of an active enzyme, hydroxyl radical cleavage was used to define the solvent accessibility along the backbone of a ribozyme derived from group II intron ai5gamma. These studies show that a highly homogeneous ribozyme population folds into a catalytically compact structure with an extensively internalized catalytic core. In parallel, a model of the intron core was built based on known tertiary contacts. Although constructed independently of the footprinting data, the model implicates the same elements for involvement in the catalytic core of the intron.

Guiding ribozyme cleavage through motif recognition: the mechanism of cleavage site selection by a group ii intron ribozyme.

The mechanism by which group II introns cleave the correct phosphodiester linkage was investigated by studying the reaction of mutant substrates with a ribozyme derived from intron ai5gamma. While fidelity was found to be quite high in most cases, a single mutation on the substrate (+1C) resulted in a dramatic loss of fidelity. When this mutation was combined with a second mutation that induces a bulge in the exon binding site 1/intron binding site 1 (EBS1/IBS1) duplex, the base-pairing register of the EBS1/IBS1 duplex was shifted and the cleavage site moved to a downstream position on the substrate. Conversely, when mismatches were incorporated at the EBS1/IBS1 terminus, the duplex was effectively truncated and cleavage occurred at an upstream site. Taken together, these data demonstrate that the cleavage site of a group II intron ribozyme can be tuned at will by manipulating the thermodynamic stability and structure of the EBS1/IBS1 pairing. The results are consistent with a model in which the cleavage site is not designated through recognition of specific nucleotides (such as the 5'-terminal residue of EBS1). Instead, the ribozyme detects a structure at the junction between single and double-stranded residues on the bound substrate. This finding explains the puzzling lack of phylogenetic conservation in ribozyme and substrate sequences near group II intron target sites.

Active disruption of an RNA-protein interaction by a DExH/D RNA helicase.

All aspects of cellular RNA metabolism and the replication of many viruses require DExH/D proteins that manipulate RNA in a manner that requires nucleoside triphosphates. Although DExH/D proteins have been shown to unwind purified RNA duplexes, most RNA molecules in the cellular environment are complexed with proteins. It has therefore been speculated that DExH/D proteins may also affect RNA-protein interactions. We demonstrate that the DExH protein NPH-II from vaccinia virus can displace the protein U1A from RNA in an active adenosine triphosphate-dependent fashion. NPH-II increases the rate of U1A dissociation by more than three orders of magnitude while retaining helicase processivity. This indicates that DExH/D proteins can effectively catalyze protein displacement from RNA and thereby participate in the structural reorganization of ribonucleoprotein assemblies.

Kinetic dissection of the multistep process in L1.ltrB intron mobility.

A kinetic characterization is presented for intron insertion into a target duplex DNA site during L1.ltrB intron mobility. This reaction is catalyzed by a ribonucleoprotein particle (RNP), consisting of a lariat form of group II intron RNA and the intron-encoded LtrA protein. In the first stage of intron mobility, the RNA component of the enzyme by itself inserts directly into the target ds DNA site, and thus, the RNP enzyme does not carry out turnover. Using single-turnover kinetics, we established an in vitro kinetic assay system and investigated mechanism of the intron RNA insertion process.

Metal ion binding sites in a group II intron core.

Group II introns are catalytic RNA molecules that require divalent metal ions for folding, substrate binding, and chemical catalysis. Metal ion binding sites in the group II core have now been elucidated by monitoring the site-specific RNA hydrolysis patterns of bound ions such as Tb(3+) and Mg(2+). Major sites are localized near active site elements such as domain 5 and its surrounding tertiary interaction partners. Numerous sites are also observed at intron substructures that are involved in binding and potentially activating the splice sites. These results highlight the locations of specific metal ions that are likely to play a role in ribozyme catalysis.

A tertiary interaction that links active-site domains to the 5' splice site of a group II intron.

Group II introns are self-splicing RNAs that are commonly found in the genes of plants, fungi, yeast and bacteria. Little is known about the tertiary structure of group II introns, which are among the largest natural ribozymes. The most conserved region of the intron is domain 5 (D5), which, together with domain 1 (D1), is required for all reactions catalysed by the intron. Despite the importance of D5, its spatial relationship and tertiary contacts to other active-site constituents have remained obscure. Furthermore, D5 has never been placed directly at a site of catalysis by the intron. Here we show that a set of tertiary interactions (lambda-lambda') links catalytically essential regions of D5 and D1, creating the framework for an active-site and anchoring it at the 5' splice site. Highly conserved elements similar to components of the lambda-lambda' interaction are found in the eukaryotic spliceosome.

