Targeted pseudouridylation: An approach for suppressing nonsense mutations in disease genes.

Nonsense mutations create premature termination codons (PTCs), activating the nonsense-mediated mRNA decay (NMD) pathway to degrade most PTC-containing mRNAs. The undegraded mRNA is translated, but translation terminates at the PTC, leading to no production of the full-length protein. This work presents targeted PTC pseudouridylation, an approach for nonsense suppression in human cells. Specifically, an artificial box H/ACA guide RNA designed to target the mRNA PTC can suppress both NMD and premature translation termination in various sequence contexts. Targeted pseudouridylation exhibits a level of suppression comparable with that of aminoglycoside antibiotic treatments. When targeted pseudouridylation is combined with antibiotic treatment, a much higher level of suppression is observed. Transfection of a disease model cell line (carrying a chromosomal PTC) with a designer guide RNA gene targeting the PTC also leads to nonsense suppression. Thus, targeted pseudouridylation is an RNA-directed gene-specific approach that suppresses NMD and concurrently promotes PTC readthrough.

Pseudouridylation-mediated gene expression modulation.

RNA-guided pseudouridylation, a widespread post-transcriptional RNA modification, has recently gained recognition for its role in cellular processes such as pre-mRNA splicing and the modulation of premature termination codon (PTC) readthrough. This review provides insights into its mechanisms, functions, and potential therapeutic applications. It examines the mechanisms governing RNA-guided pseudouridylation, emphasizing the roles of guide RNAs and pseudouridine synthases in catalyzing uridine-to-pseudouridine conversion. A key focus is the impact of RNA-guided pseudouridylation of U2 small nuclear RNA on pre-mRNA splicing, encompassing its influence on branch site recognition and spliceosome assembly. Additionally, the review discusses the emerging role of RNA-guided pseudouridylation in regulating PTC readthrough, impacting translation termination and genetic disorders. Finally, it explores the therapeutic potential of pseudouridine modifications, offering insights into potential treatments for genetic diseases and cancer and the development of mRNA vaccine.

Precise small-molecule cleavage of an r(CUG) repeat expansion in a myotonic dystrophy mouse model.

Myotonic dystrophy type 1 (DM1) is an incurable neuromuscular disorder caused by an expanded CTG repeat that is transcribed into r(CUG)exp The RNA repeat expansion sequesters regulatory proteins such as Muscleblind-like protein 1 (MBNL1), which causes pre-mRNA splicing defects. The disease-causing r(CUG)exp has been targeted by antisense oligonucleotides, CRISPR-based approaches, and RNA-targeting small molecules. Herein, we describe a designer small molecule, Cugamycin, that recognizes the structure of r(CUG)exp and cleaves it in both DM1 patient-derived myotubes and a DM1 mouse model, leaving short repeats of r(CUG) untouched. In contrast, oligonucleotides that recognize r(CUG) sequence rather than structure cleave both long and short r(CUG)-containing transcripts. Transcriptomic, histological, and phenotypic studies demonstrate that Cugamycin broadly and specifically relieves DM1-associated defects in vivo without detectable off-targets. Thus, small molecules that bind and cleave RNA have utility as lead chemical probes and medicines and can selectively target disease-causing RNA structures to broadly improve defects in preclinical animal models.

Design, Optimization, and Study of Small Molecules That Target Tau Pre-mRNA and Affect Splicing.

Approximately 95% of human genes are alternatively spliced, and aberrant splicing events can cause disease. One pre-mRNA that is alternatively spliced and linked to neurodegenerative diseases is tau (microtubule-associated protein tau), which can cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and can contribute to Alzheimer's disease. Here, we describe the design of structure-specific lead small molecules that directly target tau pre-mRNA from sequence. This was followed by hit expansion and analogue synthesis to further improve upon these initial lead molecules. The emergent compounds were assessed for functional activity in a battery of assays, including binding assays and an assay that mimics molecular recognition of tau pre-mRNA by a U1 small nuclear ribonucleoprotein (snRNP) splicing factor. Compounds that emerged from these studies had enhanced potency and selectivity for the target RNA relative to the initial hits, while also having significantly improved drug-like properties. The compounds are shown to directly target tau pre-mRNA in cells, via chemical cross-linking and isolation by pull-down target profiling, and to rescue disease-relevant splicing of tau pre-mRNA in a variety of cellular systems, including primary neurons. More broadly, this study shows that lead, structure-specific compounds can be designed from sequence and then further optimized for their physicochemical properties while at the same time enhancing their activity.

Using Genome Sequence to Enable the Design of Medicines and Chemical Probes.

