Tau protein isoforms, regulated during development, are influenced by the nuclear factor TDP-43, which plays a crucial role in tau mRNA stability and exon 10 inclusion. Both tau and TDP-43 are prone to pathological phosphorylation and aggregation, with specific phosphorylated forms of TDP-43 linked to cytoplasmic mislocalization and alterations in the 3R/4R tau ratio as detected in different pathologies. In this study, we show that insulin treatment of embryonic mouse primary cortical neurons-cells that normally express only 3R-tau-induces the expression of 4R-tau, suggesting that metabolic signaling can influence tau isoform expression in a developmentally immature neuronal context. In addition, experiments in HEK293 cells revealed isoform-specific stabilization effects and showed that insulin promotes TDP-43 redistribution to the cytoplasm along with a phosphorylation pattern. These results underscore the complex interplay between TDP-43 and tau isoforms and metabolic signaling pathways that play a crucial role in their expression and localization with potential implications for understanding mechanisms of neurodegenerative disease onset and progression.

Reduction of iron overload in hereditary hemochromatosis model mice by presence of iron metabolism mutant

Background: DNA methylation, as an epigenetic modification, is crucial in the regulatory mechanism of salt resistance in plants. Methods: To gain deeper insight into the relationship between DNA methylation and mRNA exons in halophytes and their potential roles in regulating salt tolerance, this study employed whole-genome bisulfite sequencing (WGBS) and transcriptome sequencing data to analyze the leaves of the halophyte Paspalum vaginatum, widely distributed in tropical regions. Results: The findings revealed that the methylation level of 5-methylcytosine (5mC) in the genomic elements of P. vaginatum increased with prolonged salt treatment under salt stress conditions. This observation suggested that the methylation level plays a pivotal role in the salt stress response of P. vaginatum. Notably, under salt stress, the number of variants at the mRNA exon level was significantly higher than that at the DNA level. Furthermore, comparative analysis revealed sequence variants within exonic regions of mature mRNA transcripts for several genes in salt-treated samples relative to pre-stress controls, and these changes were found to be enriched in several salt-tolerance pathways, including unsaturated fatty acid metabolism and ascorbic acid metabolism, among others. Further analysis demonstrated that the occurrence of these variants changed concomitantly with the dynamic changes in CG methylation levels in the gene body of some salt-tolerant genes. Therefore, it was speculated that mRNA exon variations probably promoted the elevation of CG 5mC methylation levels at the DNA level under salt stress conditions, further enabling the plant to adapt to the salt-stress environment. Conclusions: These findings offer preliminary insights into the relationship between DNA methylation and mRNA exon variations in P. vaginatum under salt stress, providing valuable information and avenues for further investigation into the regulatory role of mRNA in DNA methylation.

The sulfonylurea receptor 1 (SUR1) has been classified as a member of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter superfamily. SUR1, unlike the classic ABC transporters, assembles with Kir6.2, forming KATP channels to regulate the flux of potassium ions. In the central nervous system, SUR1 is weakly expressed in some brain regions but is induced by pathological conditions in the different cell types of the neurovascular unit. Therefore, we first analyzed the expression of SUR1 in various rat tissues and brain regions to identify SUR1 isoforms and their mRNA exon composition under physiological conditions. Later, we focused on the SUR1 expression in the brain and heart after ischemia/reperfusion. We observed two SUR1 isoforms (170 and 60-75 kDa) abundantly expressed in most rat tissues, except for the testis and brain, where basal expression of these isoforms was relatively low and exhibit a band of 100 kDa. Every exons coding for the functional domains of SUR1 mRNA were amplified from the tissues and brain regions analyzed. Results from in vitro and in vivo experiments indicated that SUR1 isoforms previously identified (170 and 60-75 kDa) were dramatically overexpressed in the brain after middle cerebral artery occlusion followed by reperfusion. In contrast, myocardial infarction followed by reperfusion significantly reduced SUR1 isoform expression in the heart. This study demonstrates the expression of at least two SUR1 isoforms in various tissues and suggests that ischemic processes may differentially regulate SUR1 expression depending on the tissue injured.

