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.

Pseudouridine-mediated stop codon readthrough in S. cerevisiae is sequence context-independent.

We have previously shown that when the uridine of a stop codon (UAA, UAG, or UGA) is pseudouridylated, the ribosome reads through the modified stop codon. However, it is not clear as to whether or not the pseudouridine (Ψ)-mediated readthrough is dependent on the sequence context of mRNA. Here, we use several different approaches and the yeast system to address this question. We show that when a stop codon (premature termination codon, PTC) is introduced into the coding region of a reporter mRNA at several different positions (with different sequence contexts) and pseudouridylated, we detect similar levels of readthrough. Using mutational and selection/screen analyses, we also show that the upstream sequence (relative to PTC) as well as the nucleotides surrounding the PTC (upstream and downstream) play a minimal role (if at all) in Ψ-mediated ribosome readthrough. Interestingly, we detect no suppression of NMD (nonsense-mediated mRNA decay) by targeted PTC pseudouridylation in the yeast system. Our results indicate that Ψ-mediated nonsense suppression occurs at the translational level, and that the suppression is sequence context-independent, unlike some previously characterized rare stop codon readthrough events.

Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly.

Pseudouridine (Ψ) is the most abundant internal modification identified in RNA, and yet little is understood of its effects on downstream reactions. Yeast U2 snRNA contains three conserved Ψs (Ψ35, Ψ42, and Ψ44) in the branch site recognition region (BSRR), which base pairs with the pre-mRNA branch site during splicing. Here, we show that blocks to pseudouridylation at these positions reduce the efficiency of pre-mRNA splicing, leading to growth-deficient phenotypes. Restoration of pseudouridylation at these positions using designer snoRNAs results in near complete rescue of splicing and cell growth. These Ψs interact genetically with Prp5, an RNA-dependent ATPase involved in monitoring the U2 BSRR-branch site base-pairing interaction. Biochemical analysis indicates that Prp5 has reduced affinity for U2 snRNA that lacks Ψ42 and Ψ44 and that Prp5 ATPase activity is reduced when stimulated by U2 lacking Ψ42 or Ψ44 relative to wild type, resulting in inefficient spliceosome assembly. Furthermore, in vivo DMS probing analysis reveals that pseudouridylated U2, compared to U2 lacking Ψ42 and Ψ44, adopts a slightly different structure in the branch site recognition region. Taken together, our results indicate that the Ψs in U2 snRNA contribute to pre-mRNA splicing by directly altering the binding/ATPase activity of Prp5.

Translational homeostasis via the mRNA cap-binding protein, eIF4E.

Translational control of gene expression plays a key role in many biological processes. Consequently, the activity of the translation apparatus is under tight homeostatic control. eIF4E, the mRNA 5' cap-binding protein, facilitates cap-dependent translation and is a major target for translational control. eIF4E activity is controlled by a family of repressor proteins, termed 4E-binding proteins (4E-BPs). Here, we describe the surprising finding that despite the importance of eIF4E for translation, a drastic knockdown of eIF4E caused only minor reduction in translation. This conundrum can be explained by the finding that 4E-BP1 is degraded in eIF4E-knockdown cells. Hypophosphorylated 4E-BP1, which binds to eIF4E, is degraded, whereas hyperphosphorylated 4E-BP1 is refractory to degradation. We identified the KLHL25-CUL3 complex as the E3 ubiquitin ligase, which targets hypophosphorylated 4E-BP1. Thus, the activity of eIF4E is under homeostatic control via the regulation of the levels of its repressor protein 4E-BP1 through ubiquitination.

Suppression of Nonsense Mutations by New Emerging Technologies.