Rapid progress in genome sequencing technology has put us firmly into a postgenomic era. A key challenge in biomedical research is harnessing genome sequence to fulfill the promise of personalized medicine. This Review describes how genome sequencing has enabled the identification of disease-causing biomolecules and how these data have been converted into chemical probes of function, preclinical lead modalities, and ultimately U.S. Food and Drug Administration (FDA)-approved drugs. In particular, we focus on the use of oligonucleotide-based modalities to target disease-causing RNAs; small molecules that target DNA, RNA, or protein; the rational repurposing of known therapeutic modalities; and the advantages of pharmacogenetics. Lastly, we discuss the remaining challenges and opportunities in the direct utilization of genome sequence to enable design of medicines.

Improving RNA nearest neighbor parameters for helices by going beyond the two-state model.

RNA folding free energy change nearest neighbor parameters are widely used to predict folding stabilities of secondary structures. They were determined by linear regression to datasets of optical melting experiments on small model systems. Traditionally, the optical melting experiments are analyzed assuming a two-state model, i.e. a structure is either complete or denatured. Experimental evidence, however, shows that structures exist in an ensemble of conformations. Partition functions calculated with existing nearest neighbor parameters predict that secondary structures can be partially denatured, which also directly conflicts with the two-state model. Here, a new approach for determining RNA nearest neighbor parameters is presented. Available optical melting data for 34 Watson-Crick helices were fit directly to a partition function model that allows an ensemble of conformations. Fitting parameters were the enthalpy and entropy changes for helix initiation, terminal AU pairs, stacks of Watson-Crick pairs and disordered internal loops. The resulting set of nearest neighbor parameters shows a 38.5% improvement in the sum of residuals in fitting the experimental melting curves compared to the current literature set.

Structure and Dynamics of RNA Repeat Expansions That Cause Huntington's Disease and Myotonic Dystrophy Type 1.

RNA repeat expansions cause a host of incurable, genetically defined diseases. The most common class of RNA repeats consists of trinucleotide repeats. These long, repeating transcripts fold into hairpins containing 1 × 1 internal loops that can mediate disease via a variety of mechanism(s) in which RNA is the central player. Two of these disorders are Huntington's disease and myotonic dystrophy type 1, which are caused by r(CAG) and r(CUG) repeats, respectively. We report the structures of two RNA constructs containing three copies of a r(CAG) [r(3×CAG)] or r(CUG) [r(3×CUG)] motif that were modeled with nuclear magnetic resonance spectroscopy and simulated annealing with restrained molecular dynamics. The 1 × 1 internal loops of r(3×CAG) are stabilized by one-hydrogen bond (cis Watson-Crick/Watson-Crick) AA pairs, while those of r(3×CUG) prefer one- or two-hydrogen bond (cis Watson-Crick/Watson-Crick) UU pairs. Assigned chemical shifts for the residues depended on the identity of neighbors or next nearest neighbors. Additional insights into the dynamics of these RNA constructs were gained by molecular dynamics simulations and a discrete path sampling method. Results indicate that the global structures of the RNA are A-form and that the loop regions are dynamic. The results will be useful for understanding the dynamic trajectory of these RNA repeats but also may aid in the development of therapeutics.

Structural features of a 3' splice site in influenza a.

Influenza A is an RNA virus with a genome of eight negative sense segments. Segment 7 mRNA contains a 3' splice site for alternative splicing to encode the essential M2 protein. On the basis of sequence alignment and chemical mapping experiments, the secondary structure surrounding the 3' splice site has an internal loop, adenine bulge, and hairpin loop when it is in the hairpin conformation that exposes the 3' splice site. We report structural features of a three-dimensional model of the hairpin derived from nuclear magnetic resonance spectra and simulated annealing with restrained molecular dynamics. Additional insight was provided by modeling based on (1)H chemical shifts. The internal loop containing the 3' splice site has a dynamic guanosine and a stable imino (cis Watson-Crick/Watson-Crick) GA pair. The adenine bulge also appears to be dynamic with the A either stacked in the stem or forming a base triple with a Watson-Crick GC pair. The hairpin loop is a GAAA tetraloop closed by an AC pair.

Nuclear Magnetic Resonance-Assisted Prediction of Secondary Structure for RNA: Incorporation of Direction-Dependent Chemical Shift Constraints.