Long-read sequencing (LRS) technologies have the potential to revolutionize scientific discoveries in RNA biology through the comprehensive identification and quantification of full-length mRNA isoforms. Despite great promise, challenges remain in the widespread implementation of LRS technologies for RNA-based applications, including concerns about low coverage, high sequencing error, and robust computational pipelines. Although much focus has been placed on defining mRNA exon composition and structure with LRS data, less careful characterization has been done of the ability to assess the terminal ends of isoforms, specifically, transcription start and end sites. Such characterization is crucial for completely delineating full mRNA molecules and regulatory consequences. However, there are substantial inconsistencies in both start and end coordinates of LRS reads spanning a gene, such that LRS reads often fail to accurately recapitulate annotated or empirically derived terminal ends of mRNA molecules. Here, we describe the specific challenges of identifying and quantifying mRNA terminal ends with LRS technologies and how these issues influence biological interpretations of LRS data. We then review recent experimental and computational advances designed to alleviate these problems, with ideal use cases for each approach. Finally, we outline anticipated developments and necessary improvements for the characterization of terminal ends from LRS data.

RTP801/REDD1 is a stress-responsive protein overexpressed in neurodegenerative diseases such as Alzheimer’s disease (AD) that contributes to cognitive deficits and neuroinflammation. Here, we found that RTP801 interacts with HSPC117, DDX1 and CGI-99, three members of the tRNA ligase complex (tRNA-LC), which ligates the excised exons of intron-containing tRNAs and the mRNA exons of the transcription factor XBP1 during the unfolded protein response (UPR). We also found that RTP801 modulates the mRNA ligase activity of the complex in vitro since RTP801 knockdown promoted XBP1 splicing and the expression of its transcriptional target, SEC24D. Conversely, RTP801 overexpression inhibited the splicing of XBP1. Similarly, in human AD postmortem hippocampal samples, where RTP801 is upregulated, we found that XBP1 splicing was dramatically decreased. In the 5xFAD mouse model of AD, silencing RTP801 expression in hippocampal neurons promoted Xbp1 splicing and prevented the accumulation of intron-containing pre-tRNAs. Finally, the tRNA-enriched fraction obtained from 5xFAD mice promoted abnormal dendritic arborization in cultured hippocampal neurons, and RTP801 silencing in the source neurons prevented this phenotype. Altogether, these results show that elevated RTP801 impairs RNA processing in vitro and in vivo in the context of AD and suggest that RTP801 inhibition could be a promising therapeutic approach.

The methyltransferase SETD3 regulates mRNA alternative splicing through interacting with hnRNPK

Double pH-responsive xenopeptide carriers containing succinoyl tetraethylene pentamine (Stp) and lipo amino fatty acids (LAFs) were evaluated for CRISPR/Cas9 based genome editing. Different carrier topologies, variation of LAF/Stp ratios and LAF types as Cas9 mRNA/sgRNA polyplexes were screened in three different reporter cell lines using three different genomic targets (Pcsk9, eGFP, mdx exon 23). One U-shaped and three bundle (B2)-shaped lipo-xenopeptides exhibiting remarkable efficiencies were identified. Genome editing potency of top carriers were observed at sub-nanomolar EC50 concentrations of 0.4 nM sgRNA and 0.1 nM sgRNA for the top U-shape and top B2 carriers, respectively, even after incubation in full (≥ 90%) serum. Polyplexes co-delivering Cas9 mRNA/sgRNA with a single stranded DNA template for homology directed gene editing resulted in up to 38% conversion of eGFP to BFP in reporter cells. Top carriers were formulated as polyplexes or lipid nanoparticles (LNPs) for subsequent in vivo administration. Formulations displayed long-term physicochemical and functional stability upon storage at 4 °C. Importantly, intravenous administration of polyplexes or LNPs mediated in vivo editing of the dystrophin gene, triggering mRNA exon 23 splicing modulation in dystrophin-expressing cardiac muscle, skeletal muscle and brain tissue.