The DExH protein NPH-II is a processive and directional motor for unwinding RNA.

All aspects of cellular RNA metabolism and processing involve DExH/D proteins, which are a family of enzymes that unwind or manipulate RNA in an ATP-dependent fashion. DExH/D proteins are also essential for the replication of many viruses, and therefore provide targets for the development of therapeutics. All DExH/D proteins characterized to date hydrolyse nucleoside triphosphates and, in most cases, this activity is stimulated by the addition of RNA or DNA. Several members of the family unwind RNA duplexes in an NTP-dependent fashion in vitro; therefore it has been proposed that DExH/D proteins couple NTP hydrolysis to RNA conformational change in complex macromolecular assemblies. Despite the central role of DExH/D proteins, their mechanism of RNA helicase activity remains unknown. Here we show that the DExH protein NPH-II unwinds RNA duplexes in a processive, unidirectional fashion with a step size of roughly one-half helix turn. We show that there is a quantitative connection between ATP utilization and helicase processivity, thereby providing direct evidence that DExH/D proteins can function as molecular motors on RNA.

Calculating the electrostatic properties of RNA provides new insights into molecular interactions and function.

Solutions to the nonlinear Poisson-Boltzmann equation were used to obtain the electrostatic potentials of RNA molecules that have known three-dimensional structures. The results are described in terms of isopotential contours and surface electrostatic potential maps. Both representations have unexpected features: 'cavities' within isopotential contours and areas of enhanced negative potential on molecular surfaces. Intriguingly, the sites of unusual electrostatic features correspond to functionally important regions, suggesting that electrostatic properties play a key role in RNA recognition and stabilization. These calculations reveal that the electrostatic potentials generated by RNA molecules have a variety of functionally important characteristics that cannot be discerned by simple visual inspection of the molecular structure.

Antagonistic substrate binding by a group II intron ribozyme.

In this study, the thermodynamic properties of substrate-ribozyme recognition were explored using a system derived from group II intron ai5gamma. Substrate recognition by group II intron ribozymes is of interest because any nucleic ac?id sequence can be targeted, the recognition sequence can be quite long (>/=13 bp), and reaction can proceed with a very high degree of sequence specificity. Group II introns target their substrates throug?h the formation of base-pairing interactions with two regions of the intron (EBS1 and EBS2), which are usually located far apart in the secondary structure. These structures pair with adjacent, corresponding sites (IBS1 and IBS2) on the substrate. In order to understand the relative energetic contribution of each base-pairing interaction (EBS1-IBS1 or EBS2-IBS2) to substrate binding energy, the free energy of each helix was measured. The individual helices were found to have base-pairing free energies similar to those calculated for regular RNA duplexes of the same sequence, suggesting that each recognition helix derives its binding energy from base-pairing interactions alone and that each helix can form independently. Most interestingly, it was found that the sum of the measured individual free energies (approximately 20 kcal/mol) was much higher than the known free energy for substrate binding (approximately 12 kcal/mol). This indicates that certain group II intron ribozymes can bind their substrates in an antagonistic fashion, paying a net energetic penalty upon binding the full-length substrate. This loss of binding energy is not due to weakening of individual helices, but appears to be linked to ribozyme conformational changes induced by substrate binding. This coupling between substrate binding and ribozyme conformational rearrangement may provide a mechanism for lowering overall substrate binding energy while retaining the full information content of 13 bp, thus resulting in a mechanism for ensuring sequence specificity.

Site-specific labeling of RNA with fluorophores and other structural probes.

Site-specific probes provide a powerful tool for structure and function studies of nucleic acids, especially in elucidating tertiary structures of large ribozymes and other folded RNA molecules. Among many types of extrinsic labels, fluorophores are most attractive because they can provide structural information at millisecond time resolution, thus allowing real-time observation of structural transition during biological function. Methods for introducing fluorophores in RNA molecules are summarized here. These methods are robust and readily applicable to the labeling of other types of probes. However, as each case of RNA modification is unique, fine tuning of the general methodology is beneficial.

Defining functional groups, core structural features and inter-domain tertiary contacts essential for group II intron self-splicing: a NAIM analysis.