Knowledge of RNA structure is necessary to determine structure-function relationships and to facilitate design of potential therapeutics. RNA secondary structure prediction can be improved by applying constraints from nuclear magnetic resonance (NMR) experiments to a dynamic programming algorithm. Imino proton walks from NOESY spectra reveal double-stranded regions. Chemical shifts of protons in GH1, UH3, and UH5 of GU pairs, UH3, UH5, and AH2 of AU pairs, and GH1 of GC pairs were analyzed to identify constraints for the 5' to 3' directionality of base pairs in helices. The 5' to 3' directionality constraints were incorporated into an NMR-assisted prediction of secondary structure (NAPSS-CS) program. When it was tested on 18 structures, including nine pseudoknots, the sensitivity and positive predictive value were improved relative to those of three unrestrained programs. The prediction accuracy for the pseudoknots improved the most. The program also facilitates assignment of chemical shifts to individual nucleotides, a necessary step for determining three-dimensional structure.

Updated Pseudo-seq Protocol for Transcriptome-Wide Detection of Pseudouridines.

Pseudouridine (Ψ), the most prevalent modified base in cellular RNAs, has been mapped to numerous sites not only in rRNAs, tRNAs, and snRNAs but also mRNAs. Although there have been multiple techniques to identify Ψs, due to the recent development of sequencing technologies some reagents are not compatible with the current sequencer. Here, we show the updated Pseudo-seq, a technique enabling the genome-wide identification of pseudouridylation sites with single-nucleotide precision. We provide a comprehensive description of Pseudo-seq, covering protocols for RNA isolation from human cells, library preparation, and detailed data analysis procedures. The methodology presented is easily adaptable to any cell or tissue type with high-quality mRNA isolation. It can be used for discovering novel pseudouridylation sites, thus constituting a crucial initial step toward understanding the regulation and function of this modification. Key features • Identification of Ψ sites on mRNAs. • Updated Pseudo-seq provides precise positional and quantitative information of Ψ. • Uses a more efficient library preparation with the latest, currently available materials.

NMR structures of small molecules bound to a model of an RNA CUG repeat expansion.

Trinucleotide repeat expansions fold into long, stable hairpins and cause a variety of incurable RNA gain-of-function diseases such as Huntington's disease, the myotonic dystrophies, and spinocerebellar ataxias. One approach for treating these diseases is to bind small molecules to the structured RNAs. Both Huntington's disease-like 2 (HDL2) and myotonic dystrophy type 1 (DM1) are caused by a r(CUG) repeat expansion, or r(CUG) exp . The RNA folds into a hairpin structure with a periodic array of 1×1 nucleotide UU loops (5'C U G/3'G U C; where the underlined nucleotides indicate the Us in the internal loop) that sequester various RNA-binding proteins (RBP) and hence the source of its gain-of-function. Here, we report NMR-refined structures of single 5'C U G/3'G U C motifs in complex with three different small molecules, a diguandinobenzoate ( 1 ), a derivative of 1 where the guanidino groups have been exchanged for imidazole ( 2 ), and a quinoline with improved drug-like properties ( 3 ). These structures were determined using nuclear magnetic resonance (NMR) spectroscopy and simulated annealing with restrained molecular dynamics (MD). Compounds 1 , 2 , and 3 formed stacking and hydrogen bonding interactions with the 5'C U G/3'G U C motif. Compound 3 also formed van der Waals interactions with the internal loop. The global structure of each RNA-small molecule complexes retains an A-form conformation, while the internal loops are still dynamic but to a lesser extent compared to the unbound form. These results aid our understanding of ligand-RNA interactions and enable structure-based design of small molecules with improved binding affinity for and biological activity against r(CUG) exp . As the first ever reported structures of RNA r(CUG) repeats bound to ligands, these structures can enable virtual screening campaigns combined with machine learning assisted de novo design.

In Vitro Reconstitution of Pseudouridylation Catalyzed by Human Box H/ACA Ribonucleoprotein Particles.

Pseudouridine (Ψ) is the most common chemical modification in RNA. In eukaryotes and archaea, pseudouridine synthases, mainly guided by box H/ACA snoRNAs, convert uridine to Ψ. Ψ stabilizes RNA structure and alters RNA-RNA and RNA-protein interactions, conferring important roles in gene expression. Notably, several Ψ-linked human diseases have been identified over the years. In addition, Ψ has also been extensively used in developing mRNA vaccines. Furthermore, it has been shown that pseudouridylation can be site-specifically directed to modify specific nonsense codons, leading to nonsense suppression. All of these, together with a need to better understand the specific functions of Ψs, have motivated the development of in vitro pseudouridylation assays using purified and reconstituted box H/ACA RNPs. Here, we describe an in vitro system for box H/ACA RNA-guided RNA pseudouridylation using human cell extracts. We show that a half guide RNA (only one hairpin) is just as functionally competent as the full-length guide RNA (two hairpins) in guiding site-specific pseudouridylation in the human cell extracts. This discovery offers the opportunity for direct delivery of a short guide RNA to human cells to promote site-specific nonsense suppression and therefore has potential clinical applications.

