The cannabinoid receptor type 1 (CB1R), a G protein-coupled receptor (GPCR), plays an essential role in the control of many physiological processes such as hunger, memory loss, gastrointestinal activity, catalepsy, fear, depression, and chronic pain. Therefore, it is an attractive target for drug discovery to manage pain, neurodegenerative disorders, obesity, and substance abuse. However, the psychoactive adverse effects, generated by CB1R activation in the brain, limit the use of the orthosteric CB1R ligands as drugs. The discovery of CB1R allosteric modulators during the last decade provided new tools to target the CB1R. Moreover, application of the site-directed mutagenesis in combination with advanced physical methods, especially X-ray crystallography and computational modeling, has opened new horizons for understanding the complexity of the structure, function, and activity of cannabinoid receptors. In this paper, we present the latest advances in research on the CB1R, its allosteric modulation and allosteric ligands, and their translational potential. We focused on structural essentials of the cannabinoid 1 receptor- ligand (drug) interactions, as well as modes of CB1R signaling regulation.

RNAs adopt specific structures in order to perform their biological activities. The structure of RNA is an important layer of gene expression regulation, and can impact a plethora of cellular processes, starting with transcription, RNA processing, and translation, and ending with RNA turnover. The development of high-throughput technologies has enabled a deeper insight into the sophisticated interplay between the structure of the cellular transcriptome and the living cells environment. In this review, we present the current view on the RNA structure in vivo resulting from the most recent transcriptome-wide studies in different organisms, including mammalians, yeast, plants, and bacteria. We focus on the relationship between the mRNA structure and translation, mRNA stability and degradation, protein binding, and RNA posttranscriptional modifications.

Long terminal repeat (LTR)-retrotransposons constitute a significant part of eukaryotic genomes and influence their function and evolution. Like other RNA viruses, LTR-retrotransposons efficiently utilize their RNA genome to interact with host cell machinery during replication. Here, we provide the first genome-wide RNA secondary structure model for a LTR-retrotransposon in living cells. Using SHAPE probing, we explore the secondary structure of the yeast Ty1 retrotransposon RNA genome in its native in vivo state and under defined in vitro conditions. Comparative analyses reveal the strong impact of the cellular environment on folding of Ty1 RNA. In vivo, Ty1 genome RNA is significantly less structured and more dynamic but retains specific well-structured regions harboring functional cis-acting sequences. Ribosomes participate in the unfolding and remodeling of Ty1 RNA, and inhibition of translation initiation stabilizes Ty1 RNA structure. Together, our findings support the dual role of Ty1 genomic RNA as a template for protein synthesis and reverse transcription. This study also contributes to understanding how a complex multifunctional RNA genome folds in vivo, and strengthens the need for studying RNA structure in its natural cellular context.

A universal feature of retroelement propagation is the formation of distinct nucleoprotein complexes mediated by the Gag capsid protein. The Ty1 retrotransposon Gag protein from Saccharomyces cerevisiae lacks sequence homology with retroviral Gag, but is functionally related. In addition to capsid assembly functions, Ty1 Gag promotes Ty1 RNA dimerization and cyclization and initiation of reverse transcription. Direct interactions between Gag and retrotransposon genomic RNA (gRNA) are needed for Ty1 replication, and mutations in the RNA-binding domain disrupt nucleation of retrosomes and assembly of functional virus-like particles (VLPs). Unlike retroviral Gag, the specificity of Ty1 Gag-RNA interactions remain poorly understood. Here we use microscale thermophoresis (MST) and electrophoretic mobility shift assays (EMSA) to analyze interactions of immature and mature Ty1 Gag with RNAs. The salt-dependent experiments showed that Ty1 Gag binds with high and similar affinity to different RNAs. However, we observed a preferential interaction between Ty1 Gag and Ty1 RNA containing a packaging signal (Psi) in RNA competition analyses. We also uncover a relationship between Ty1 RNA structure and Gag binding involving the pseudoknot present on Ty1 gRNA. In all likelihood, the differences in Gag binding affinity detected in vitro only partially explain selective Ty1 RNA packaging into VLPs in vivo.

Activity-regulated cytoskeleton-associated protein (ARC) is an RNA-binding protein that also serves as a central hub for neuronal protein–protein interactions. It is essential for intercellular signaling and contributes to synaptic plasticity. ARC includes Gag-like sequences of Ty3/Gypsy retrotransposons and retains the ability to self-assemble into capsid-like structures containing Arc mRNA. Here, we employ an integrative approach to provide the first detailed in vitro analysis of ARC–RNA interactions. Using quantitative binding assays, RNA structure mapping, and ribonucleoprotein (RNP) footprinting, complemented by extensive computational analyses, we identified Arc mRNA regions specifically and non-specifically bound by ARC, as well as ARC amino acid residues involved in RNA interactions. We show that ARC recognizes RNA sequence and structure. A specific GC-rich motif is common to all bound RNA sequences, and the binding preferentially occurs near highly stable, solvent-exposed helices in the 5′ region of Arc mRNA. Surprisingly, the conserved coding sequence seems more relevant to binding specificity than the 5′-untranslated region. Our predictions suggest that ARC binding to RNA through positively charged regions of matrix and capsid domains exposes an oligomerization motif, enhancing binding cooperativity. We also provide evidence that ARC acts as a nucleic acid chaperone and can locally destabilize Arc mRNA structure.

Long terminal repeat (LTR)-retrotransposons are significant contributors to the evolution and diversity of eukaryotic genomes. Their RNA genomes (gRNA) serve as a template for protein synthesis and reverse transcription to a DNA copy, which can integrate into the host genome. Here, we used the SHAPE-MaP strategy to explore Ty3 retrotransposon gRNA structure in yeast and under cell-free conditions. Our study reveals the structural dynamics of Ty3 gRNA and the well-folded core, formed independently of the cellular environment. Based on the detailed map of Ty3 gRNA structure, we characterized the structural context of cis-acting sequences involved in reverse transcription and frameshifting. We also identified a novel functional sequence as a potential initiator for Ty3 gRNA dimerization. Our data indicate that the dimer is maintained by direct interaction between short palindromic sequences at the 5′ ends of the two Ty3 gRNAs, resembling the model characteristic for other retroelements like HIV-1 and Ty1. This work points out a range of cell-dependent and -independent Ty3 gRNA structural changes that provide a solid background for studies on RNA structure-function relationships important for retroelement biology.