Nonsense mutations often result from single nucleotide substitutions that change a sense codon (coding for an amino acid) to a nonsense or premature termination codon (PTC) within the coding region of a gene. The impact of nonsense mutations is two-fold: (1) the PTC-containing mRNA is degraded by a surveillance pathway called nonsense-mediated mRNA decay (NMD) and (2) protein translation stops prematurely at the PTC codon, and thus no functional full-length protein is produced. As such, nonsense mutations result in a large number of human diseases. Nonsense suppression is a strategy that aims to correct the defects of hundreds of genetic disorders and reverse disease phenotypes and conditions. While most clinical trials have been performed with small molecules, there is an increasing need for sequence-specific repair approaches that are safer and adaptable to personalized medicine. Here, we discuss recent advances in both conventional strategies as well as new technologies. Several of these will soon be tested in clinical trials as nonsense therapies, even if they still have some limitations and challenges to overcome.

The TOR signaling pathway regulates starvation-induced pseudouridylation of yeast U2 snRNA.

Pseudouridine (Ψ) has been identified in various types of RNAs, including mRNA, rRNA, tRNA, snRNA, and many other noncoding RNAs. We have previously shown that RNA pseudouridylation, like DNA and protein modifications, can be induced by stress. For instance, growing yeast cells to saturation induces the formation of Ψ93 in U2 snRNA. Here, we further investigate this inducible RNA modification. We show that switching yeast cells from nutrient-rich medium to different nutrient-deprived media (including water) results in the formation of Ψ93 in U2 snRNA. Using gene deletion/conditional depletion as well as rapamycin treatment, we further show that the TOR signaling pathway, which controls cell entry into stationary phase, regulates Ψ93 formation. The RAS/cAMP signaling pathway, which parallels the TOR pathway, plays no role in this inducible modification.

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.

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.

Therapeutic potential of anti-interleukin-17A aptamer: suppression of interleukin-17A signaling and attenuation of autoimmunity in two mouse models.

OBJECTIVE: The proinflammatory cytokine interleukin-17A (IL-17A) is produced primarily by the CD4+ T cell subset called Th17 cells, which is involved in host defense, inflammation, and autoimmune disorders. This study was undertaken to investigate the effect of a high-affinity RNA molecule, called an aptamer, against human IL-17A on IL-17A-induced signal transduction in vitro and its anti-autoimmune efficacy in vivo in 2 mouse models of inflammation. METHODS: By screening a large library of nuclease-resistant RNA oligonucleotides, we selected an RNA aptamer, Apt21-2, that binds human and mouse IL-17 and blocks the interaction between IL-17A and its receptor. The inhibition of IL-17A-mediated phosphorylation and marker protein production was analyzed in human and mouse cells. Mice with glucose-6-phosphate isomerase (GPI)-induced rheumatoid arthritis and myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis were used to assess efficacy. RESULTS: Apt21-2 prevented efficient phosphorylation of the IL-17A signaling factors IκB and JNK and inhibited the production of IL-6 in human and mouse cells. A PEGylated form of Apt21-2 (PEG21-2idT) exhibited a 50% inhibition concentration (IC(50) ) in the range of 1-2 nM and 70-80 nM in human and mouse cells, respectively. When administered immediately after immunization with GPI or MOG, PEG21-2idT inhibited in a dose-dependent manner the development of arthritic or neurologic symptoms. Significantly, PEG21-2idT slowed the progression of arthritis when administered after the onset of GPI-induced arthritis. CONCLUSION: Our findings indicate that the chemically processed anti-IL-17A aptamer PEG21-2idT inhibits the actions of IL-17A as well as the development of autoimmunity in 2 mouse models of inflammation. These results offer for the first time an aptamer-based therapeutic approach to the treatment of Th17 cell-mediated autoimmune disorders.

Angptl4 deficiency decreases serum triglyceride levels in low-density lipoprotein receptor knockout mice and streptozotocin-induced diabetic mice.

