The complexity of eukaryotic organisms is intricately tied to transcriptome-level processes, notably alternative splicing and the precise modulation of gene expression through a sophisticated interplay involving RNA-binding protein (RBP) networks and their RNA targets. Recent advances in our understanding of the molecular pathways responsible for this control have paved the way for the development of tools capable of steering and managing RNA regulation and gene expression. The fusion between a rapidly developing understanding of endogenous RNA regulation and the burgeoning capabilities of CRISPR-Cas and other programmable RBP platforms has given rise to an exciting frontier in engineered RNA regulators. This review offers an overview of the existing toolkit for constructing synthetic RNA regulators using programmable RBPs and effector domains, capable of altering RNA sequence composition or fate, and explores their diverse applications in both basic research and therapeutic contexts.
The biological role of the fat mass and obesity-associated protein (FTO) in the initiation and progress of acute myeloid leukemia (AML) has been elucidated, and several representative FTO inhibitors can markedly suppress the proliferation of AML cells. We previously developed FTO inhibitors including FB23. In this study, we adopted bioisosteric replacement of the intramolecular hydrogen bond in FB23 with a sulfur-oxygen interaction to generate a series of 2-(arylthio)benzoic acid FTO inhibitors and established their structure-activity relationships. Compound 8c was the most potent 2-(arylthio)benzoic acid FTO inhibitor with an IC50 value of 0.3±0.1 μM, which was comparable with that of FB23 in vitro. To enhance the antiproliferative effects in AML cell lines, we applied a prodrug strategy and prepared some esters. 7l, the methyl ester of 8l, exerted a superior inhibitory effect on a panel of AML cancer cell lines. Additionally, 7l treatment notably increased global m6A abundance in AML cells. Collectively, our data suggest that 2-(arylthio)benzoic acid may be a new lead compound for inhibition of FTO, and the prodrug analog exhibit potential in the treatment of AML.
The widespread involvement of 5-formylcytosine f5C RNA in gene function regulation and its impact on crucial life processes like cell differentiation, embryonic development, and disease development underscores the significance of detecting this specific base modification. This detection holds great importance for basic epigenetics research and the early diagnosis and pathogenesis research of various diseases. This review aims to summarize recent research progress in f5C detection methods using selective chemical labeling, with the hope of aiding future research endeavors.
Protein post-translational modifications, including phosphorylation, acetylation, ubiquitylation, and more, confer key levels of regulation and can dramatically alter the structure and function of proteins, acting as molecular switches or rheostats for tuning activity.1 Many post-translational modifications are specialized to specific subcellular compartments and clientele, such as the sophisticated pathways for protein N-glycosylation in the endoplasmic reticulum (ER) and Golgi mediated by a suite of glycosyltransferase and glycosidase enzymes. Protein glycosylation involves covalent modification of amino acid sidechains with sugars to yield linear or branched structures (glycans; Figure 1). The consequences of glycosylation shape protein function, cell–cell recognition, cell–matrix interactions, and more.2
Protein N-linked glycosylation is a co- and post-translational modification that involves the installation of glycans on asparagine side chains in specific amino acid sequons in proteins traversing the secretory pathway. A: A 14-residue precursor oligosaccharide is first synthesized in a step-wise fashion while attached to a dolichol pyrophosphate molecule on the ER membrane. Monosaccharide substrates in the form of nucleotide sugars are each added to the growing sugar chain by their respective transferase enzymes. The dolichol-linked precursor then requires the action of flippase enzymes, prior to being added to a nascent ER client protein by the oligosaccharyltransferase (OST) complex as the polypeptide translocates from the ribosome to the ER. Note that N-glycans can also be installed post-translationally by OST. After installation of the precursor, folding and initial trimming occurs in the ER and the nascent glycoprotein is trafficked to the Golgi for further processing. B: Glycan-modifying enzymes in the ER and Golgi process the N-glycan via sequential removal and addition of monosaccharides by specific enzymes, ultimately yielding a vast array of potential glycan structures, including hybrid glycans, complex glycans, core fucosylated glycans, and sialylated glycans. The specific identity of the glycan has important and varied consequences for cellular communication and the function of
Sequencing for RNA modifications with the nanopore direct RNA sequencing platform provides ionic current levels, helicase dwell times, and base call data that differentiate the modifications from the canonical form. Herein, model RNAs were synthesized with site-specific uridine (U) base modifications that enable the study of increasing an alkyl group size, halogen identity, or a change in base acidity to impact the nanopore data. The analysis concluded that increases in alkyl size trend with greater current blockage but a similar change in base-call error was not found. The addition of a halogen series to C5 of U revealed that the current levels recorded a trend with the water-octanol partition coefficient of the base, as well as the base call error. Studies with U modifications that are deprotonated (i. e., anionic) under the sequencing conditions gave broad current levels that influenced the base call error. Some modifications led to helicase dwell time changes. These insights provide design parameters for modification-specific chemical reagents that can shift nanopore signatures to minimize false positive reads, a known issue with this sequencing approach.