Broad-spectrum metastasis suppressing compounds and therapeutic uses thereof in human tumors.

Previously, we have identified a novel human metastasis-inducing lncRNA (named SKAI1BC), that suppresses the KAI1/CD82 metastasis-suppressing gene and is upregulated in triple negative breast cancer and melanoma derived cell lines. Modeling of the SKAI1BC lncRNA secondary structure and its potential interaction with Inforna compounds, led us to identify several compounds that might bind the SKAI1BC lncRNA. We found that these compounds inhibit metastasis invasion and cell migration in culture, in all eight types of solid human cancers tested: several of which are the most lethal and/or frequent human malignancies. Moreover, in most cases, the mechanism of action of several of our compounds involves enhancement of KAI1/CD82 RNA level depending on the specific compound and the human tumor type. With the epigenetic inactivation of KAI1/CD82 in at least ten additional solid human cancers, this implies a very good chance to broaden the spectrum of human cancers affected by our compounds. This is the first time that modeling of a large lncRNA (> 700 bp) secondary structure followed by its potential interaction with Inforna like compounds database has led to the identification of potential biologically active small molecule drugs.

Testing the nearest neighbor model for canonical RNA base pairs: revision of GU parameters.

Thermodynamic parameters for GU pairs are important for predicting the secondary structures of RNA and for finding genomic sequences that code for structured RNA. Optical melting curves were measured for 29 RNA duplexes with GU pairs to improve nearest neighbor parameters for predicting stabilities of helixes. The updated model eliminates a prior penalty assumed for terminal GU pairs. Six additional duplexes with the 5'GG/3'UU motif were added to the single representation in the previous database. This revises the ΔG°(37) for the 5'GG/3'UU motif from an unfavorable 0.5 kcal/mol to a favorable -0.2 kcal/mol. Similarly, the ΔG°(37) for the 5'UG/3'GU motif changes from 0.3 to -0.6 kcal/mol. The correlation coefficients between predicted and experimental ΔG°(37), ΔH°, and ΔS° for the expanded database are 0.95, 0.89, and 0.87, respectively. The results should improve predictions of RNA secondary structure.

Biophysical analysis of influenza A virus RNA promoter at physiological temperatures.

Each segment of the influenza A virus (IAV) genome contains conserved sequences at the 5'- and 3'-terminal ends, which form the promoter region necessary for polymerase binding and initiation of RNA synthesis. Although several models of interaction have been proposed it remains unclear if these two short, partially complementary, and highly conserved sequences can form a stable RNA duplex at physiological temperatures. First, our time-resolved FRET analysis revealed that a 14-mer 3'-RNA and a 15-mer 5'-RNA associate in solution, even at 42 °C. We also found that a nonfunctional RNA promoter containing the 3'-G3U mutation, as well as a promoter containing the compensatory 3'-G3U/C8A mutations, was able to form a duplex as efficiently as wild type. Second, UV melting analysis demonstrated that the wild-type and mutant RNA duplexes have similar stabilities in solution. We also observed an increase in thermostability for a looped promoter structure. The absence of differences in the stability and binding kinetics between wild type and a nonfunctional sequence suggests that the IAV promoter can be functionally inactivated without losing the capability to form a stable RNA duplex. Finally, using uridine specific chemical probing combined with mass spectrometry, we confirmed that the 5' and 3' sequences form a duplex which protects both RNAs from chemical modification, consistent with the previously published panhandle structure. These data support that these short, conserved promoter sequences form a stable complex at physiological temperatures, and this complex likely is important for polymerase recognition and viral replication.

A Druglike Small Molecule that Targets r(CCUG) Repeats in Myotonic Dystrophy Type 2 Facilitates Degradation by RNA Quality Control Pathways.

Myotonic dystrophy type 2 (DM2) is one of >40 microsatellite disorders caused by RNA repeat expansions. The DM2 repeat expansion, r(CCUG)exp (where "exp" denotes expanded repeating nucleotides), is harbored in intron 1 of the CCHC-type zinc finger nucleic acid binding protein (CNBP). The expanded RNA repeat causes disease by a gain-of-function mechanism, sequestering various RNA-binding proteins including the pre-mRNA splicing regulator MBNL1. Sequestration of MBNL1 results in its loss-of-function and concomitant deregulation of the alternative splicing of its native substrates. Notably, this r(CCUG)exp causes retention of intron 1 in the mature CNBP mRNA. Herein, we report druglike small molecules that bind the structure adopted by r(CCUG)exp and improve DM2-associated defects. These small molecules were optimized from screening hits from an RNA-focused small-molecule library to afford a compound that binds r(CCUG)exp specifically and with nanomolar affinity, facilitates endogenous degradation of the aberrantly retained intron in which it is harbored, and rescues alternative splicing defects.

