Structural insights into RNA recognition by RIG-I.

Intracellular RIG-I-like receptors (RLRs, including RIG-I, MDA-5, and LGP2) recognize viral RNAs as pathogen-associated molecular patterns (PAMPs) and initiate an antiviral immune response. To understand the molecular basis of this process, we determined the crystal structure of RIG-I in complex with double-stranded RNA (dsRNA). The dsRNA is sheathed within a network of protein domains that include a conserved "helicase" domain (regions HEL1 and HEL2), a specialized insertion domain (HEL2i), and a C-terminal regulatory domain (CTD). A V-shaped pincer connects HEL2 and the CTD by gripping an α-helical shaft that extends from HEL1. In this way, the pincer coordinates functions of all the domains and couples RNA binding with ATP hydrolysis. RIG-I falls within the Dicer-RIG-I clade of the superfamily 2 helicases, and this structure reveals complex interplay between motor domains, accessory mechanical domains, and RNA that has implications for understanding the nanomechanical function of this protein family and other ATPases more broadly.

Dicer-related helicase 3 forms an obligate dimer for recognizing 22G-RNA.

Dicer is a specialized nuclease that produces RNA molecules of specific lengths for use in gene silencing pathways. Dicer relies on the correct measurement of RNA target duplexes to generate products of specific lengths. It is thought that Dicer uses its multidomain architecture to calibrate RNA product length. However, this measurement model is derived from structural information from a protozoan Dicer, and does not account for the helicase domain present in higher organisms. The Caenorhabditis elegans Dicer-related helicase 3 (DRH-3) is an ortholog of the Dicer and RIG-I family of double-strand RNA activated ATPases essential for secondary siRNA production. We find that DRH-3 specifies 22 bp RNAs by dimerization of the helicase domain, a process mediated by ATPase activity and the N-terminal domain. This mechanism for RNA length discrimination by a Dicer family protein suggests an alternative model for RNA length measurement by Dicer, with implications for recognition of siRNA and miRNA targets.

The thermodynamic basis for viral RNA detection by the RIG-I innate immune sensor.

RIG-I is a cytoplasmic surveillance protein that contributes to the earliest stages of the vertebrate innate immune response. The protein specifically recognizes 5'-triphosphorylated RNA structures that are released into the cell by viruses, such as influenza and hepatitis C. To understand the energetic basis for viral RNA recognition by RIG-I, we studied the binding of RIG-I domain variants to a family of dsRNA ligands. Thermodynamic analysis revealed that the isolated RIG-I domains each make important contributions to affinity and that they interact using different strategies. Covalent linkage between the domains enhances RNA ligand specificity while reducing overall binding affinity, thereby providing a mechanism for discriminating virus from host RNA.

A nuclear magnetic resonance-based structural rationale for contrasting stoichiometry and ligand binding site(s) in fatty acid-binding proteins.

Liver fatty acid-binding protein (LFABP) is a 14 kDa cytosolic polypeptide, differing from other family members in the number of ligand binding sites, the diversity of bound ligands, and the transfer of fatty acid(s) to membranes primarily via aqueous diffusion rather than direct collisional interactions. Distinct two-dimensional (1)H-(15)N nuclear magnetic resonance (NMR) signals indicative of slowly exchanging LFABP assemblies formed during stepwise ligand titration were exploited, without determining the protein-ligand complex structures, to yield the stoichiometries for the bound ligands, their locations within the protein binding cavity, the sequence of ligand occupation, and the corresponding protein structural accommodations. Chemical shifts were monitored for wild-type LFABP and an R122L/S124A mutant in which electrostatic interactions viewed as being essential to fatty acid binding were removed. For wild-type LFABP, the results compared favorably with the data for previous tertiary structures of oleate-bound wild-type LFABP in crystals and in solution: there are two oleates, one U-shaped ligand that positions the long hydrophobic chain deep within the cavity and another extended structure with the hydrophobic chain facing the cavity and the carboxylate group lying close to the protein surface. The NMR titration validated a prior hypothesis that the first oleate to enter the cavity occupies the internal protein site. In contrast, (1)H and (15)N chemical shift changes supported only one liganded oleate for R122L/S124A LFABP, at an intermediate location within the protein cavity. A rationale based on protein sequence and electrostatics was developed to explain the stoichiometry and binding site trends for LFABPs and to put these findings into context within the larger protein family.