Angiopoietin-like protein family 4 (Angptl4) has been shown to regulate lipoprotein metabolism through the inhibition of lipoprotein lipase (LPL). In familial hypercholesterolemia (FH), individuals lacking low-density lipoprotein receptor (LDLR) present not only hypercholesterolemia, but also increased plasma triglyceride (TG)-rich lipoprotein remnants, and develop atherosclerosis. In addition, the most common type of dyslipidemia in individuals with diabetes is increased TG levels. We first generated LDLR(-/-)Angptl4(-/-) mice to study the effect of Angptl4 deficiency on the lipid metabolism. Fasting total cholesterol, VLDL-C, LDL-C, HDL-C and TG levels were decreased in LDLR(-/-)Angptl4(-/-) mice compared with LDLR(-/-)Angptl4(+/+) mice. In particular, post olive oil-loaded TG excursion were largely attenuated in LDLR(-/-)Angptl4(-/-) mice compared with LDLR(-/-)Angptl4(+/+) mice. We next introduced diabetes by streptozotocin (STZ) treatment in Angptl4(-/-) mice and examined the impacts of Angptl4 deficiency. Compared with diabetic Angptl4(+/+) mice, diabetic Angptl4(-/-) mice showed the improvement of fasting and postprandial hypertriglyceridemia as well. Thus, targeted silencing of Angptl4 offers a potential therapeutic strategy for the treatment of dyslipidemia in individuals with FH and insulin deficient diabetes.

The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines.

The current COVID-19 pandemic is a massive source of global disruption, having led so far to two hundred and fifty million COVID-19 cases and almost five million deaths worldwide. It was recognized in the beginning that only an effective vaccine could lead to a way out of the pandemic, and therefore the race for the COVID-19 vaccine started immediately, boosted by the availability of the viral sequence data. Two novel vaccine platforms, based on mRNA technology, were developed in 2020 by Pfizer-BioNTech and Moderna Therapeutics (comirnaty® and spikevax®, respectively), and were the first ones presenting efficacies higher than 90%. Both consisted of N1-methyl-pseudouridine-modified mRNA encoding the SARS-COVID-19 Spike protein and were delivered with a lipid nanoparticle (LNP) formulation. Because the delivery problem of ribonucleic acids had been known for decades, the success of LNPs was quickly hailed by many as the unsung hero of COVID-19 mRNA vaccines. However, the clinical trial efficacy results of the Curevac mRNA vaccine (CVnCoV) suggested that the delivery system was not the only key to the success. CVnCoV consisted of an unmodified mRNA (encoding the same spike protein as Moderna and Pfizer-BioNTech's mRNA vaccines) and was formulated with the same LNP as Pfizer-BioNTech's vaccine (Acuitas ALC-0315). However, its efficacy was only 48%. This striking difference in efficacy could be attributed to the presence of a critical RNA modification (N1-methyl-pseudouridine) in the Pfizer-BioNTech and Moderna's mRNA vaccines (but not in CVnCoV). Here we highlight the features of N1-methyl-pseudouridine and its contributions to mRNA vaccines.

Spliceosomal snRNA Epitranscriptomics.

Small nuclear RNAs (snRNAs) are critical components of the spliceosome that catalyze the splicing of pre-mRNA. snRNAs are each complexed with many proteins to form RNA-protein complexes, termed as small nuclear ribonucleoproteins (snRNPs), in the cell nucleus. snRNPs participate in pre-mRNA splicing by recognizing the critical sequence elements present in the introns, thereby forming active spliceosomes. The recognition is achieved primarily by base-pairing interactions (or nucleotide-nucleotide contact) between snRNAs and pre-mRNA. Notably, snRNAs are extensively modified with different RNA modifications, which confer unique properties to the RNAs. Here, we review the current knowledge of the mechanisms and functions of snRNA modifications and their biological relevance in the splicing process.

From Antisense RNA to RNA Modification: Therapeutic Potential of RNA-Based Technologies.