The tight control of fate transitions during stem cell differentiation is essential for proper tissue development and maintenance. However, the challenges in studying sparsely distributed adult stem cells in a systematic manner have hindered efforts to identify how the multilayered regulation of gene expression programs orchestrates stem cell differentiation in vivo. Here, we synchronised Drosophila female germline stem cell (GSC) differentiation in vivo to perform in-depth transcriptome and translatome analyses at high temporal resolution. This characterisation revealed widespread and dynamic changes in mRNA level, promoter usage, exon inclusion, and translation efficiency. Transient expression of the master regulator, Bam, drives a first wave of expression changes, primarily modifying the cell cycle program. Surprisingly, as Bam levels recede, differentiating cells return to a remarkably stem cell-like transcription and translation program, with a few crucial changes feeding into a second phase driving terminal differentiation to form the oocyte. Altogether, these findings reveal that rather than a unidirectional accumulation of changes, the in vivo differentiation of stem cells relies on distinctly regulated and developmentally sequential waves.

Benzene and its metabolite hydroquinone (HQ) are widely used in daily life, and long-term exposure to benzene or HQ can induce acute myeloid leukemia (AML). Circular RNAs (circRNAs) are mostly produced by reverse splicing of gene exon mRNA precursors. The modulation of circRNA expression is connected to leukemia progression; however, the molecular mechanism is still unknown. In this study, the cells were divided into four groups: PBS control group (PBS-TK6), TK6 malignantly transformed cells induced by 10.0 μmol/L HQ (HQ-TK6), and HQ-TK6 cells treated with 5 μmol/L 5-AzaC (DNA methyltransferase inhibitor) for 24 h (HQ + 5-AzaC). HQ-TK6 cells were treated with 200 nmol/L TSA (histone deacetylation inhibitor) for 24 h (HQ + TSA). qRT-PCR was used to identify the differential hsa_circ_401351 expression between the four groups. We further determined the hsa_circ_401351 promoter methylation level with methylation-specific PCR. DNMT1 and DNMT3b were knocked down by CRISPR/Cas9 to elucidate the specific molecular mechanism of hsa_circ_401351 in HQ-TK6 cells. CCK-8 and flow cytometry detected cell proliferation and apoptosis, respectively, after hsa_circ_401351 was overexpressed in HQ-TK6 cells. Compared with the PBS-TK6 group, the expression of hsa_circ_401351 was found to be lower in the HQ-TK6 group. Nevertheless, treatment with 5-AzaC or TSA increased hsa_circ_401351 expression, with the upregulation being more pronounced in the TSA group. The expression of hsa_circ_401351 in the DNMT1 knockdown group was dramatically increased by 50% compared to that in the control group, and the DNA methylation level of the hsa_circ_401351 promoter region was decreased. When hsa_circ_401351 was overexpressed, HQ-TK6 cell proliferation was significantly slowed after 48 h compared with the control group. Flow cytometry showed that cells were mainly arrested in G1 phase, and apoptosis was significantly enhanced. Similarly, qRT-PCR and Western blot data showed significant reductions in Caspase-3 mRNA and protein production, and Bcl-2 mRNA levels were also elevated. Overall, our research showed that elevated DNMT1 expression in HQ-TK6 cells increased methylation levels and decreased expression of the hsa_circ_401351 promoter region, limiting its ability to suppress HQ-TK6 cell growth and enhance apoptosis.