Group II introns are self-splicing RNA molecules that are of considerable interest as ribozymes, mobile genetic elements and examples of folded RNA. Although these introns are among the most common ribozymes, little is known about the chemical and structural determinants for their reactivity. By using nucleotide analog interference mapping (NAIM), it has been possible to identify the nucleotide functional groups (Rp phosphoryls, 2'-hydroxyls, guanosine exocyclic amines, adenosine N7 and N6) that are most important for composing the catalytic core of the intron. The majority of interference effects occur in clusters located within the two catalytically essential Domains 1 and 5 (D1 and D5). Collectively, the NAIM results indicate that key tetraloop-receptor interactions display a specific chemical signature, that the epsilon-epsilon' interaction includes an elaborate array of additional features and that one of the most important core structures is an uncharacterized three-way junction in D1. By combining NAIM with site-directed mutagenesis, a new tertiary interaction, kappa-kappa', was identified between this region and the most catalytically important section of D5, adjacent to the AGC triad in stem 1. Together with the known zeta-zeta' interaction, kappa-kappa' anchors D5 firmly into the D1 scaffold, thereby presenting chemically essential D5 functionalities for participation in catalysis.

A map of the binding site for catalytic domain 5 in the core of a group II intron ribozyme.

Group II introns are ribozymes with a complex tertiary architecture that is of great interest as a model for RNA folding. Domain 5 (D5) is a highly conserved region of the intron that is considered one of the most critical structures in the catalytic core. Despite its central importance, the means by which D5 interacts with other core elements is unclear. To obtain a map of potential interaction sites, dimethyl sulfate was used to footprint regions of the intron that are involved in D5 binding. These studies were complemented by measurements of D5 binding to a series of truncated intron derivatives. In this way, the minimal region of the intron required for strong D5 association was defined and the sites most likely to represent thermodynamically significant positions of tertiary contact were identified. These studies show that ground-state D5 binding is mediated by tertiary contacts to specific regions of D1, including a tetraloop receptor and an adjacent three-way junction. In contrast, D2 and D3 are not found to stabilize D5 association. These data highlight the significance of D1-D5 interactions and will facilitate the identification of specific tertiary contacts between them.

More than one way to splice an RNA: branching without a bulge and splicing without branching in group II introns.

Domain 6 (D6) of group II introns contains a bulged adenosine that serves as the branch-site during self-splicing. In addition to this adenosine, other structural features in D6 are likely to contribute to the efficiency of branching. To understand their role in promoting self-splicing, the branch-site and surrounding nucleotides were mutagenized. Detailed kinetic analysis on the self-splicing efficiency of the mutants revealed several interesting features. First, elimination of the branch-site does not preclude efficient splicing, which takes place instead through a hydrolytic first step. Second, pairing of the branch-site does not eliminate branching, particularly if the adenosine is involved in a mispair. Third, the G-U pairs that often surround group II intron branch-points contribute to the efficiency of branching. These results suggest that there is a strong driving force for promoting self-splicing by group II introns, which employ a versatile set of different mechanisms for ensuring that splicing is successful. In addition, the behavior of these mutants indicates that a bulged adenosine per se is not the important determinant for branch-site recognition in group II introns. Rather, the data suggest that the branch-site adenosine is recognized as a flipped base, a conformation that can be promoted by a variety of different substructures in RNA and DNA.

Ribozyme catalysis from the major groove of group II intron domain 5.

The most highly conserved nucleotides in D5, an essential active site component of group II introns, consist of an AGC triad, of which the G is invariant. To understand how this G participates in catalysis, the mechanistic contribution of its functional groups was examined. We observed that the exocyclic amine of G participates in ground state interactions that stabilize D5 binding from the minor groove. In contrast, each major groove heteroatom of the critical G (specifically N7 or O6) is essential for chemistry. Thus, major groove atoms in an RNA helix can participate in catalysis, despite their presumed inaccessibility. N7 or O6 of the critical G could engage in critical tertiary interactions with the rest of the intron or they could, together with phosphate oxygens, serve as a binding site for catalytic metal ions.

The architectural organization and mechanistic function of group II intron structural elements.

Group II introns are large, self-splicing RNAs and mobile genetic elements that provide good model systems for studies of RNA folding. The structures and mechanistic functions of individual domains are being elucidated, and long-range tertiary interactions between the domains are being identified, thus helping to define the three-dimensional architecture of the intron.