Epilepsy is one of the most common neurological diseases in the world with a broad phenotypic spectrum.1 Recent advances in genome sequencing identified an increasing number of genes that are associated with epilepsy.2 According to protein functions, epilepsy-associated genes can be grouped to ion channels, enzymes and enzyme modulators, transports and receptors, and others.3 Genetic epilepsy is often linked to developmental delay, movement disorder, and other comorbidities.4 Due to the important role of neurotransmitter-gated ion channels in controlling the excitation-inhibition balance in the central nervous system, genes encoding these ion channels, including excitatory N-methyl-D-aspartate (NMDA) receptors and inhibitory γ-aminobutyric acid type A (GABAA) receptors, are recognized as prominent epilepsy-causing genes.5 Here, we focus on GABAA receptors, the primary inhibitory neurotransmitter-gated ion channels in the human brain.6 They mediate the fast inhibitory GABA-induced chloride currents and hyperpolarize the postsynaptic membranes to reduce neuronal firing.
Proteostasis maintenance of GABAA receptors is essential for their function in the central nervous system.7 GABAA receptors are assembled as pentamers from a specific combination of 19 subunits, including α1-α6 (GABRA1-A6), β1-β3 (GABRB1-B3), γ1-γ3 (GABRG1-G3), δ (GABRD), ϵ (GABRE), θ (GABRQ), π (GABRP), and ρ1-ρ3 (GABRR1-R3). The distribution of GABAA receptors is throughout the brain regions, and the most abundant subtype is composed of two α1 subunits, two β2 subunits, and one γ2 subunit.8 To function, GABAA receptor subunits need to fold in the endoplasmic reticulum (ER) with the assistance of molecular chaperones and subsequently assemble with other subunits to form heteropentamers. The properly assembled receptors exit the ER and traffic to the plasma membrane to act as chloride channels. Unassembled and misfolded subunits are retained in the ER, which could be routed to the degradation pathway by the ER-associated degradation.9 Recent quantitative proteomics analysis identified the proteostasis network that regulates the folding, assembly, trafficking, and degradation of GABAA receptors.10
Recent cryo-electron microscopy (cryo-EM) studies solved the high-resolution structures of pentameric GABAA receptors, including α1β2γ2 receptors11 and α1β3γ2 receptors.12 The pentameric receptors are arranged as β-α1-β-α1-γ2 counterclockwise when viewed from the synaptic cleft (Figure 1A). Each pentamer has two binding sites for the neurotransmitter, GABA, at the interfaces between
No conflicts to declare.
Genetically encoded peptide libraries have become powerful resources for de novo drug discovery.1, 2 Embedded within state-of-the-art display technologies, they facilitate the identification of high-affinity peptide ligands from a vast sequence space.1-3 Chemical modification of the library, including the incorporation of non-canonical amino acids, can greatly enhance the diversity and drug-likeness of the screened peptides.4-6 Hence, most peptide displays favour the use of modified constrained peptides over their linear counterparts,7 as they possess beneficial pharmaceutical properties.8
Macrocyclic peptides are a diverse class of molecules characterised by their structural constraint.9, 10 Featuring a variety of topologies,11-14 they possess architectures particularly suited to mimic and disrupt protein-protein interactions.15 Likewise, the inherent rigidity of constrained peptides enhances their target affinity and metabolic stability.16, 17 The remarkable targeting and exceptional binding affinity of constrained peptides have invited comparisons with antibodies, which are renowned for these properties.18, 19 With a comparatively low molecular weight, however, macrocyclic peptides maintain synthetic accessibility similar to small molecules.11, 17 Consequently, constrained peptides strike a balance that situates them in the ‘Goldilocks zone’ between small molecules and protein therapeutics (Figure 1),6, 20 rendering them highly relevant for future therapeutic development.8, 21
Constrained peptides occupy the ‘Goldilocks zone’ between small molecules and biologics8-10, 20 (molecules not to scale; structures obtained from PDB: 7Y4G, 4DGC, 5DK3).22-24
The RaPID (Random Nonstandard Peptides Integrated Discovery) platform25 (Figure 2) facilitates the identification of selective and high-affinity binding peptide macrocycles.26
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