Mammalian mRNA and lncRNA exons are often small compared to introns. The exon definition model predicts that exons splice autonomously, dependent on proximal exon sequence features, explaining their delineation within large introns. This model has not been examined on a genome-wide scale, however, leaving open the question of how often mRNA and lncRNA exons are autonomous. It is also unknown how frequently such exons can arise by chance. Here, we directly assayed large fragments (500-1000 bp) of the human genome by exon trapping, which detects exons spliced into a heterologous transgene, here designed with a large intron context. We define the trapped exons as "autonomous." We obtained ∼1.25 million trapped exons, including most known mRNA and well-annotated lncRNA internal exons, demonstrating that human exons are predominantly autonomous. mRNA exons are trapped with the highest efficiency. Nearly a million of the trapped exons are unannotated, most located in intergenic regions and antisense to mRNA, with depletion from the forward strand of introns. These exons are not conserved, suggesting they are nonfunctional and arose from random mutations. They are nonetheless highly enriched with known splicing promoting sequence features that delineate known exons. Novel autonomous exons are more numerous than annotated lncRNA exons, and computational models also indicate they will occur with similar frequency in any randomly generated sequence. These results show that most human coding exons splice autonomously, and provide an explanation for the existence of many unconserved lncRNAs, as well as a new annotation and inclusion levels of spliceable loci in the human genome.

Abstract: Lung adenocarcinoma, a major cause of cancer-related deaths globally, was examined through a comprehensive analysis of 230 resected tumors. This study integrated various sequencing techniques to profile messenger RNA, microRNA, and DNA, along with evaluating copy number variations, methylation patterns, and proteomic data. The findings revealed a notable rate of somatic mutations (average of 8.9 mutations per megabase). Among the 18 significantly mutated genes were RIT1 activating mutations and newly identified MGA loss-of-function mutations, which were mutually exclusive with MYC amplification. Gender-based differences were observed, with EGFR mutations being more common in females and RBM10 mutations in males. Genetic abnormalities in NF1, MET, ERBB2, and RIT1 were detected in 13% of cases, often in tumors lacking other activated oncogenes, suggesting their potential driver roles. Splicing alterations were driven by somatic genomic changes, as evidenced by DNA and mRNA sequencing from the same tumor, including MET mRNA exon 14 skipping in 4% of cases. While MAPK and PI(3)K pathway activity was explained by known mutations in some cases, unexplained mechanisms appeared to activate the pathways in others. These findings lay the groundwork for understanding lung adenocarcinoma’s molecular basis and offer insights for classification and further investigations. Molecular profiling is crucial for identifying actionable mutations, guiding treatment choices in advanced adenocarcinoma and acquired resistance to tyrosine kinase inhibitors. Circulating tumor DNA sequencing gains importance in NSCLC diagnostics due to its accessibility, allowing longitudinal disease monitoring. Notably, the overall cancer death rate dropped by 27% between 1991 and 2016, resulting in around 2,629,200 fewer expected cancer deaths compared to peak death rates.

BackgroundAlmost half of severe hemophilia A (HA) is caused by an intron 22 inversion mutation (Int22Inv), which disrupts the 26-exon F8 gene. Inverted F8 mRNA exons 1-22 are transcribed, while F8B mRNA, containing F8 exons 23-26, is transcribed from a promoter within intron 22. Neither FVIII activity nor FVIII antigen (cross-reacting material, CRM) are detectable in plasma of patients with an intron-22 inversion.ObjectivesTo test the hypothesis that (putative) intracellular synthesis of FVIII proteins encoded by inverted F8 and F8B mRNAs confers T-cell tolerance to almost the entire FVIII sequence, and to evaluate the immunogenicity of the region encoded by the F8 exon 22-23 junction sequence.Patients/MethodsPeripheral blood mononuclear cells (PBMCs) from 30 severe or moderate HA subjects (17 with an Int22Inv mutation) were tested by ELISPOT assays to detect cytokine secretion in response to FVIII proteins and peptides and to map immunodominant T-cell epitopes. Potential immunogenicity of FVIII sequences encoded by the F8 exon 22-23 junction region was also tested using peptide-MHCII binding assays.ResultsEight of the Int22Inv subjects showed robust cytokine secretion from PBMCs stimulated with FVIII proteins and/or peptides, consistent with earlier publications from the Conti-Fine group. Peptide ELISPOT assays identified immunogenic regions of FVIII. Specificity for sequences encoded within F8 mRNA exons 1-22 and F8B mRNA was confirmed by staining Int22Inv CD4+ T cells with peptide-loaded HLA-Class II tetramers. FVIII peptides spanning the F8 exon 22-23 junction (encoding M2124-V2125) showed limited binding to MHCII proteins and low immunogenicity, with cytokine secretion from only one Int22Inv subject.ConclusionsPBMCs from multiple subjects with an Int22Inv mutation, with and without a current FVIII inhibitor, responded to FVIII epitopes. Furthermore, the FVIII region encoded by the exon 22-23 junction sequence was not remarkably immunoreactive and is therefore unlikely to contain an immunodominant, promiscuous CD4+ T-cell epitope. Our results indicate that putative intracellular expression of partial FVIII proteins does not confer T-cell tolerance to FVIII regions encoded by inverted F8 mRNA or F8B mRNA.