Therapeutic oligonucleotides interact with a target RNA via Watson-Crick complementarity, affecting RNA-processing reactions such as mRNA degradation, pre-mRNA splicing, or mRNA translation. Since they were proposed decades ago, several have been approved for clinical use to correct genetic mutations. Three types of mechanisms of action (MoA) have emerged: RNase H-dependent degradation of mRNA directed by short chimeric antisense oligonucleotides (gapmers), correction of splicing defects via splice-modulation oligonucleotides, and interference of gene expression via short interfering RNAs (siRNAs). These antisense-based mechanisms can tackle several genetic disorders in a gene-specific manner, primarily by gene downregulation (gapmers and siRNAs) or splicing defects correction (exon-skipping oligos). Still, the challenge remains for the repair at the single-nucleotide level. The emerging field of epitranscriptomics and RNA modifications shows the enormous possibilities for recoding the transcriptome and repairing genetic mutations with high specificity while harnessing endogenously expressed RNA processing machinery. Some of these techniques have been proposed as alternatives to CRISPR-based technologies, where the exogenous gene-editing machinery needs to be delivered and expressed in the human cells to generate permanent (DNA) changes with unknown consequences. Here, we review the current FDA-approved antisense MoA (emphasizing some enabling technologies that contributed to their success) and three novel modalities based on post-transcriptional RNA modifications with therapeutic potential, including ADAR (Adenosine deaminases acting on RNA)-mediated RNA editing, targeted pseudouridylation, and 2'-O-methylation.

An acylic polyisoprenoid derivative, geranylgeranylacetone protects against visceral adiposity and insulin resistance in high-fat-fed mice.

Induction of heat shock protein (HSP)72 improves insulin resistance and obesity in diabetic animal models. Geranylgeranylacetone (GGA), known as an antiulcer drug, induces HSP72 and protects organs against several cellular stresses. This study investigated whether GGA administration would induce HSP72 in liver and render physiological protection against high-fat feeding in mice. A single and 4-wk oral administration of 200 mg/kg GGA was performed in high-fat diet (HFD)-fed mice. Metabolic parameters, cytokines, and gene expressions related to insulin signaling were evaluated. A single administration of GGA induced HSP72 in liver of normal chow-fed and HFD-fed mice. Insulin resistance after HFD was slightly ameliorated. Four weeks of GGA administration also increased HSP72 in liver and significantly improved insulin resistance and glucose homeostasis upon glucose challenge. Activation of c-jun NH2-terminal kinase (JNK) was attenuated, and insulin signaling was improved in the liver of HFD mice. Visceral adiposity was decreased in GGA-treated mice, accompanied by reduced leptin and increased adiponectin levels. GGA can be a novel therapeutic approach to treat metabolic syndrome as well as type 2 diabetes by improving insulin signaling and reducing adiposity. These beneficial effects of GGA could be mediated through HSP72 induction and JNK inactivation in the liver.

Angptl 4 deficiency improves lipid metabolism, suppresses foam cell formation and protects against atherosclerosis.

Angiopoietin-like protein family 4 (Angptl 4) has been shown to regulate lipoprotein metabolism through the inhibition of lipoprotein lipase (LPL). We generated ApoE(-/-)Angptl 4(-/-) mice to study the effect of Angptl 4 deficiency on lipid metabolism and atherosclerosis. Fasting and postolive oil-loaded triglyceride (TG) levels were largely decreased in ApoE(-/-)Angptl 4(-/-) mice compared with and ApoE(-/-)Angptl 4(+/+) mice. There was a significant (75+/-12%) reduction in atherosclerotic lesion size in ApoE(-/-)Angptl 4(-/-) mice compared with ApoE(-/-) Angptl 4(+/+) mice. Peritoneal macrophages, isolated from Angptl 4(-/-) mice to investigate the foam cell formation, showed a significant decrease in newly synthesized cholesteryl ester (CE) accumulation induced by acetyl low-density lipoprotein (acLDL) compared with those from Angptl 4(+/+) mice. Thus, genetic knockout of Angptl 4 protects ApoE(-/-) mice against development and progression of atherosclerosis and strongly suppresses the ability of the macrophages to become foam cells in vitro.