A Small Molecule that Binds an RNA Repeat Expansion Stimulates Its Decay via the Exosome Complex.

Many diseases are caused by toxic RNA repeats. Herein, we designed a lead small molecule that binds the structure of the r(CUG) repeat expansion [r(CUG)exp] that causes myotonic dystrophy type 1 (DM1) and Fuchs endothelial corneal dystrophy (FECD) and rescues disease biology in patient-derived cells and in vivo. Interestingly, the compound's downstream effects are different in the two diseases, owing to the location of the repeat expansion. In DM1, r(CUG)exp is harbored in the 3' untranslated region, and the compound has no effect on the mRNA's abundance. In FECD, however, r(CUG)exp is located in an intron, and the small molecule facilitates excision of the intron, which is then degraded by the RNA exosome complex. Thus, structure-specific, RNA-targeting small molecules can act disease specifically to affect biology, either by disabling the gain-of-function mechanism (DM1) or by stimulating quality control pathways to rid a disease-affected cell of a toxic RNA (FECD).

Ribonuclease recruitment using a small molecule reduced c9ALS/FTD r(G4C2) repeat expansion in vitro and in vivo ALS models.

The most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) is an expanded G4C2 RNA repeat [r(G4C2)exp] in chromosome 9 open reading frame 72 (C9orf72), which elicits pathology through several mechanisms. Here, we developed and characterized a small molecule for targeted degradation of r(G4C2)exp. The compound was able to selectively bind r(G4C2)exp's structure and to assemble an endogenous nuclease onto the target, provoking removal of the transcript by native RNA quality control mechanisms. In c9ALS patient­derived spinal neurons, the compound selectively degraded the mutant C9orf72 allele with limited off-targets and reduced quantities of toxic dipeptide repeat proteins (DPRs) translated from r(G4C2)exp. In vivo work in a rodent model showed that abundance of both the mutant allele harboring the repeat expansion and DPRs were selectively reduced by this compound. These results demonstrate that targeted small-molecule degradation of r(G4C2)exp is a strategy for mitigating c9ALS/FTD-associated pathologies and studying disease-associated pathways in preclinical models.

Ab initio/GIAO-CCSD(T) study of the trimethyl- and dimethylsilicenium ions: structures, (29)Si, (13)C, and (1)H NMR chemical shifts, and IR spectra.

The C(3h) conformation of the trimethylsilicenium ion 1 was established to be the preferred global energy minimum structure based on energy calculations. Because C-H hyperconjugation occurs least favorably in this conformation of the analogous tert-butyl cation, it may not contribute in large part to the stabilization of this cation, especially given the ineffectiveness of the 3p-2sp(3) overlap that would need to be involved. This is in contrast with the preferred C(s) global energy conformers of the tert-butyl cation. The C(2v) structure 4 and C(2) enantiomers 6 and 7 are the preferred conformations of the dimethylsilicenium ion based on energy comparison. None of these structures have C-H bonds ideally oriented for hyperconjugation with the empty p orbital of the cationic silicon, indicating that it does not likely stabilize the ion to any significant extent. The computed IR spectra and (29)Si, (13)C, and (1)H NMR chemical shifts of the isomers were also discussed. Whereas the studied alkylsilicenium ions are thermodynamically stable, their observation as persistent ions in solution is much more difficult because of their kinetic instability toward varied electron-donating solvents.

Small molecule targeting of RNA structures in neurological disorders.

Aberrant RNA structure and function operate in neurological disease progression and severity. As RNA contributes to disease pathology in a complex fashion, that is, via various mechanisms, it has become an attractive therapeutic target for small molecules and oligonucleotides. In this review, we discuss the identification of RNA structures that cause or contribute to neurological diseases as well as recent progress toward the development of small molecules that target them, including small molecule modulators of pre-mRNA splicing and RNA repeat expansions that cause microsatellite disorders such as Huntington's disease and amyotrophic lateral sclerosis. The use of oligonucleotide-based modalities is also discussed. There are key differences between small molecule and oligonucleotide targeting of RNA. The former targets RNA structure, while the latter prefers unstructured regions. Thus, some targets will be preferentially targeted by oligonucleotides and others by small molecules.