The RNA-binding proteins CELF1 and ELAVL1 cooperatively control the alternative splicing of CD44

The market for large molecule biologic drugs has grown rapidly, including antisense oligonucleotide (ASO) drugs. ASO drugs work as single-stranded synthetic oligonucleotides that reduce production or alter functions of disease-causing proteins through various mechanisms, such as mRNA degradation, exon skipping, and ASO-protein interactions. Since the first ASO drug, fomivirsen, was approved in 1998, the U.S. Food and Drug Administration (FDA) has approved 10 ASO drugs to date. Although ASO drugs are efficacious in treating some diseases that are untargetable by small-molecule chemical drugs, concerns on adverse drug reactions (ADRs) and toxicity cannot be ignored. Illustrative of this, mipomersen was recently taken off the market due to its hepatotoxicity risk. This paper reviews ADRs and toxicity from FDA drug labeling, preclinical studies, clinical trials, and postmarketing real-world studies on the 10 FDA-approved ASO drugs, including fomivirsen and pegaptanib, mipomersen, nusinersen, inotersen, defibrotide, eteplirsen, golodirsen, viltolarsen, and casimersen. Unique and common ADRs and toxicity for each ASO drug are summarized here. The risk of developing hepatotoxicity, kidney toxicity, and hypersensitivity reactions co-exists for multiple ASO drugs. Special precautions need to be in place when certain ASO drugs are administrated. Further discussion is extended on studying the mechanisms of ADRs and toxicity of these drugs, evaluating the existing physiologic and pathologic states of patients, optimizing the dose and route of administration, and formulating personalized treatment plans to improve the clinical utility of FDA-approved ASO drugs and discovery and development of new ASO drugs with reduced ADRs. SIGNIFICANCE STATEMENT: The current review provides a comprehensive analysis of unique and common ADRs and the toxicity of FDA-approved ASO drugs. The information can help better manage the risk of severe hepatotoxicity, kidney toxicity, and hypersensitivity reactions in the usage of currently approved ASO drugs and the discovery and development of new and safer ASO drugs.