Conocimiento del parto humanizado en Colombia en residentes y ginecólogos / Knowledge of humanized childbirth in Colombian residents and gynecologists

Resumen OBJETIVO: Identificar, mediante una encuesta, el conocimiento de especialistas y residentes de Ginecología y Obstetricia de los beneficios de la atención respetuosa y digna a la paciente en el momento del parto. MATERIALES Y MÉTODOS: Estudio observacional, descriptivo, transversal y prospectivo efectuado en una muestra de ginecoobstetras y residentes de diferentes universidades e instituciones de Colombia a quienes se aplicó, entre los meses de diciembre de 2021 a agosto de 2022, un instrumento estructurado de manera individual, presencial o virtual. RESULTADOS: Se obtuvieron 343 respuestas. El 51% de la muestra fue de especialistas con más de dos años de experiencia. El 37.2% de los residentes cursaba el tercer y 20.2% el segundo año. Más de la mitad de los encuestados no recibió ni recibe educación del tema. Se identificó un vacío en el conocimiento y su implementación. La totalidad de la muestra no reconoció algún beneficio materno o neonatal. El 86.3% consideró que no existe contraindicación para la implementación del parto digno y respetuoso y el 94.8% manifestó una correlación entre la desinformación y su baja implementación. El 69.4% de la muestra conocía y ponía en práctica el plan de parto en su práctica diaria. CONCLUSIONES: De la muestra analizada se concluye que hay desinformación acerca de los beneficios maternos y neonatales del parto digno y respetuoso por parte de residentes y especialistas de Ginecología y Obstetricia. Por lo tanto, es necesario aumentar los contenidos del tema a los programas de educación. Además, estandarizar los protocolos ajustados que faciliten su ejecución e implementación.

Abstract OBJECTIVE: To identify, by means of a survey, the knowledge of specialists and residents in Gynaecology and Obstetrics of the benefits of respectful and dignified care for the patient at the time of delivery. MATERIALSAND METHODS: Observational, descriptive, cross-sectional, and prospective study carried out on a sample of obstetrician-gynecologists and residents from different universities and institutions in Colombia to whom a structured instrument was applied individually, in person or online, between December 2021 and August 2022. RESULTS: 343 responses were obtained. Fifty-one percent of the sample were specialists with more than two years of experience. 37.2% of the residents were in their third year and 20.2% were in their second year. More than half of the respondents did not and do not receive any education on the subject. A gap in knowledge and implementation were identified. The entire sample did not recognize any maternal or neonatal benefit. 86.3% considered that there is no contraindication to the implementation of respectful and dignified childbirth and 94.8% expressed a correlation between misinformation and low implementation of respectful and dignified childbirth. 69.4% of the sample were aware of and implemented the birth plan in their daily practice. CONCLUSIONS: From the sample analyzed, it is concluded that there is misinformation about the maternal and neonatal benefits of dignified and respectful childbirth on the part of Gynaecology and Obstetrics residents and specialists. Therefore, it is necessary to increase the content of the subject in education programs. In addition, it is necessary to standardize adjusted protocols that facilitate their execution and implementation.

An evolving arsenal: viral RNA detection by RIG-I-like receptors.

RIG-I-like receptors (RLRs) utilize a specialized, multi-domain architecture to detect and respond to invasion by a diverse set of viruses. Structural similarities among these receptors provide a general mechanism for double strand RNA recognition and signal transduction. However, each RLR has developed unique strategies for sensing the specific molecular determinants on subgroups of viral RNAs. As a means to circumvent the antiviral response, viruses escape RLR detection by degrading, or sequestering or modifying their RNA. Patterns of variation in RLR sequence reveal a continuous evolution of the protein domains that contribute to RNA recognition and signaling.

Visualizing the determinants of viral RNA recognition by innate immune sensor RIG-I.

Retinoic acid inducible gene-I (RIG-I) is a key intracellular immune receptor for pathogenic RNAs, particularly from RNA viruses. Here, we report the crystal structure of human RIG-I bound to a 5' triphosphorylated RNA hairpin and ADP nucleotide at 2.8 Å resolution. The RNA ligand contains all structural features that are essential for optimal recognition by RIG-I, as it mimics the panhandle-like signatures within the genome of negative-stranded RNA viruses. RIG-I adopts an intermediate, semiclosed conformation in this product state of ATP hydrolysis. The structure of this complex allows us to visualize the first steps in RIG-I recognition and activation upon viral infection.