Post-transcriptional pseudouridylation in mRNA as well as in some major types of noncoding RNAs.

Pseudouridylation is a post-transcriptional isomerization reaction that converts a uridine to a pseudouridine (Ψ) within an RNA chain. Ψ has chemical properties that are distinct from that of uridine and any other known nucleotides. Experimental data accumulated thus far have indicated that Ψ is present in many different types of RNAs, including coding and noncoding RNAs. Ψ is particularly concentrated in rRNA and spliceosomal snRNAs, and plays an important role in protein translation and pre-mRNA splicing, respectively. Ψ has also been found in mRNA, but its function there remains essentially unknown. In this review, we discuss the mechanisms and functions of RNA pseudouridylation, focusing on rRNA, snRNA and mRNA. We also discuss the methods, which have been developed to detect Ψs in RNAs. This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.

Detection and Quantification of Pseudouridine in RNA.

Pseudouridylation is the most abundant of all RNA modifications. Pseudouridylation is dynamic and widespread among many different types of RNAs in living organisms, thus drawing a lot of recent interest from the RNA and epigenetics communities. To successfully carry out an investigation into RNA pseudouridylation, it is desirable to have a convenient and effective method capable of detection and quantification of pseudouridylation. Here, we present two such methods: one relies on pseudouridine (Ψ)-specific CMCT modification followed by reverse transcription/primer-extension (semiquantitative), and the other is based on site-specific cleavage and radiolabeling followed by nuclease digestion and TLC (quantitative). Although only semiquantitative, the CMCT and reverse transcription-based method is capable of detecting multiple Ψs (present in the same RNA molecule) in one reaction. In contrast, the second method, based on site-specific cleavage/labeling, nuclease digestion, and TLC, is quantitative, but can be used to analyze only one site at a time. These two methods can be used independently or in combination.

Regulatory RNPs: a novel class of ribonucleoproteins that potentially contribute to ribosome heterogeneity.

Many ribonucleoproteins (RNPs), which are comprised of noncoding RNA and associated proteins, are involved in essential cellular processes such as translation and pre-mRNA splicing. One class of RNP is the small Cajal body-specific RNP (scaRNP), which contributes to the biogenesis of small nuclear RNPs (snRNPs) that are central components of the spliceosome. Three scaRNAs are internally processed, generating stable nucleolus-enriched RNAs of unknown function. Here, we provide data that show that these RNAs become part of RNPs we term regulatory RNPs (regRNPs). Most modifications within rRNA (predominantly pseudouridylation and ribose 2'-O-methylation) are conducted by small nucleolar RNPs (snoRNPs), and we provide evidence that the activity of at least some of these snoRNPs is under the control of regRNPs. Because modifications within rRNA can vary in different physiological or pathological situations, rRNA modifications are thought to be the major source of ribosome heterogeneity. Our identification of regRNPs thus provides a potential mechanism for how ribosome heterogeneity may be accomplished. This work also provides additional functional connections between the Cajal body and the nucleolus.

Insight into the mechanisms and functions of spliceosomal snRNA pseudouridylation.

Pseudouridines (Ψs) are the most abundant and highly conserved modified nucleotides found in various stable RNAs of all organisms. Most Ψs are clustered in regions that are functionally important for pre-mRNA splicing. Ψ has an extra hydrogen bond donor that endows RNA molecules with distinct properties that contribute significantly to RNA-mediated cellular processes. Experimental data indicate that spliceosomal snRNA pseudouridylation can be catalyzed by both RNA-dependent and RNA-independent mechanisms. Recent work has also demonstrated that pseudouridylation can be induced at novel positions under stress conditions, suggesting a regulatory role for Ψ.