Absorption, distribution, metabolism, and excretion (ADME) are the key biologic processes for determination of a drug's pharmacokinetic parameters, which have direct impacts on efficacy and adverse drug reactions (ADRs). The chemical structures, dosage forms, and sites and routes of administration are the principal determinants of ADME profiles and consequent impacts on their efficacy and ADRs. Newly developed large molecule biologic antisense oligonucleotide (ASO) drugs have completely unique ADME that is not fully defined. ASO-based drugs are single-stranded synthetic antisense nucleic acids with diverse modes of drug actions from induction of mRNA degradation, exon skipping and restoration, and interactions with proteins. ASO drugs have a great potential to treat certain human diseases that have remained untreatable with small molecule-based drugs. The ADME of ASO drugs contributes to their unique set of ADRs and toxicity. In this review, to better understand their ADME, the 10 US Food and Drug Administration (FDA)-approved ASO drugs were selected: fomivirsen, pegaptanib, mipomersen, nusinersen, inotersen, defibrotide, eteplirsen, golodirsen, viltolarsen, and casimersen. A meta-analysis was conducted on their formulation, dosage, sites of administration, local and systematic distribution, metabolism, degradation, and excretion. Membrane permeabilization through endocytosis and nucleolytic degradation by endonucleases and exonucleases are major ADME features of the ASO drugs that differ from small-molecule drugs. The information summarized here provides comprehensive ADME characteristics of FDA-approved ASO drugs, leading to a better understanding of their therapeutic efficacy and their potential ADRs and toxicity. Numerous knowledge gaps, particularly on cellular uptake and subcellular trafficking and distribution, are identified, and future perspectives and directions are discussed. SIGNIFICANCE STATEMENT: Through a systematic analysis of the existing information of absorption, distribution, metabolism, and excretion (ADME) parameters for 10 US Food and Drug Administration (FDA)-approved antisense oligonucleotide (ASO) drugs, this review provides an overall view of the unique ADME characteristics of ASO drugs, which are distinct from small chemical drug ADME. This knowledge is useful for discovery and development of new ASO drugs as well as clinical use of current FDA-approved ASO drugs.

A hallmark pathological feature of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord1. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing2–4. Single nucleotide polymorphisms in UNC13A are among the strongest hits associated with FTD and ALS in human genome-wide association studies5,6, but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon-splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines and motor neurons derived from induced pluripotent stem cells resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. The top variants associated with FTD or ALS risk in humans are located in the intron harbouring the cryptic exon, and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD and ALS (UNC13A genetic variants), and loss of TDP-43 function.

Antenatal administration of glucocorticoids such as betamethasone (BMZ) during the late preterm period improves neonatal respiratory outcomes. However, glucocorticoids may elicit programming effects on immune function and gene regulation. Here, we test the hypothesis that exposure to antenatal BMZ alters cord blood immune cell composition in association with altered DNA methylation and alternatively expressed Exon 1 transcripts of the glucocorticoid receptor (GR) gene in cord blood CD4+ T-cells. Cord blood was collected from 51 subjects in the Antenatal Late Preterm Steroids Trial: 27 BMZ, 24 placebo. Proportions of leukocytes were compared between BMZ and placebo. In CD4+ T-cells, methylation at CpG sites in the GR promoter regions and expression of GR mRNA exon 1 variants were compared between BMZ and placebo. BMZ was associated with an increase in granulocytes (51.6% vs. 44.7% p = 0.03) and a decrease in lymphocytes (36.8% vs. 43.0% p = 0.04) as a percent of the leukocyte population vs. placebo. Neither GR methylation nor exon 1 transcript levels differed between groups. BMZ is associated with altered cord blood leukocyte proportions, although no associated alterations in GR methylation were observed.

We have found that antisense oligonucleotides and siRNA molecules modified with repeat structures of disulfide units can be directly introduced into the cytoplasm and exhibit a suppressive effect on gene expression. In this study, we analyzed the mechanism of cellular uptake of these membrane-permeable oligonucleotides (MPONs). Time-course analysis by confocal microscopy showed that the uptake of MPONs from the plasma membrane to the cytoplasm reached 50 % of the total uptake in about 5 min. In addition, analysis of the plasma membrane proteins to which MPONs bind, identified several proteins, including voltage-dependent anion channel. Next, we analyzed the behavior of MPONs in the cell and found them to be abundant in the nucleus as early as 24 h after addition with the amount increasing further after 48 and 72 h. The amount of MPONs was 2.5-fold higher than that of unmodified oligonucleotides in the nucleus after 72 h. We also designed antisense oligonucleotides and evaluated the effect of MPONs on mRNA exon skipping using DMD model cells; MPONs caused exon skipping with 69 % efficiency after 72 h, which was three times higher than the rate of the control. In summary, the high capacity for intracytoplasmic and nuclear translocation of MPONs is expected to be useful for therapeutic strategies targeting exon skipping.

Antisense oligonucleotide repress telomerase activity via manipulating alternative splicing or translation