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Insulin is a pleiotropic hormone that elicits its metabolic and mitogenic actions through numerous rapid and reversible protein phosphorylations. The temporal regulation of insulin's intracellular signaling cascade is highly complex and insufficiently understood. We conduct a time-resolved analysis of the global insulin-regulated phosphoproteome of differentiated human primary myotubes derived from satellite cells of healthy donors using high-resolution mass spectrometry. Identification and tracking of ~13,000 phosphopeptides over time reveal a highly complex and coordinated network of transient phosphorylation and dephosphorylation events that can be allocated to time-phased regulation of distinct and non-overlapping subcellular pathways. Advanced network analysis combining protein-protein-interaction (PPI) resources and investigation of donor variability in relative phosphosite occupancy over time identifies novel putative candidates in non-canonical insulin signaling and key regulatory nodes that are likely essential for signal propagation. Lastly, we find that insulin-regulated phosphorylation of the pre-catalytic spliceosome complex is associated with acute alternative splicing events in the transcriptome of human skeletal muscle. Our findings highlight the temporal relevance of protein phosphorylations and suggest that synchronized contributions of multiple signaling pathways form part of the circuitry for propagating information to insulin effector sites. The temporal regulation of intracellular insulin signaling is not well studied. Here the authors conducted a time-resolved analysis of the global insulin-regulated phosphoproteome in human muscle cells, revealing synchronized signaling pathways for propagating information to insulin effector sites. [ABSTRACT FROM AUTHOR]

The epithelial-mesenchymal transition (EMT) is a biological process whereby epithelial cells are transformed into cells with a mesenchymal phenotype. The transcription factor, X-box binding protein 1 splicing variant (XBP1s) is a key regulator of the endoplasmic reticulum stress response (ERS); but the function of XBP1s in the endometritis-induced EMT process remains unclear. Here we found that uterine tissues from goats with endometritis exhibited an EMT phenotype, with a significant decrease in the epithelial cell polarity marker E-cadherin and a significant increase in the mesenchymal markers N-cadherin and vimentin. We also found that sustained LPS treatment induced EMT in goat endometrial epithelial cells (gEECs), along with ERS and XBP1s overexpression. XBP1s KO significantly inhibited LPS-induced EMT and migration in gEECs, while XBP1s overexpression showed the opposite result. CUT & Tag experiments performed on XBP1s revealed that MAP3K2 was a downstream target gene for XBP1s regulation. We also found that expression of MAP3K2 was positively correlated with XBP1s expression in uterine tissues of goats with endometritis and in gEECs. Assays for dual luciferase reporter and molecular docking indicated that XBP1s protein regulated the transcription of MAP3K2 by modulating promoter activity. The knockdown of MAP3K2 expression significantly inhibited the migration and EMT of gEECs. XBP1s and MAP3K2 significantly promoted phosphorylation of p38 and ERK, activating the MAPK/ERK pathway. Treatment with the MAPK/ERK inhibitor, PD98059, reversed the effects of XBP1s and MAP3K2 overexpression on LPS-induced EMT. The MAPK/ERK activator, DHC, reversed the effects of XBP1s KO and MAP3K2 KD on EMT. [ABSTRACT FROM AUTHOR]

Isoform sequencing (Iso-Seq) uses long-read technology to produce highly accurate full-length reads of mRNA transcripts. Visualization of individual mRNA molecules can reveal new details of transcript variation within understudied portions of mRNA, such as the 5′ untranslated region (UTR). Differential 5′ UTRs may contain motifs, upstream open reading frames (uORFs), and secondary structures that can serve to regulate translation or further indicate changes in promoter usage, where transcriptional control may impact protein expression levels. To begin to explore isoform variation during T-cell activation, we generated the first Iso-Seq reference transcriptome of activated human CD4 T cells. Within this dataset, we discovered many novel splice- and end-variant transcripts. Remarkably, one in every eight genes expressed in our dataset was found to have a notable proportion of transcripts with 5′ UTR lengthened by over 100 bp compared to the longest corresponding UTR within the Gencode dataset. Among these end-variant transcripts, two novel isoforms were identified for CXCR5, a chemokine receptor associated with T follicular helper cell (Tfh) function and differentiation. When investigated in a model cell system, these lengthened UTR conferred reduced transcript stability and, for one of these isoforms, short uORFs introduced by the added length altered protein expression kinetics. This study highlights instances in which current reference databases are incomplete relative to the information obtained by long-read sequencing of intact mRNA. Iso-Seq is thus a promising approach to better understanding the plasticity of promoter usage, alternative splicing, and UTR sequences that influence RNA stability and translation efficiency. [ABSTRACT FROM AUTHOR]

The cellular concentrations of splicing factors (SFs) are critical for controlling alternative splicing. Most serine and arginine-enriched (SR) protein SFs regulate their own concentration via a homeostatic feedback mechanism that involves regulation of inclusion of non-coding 'poison exons' (PEs) that target transcripts for nonsense-mediated decay. The importance of SR protein PE splicing during animal development is largely unknown despite PE ultra-conservation across animal genomes. To address this, we used mouse genetics to disrupt an ultra-conserved PE in the Tra2b gene encoding the SR protein Tra2β. Focussing on germ cell development, we found that Tra2b PE deletion causes azoospermia due to catastrophic cell death during meiotic prophase. Failure to proceed through meiosis was associated with increased Tra2b expression sufficient to drive aberrant Tra2β protein hyper-responsive splice patterns. Although critical for meiotic prophase, Tra2b PE deletion spared earlier mitotically active germ cells, even though these still required Tra2b gene function. Our data indicate that PE splicing control prevents the accumulation of toxic levels of Tra2β protein that are incompatible with meiotic prophase. This unexpected connection with male fertility helps explain Tra2b PE ultra-conservation and indicates the importance of evaluating PE function in animal models. Synopsis: A non-coding "poison exon" (PE) in the Tra2b gene contains a premature termination codon and reduces Tra2β protein expression via a homeostatic feedback control pathway: at increased levels, Tra2β splicing factor binds to the poison exon and promotes its inclusion. This study shows that such regulation is essential for male gametogenesis, explaining the high conservation of this exon across vertebrate genomes. SR family splicing factor gene transcripts show high poison exon inclusion in the testis. Tra2b poison exon deletion leads to male infertility due to germ cell death at meiotic prophase. Loss of Tra2b poison exon leads to Tra2β accumulation and aberrant Tra2β-dependent splicing patterns. Fine-tuning of Tra2β splicing factor expression via conserved poison exon splicing is essential for meiosis. [ABSTRACT FROM AUTHOR]

The motor neuron disease amyotrophic lateral sclerosis (ALS) is a devastating condition with limited treatment options. The past few years have witnessed a ramping up of translational ALS research, offering the prospect of disease-modifying therapies. Although breakthroughs using gene-targeted approaches have shown potential to treat patients with specific disease-causing mutations, the applicability of such therapies remains restricted to a minority of individuals. Therapies targeting more general mechanisms that underlie motor neuron pathology in ALS are therefore of considerable interest. ALS pathology is associated with disruption to a complex array of key cellular pathways, including RNA processing, proteostasis, metabolism and inflammation. This Review details attempts to restore cellular homeostasis by targeting these pathways in order to develop effective, broadly-applicable ALS therapeutics. This Review explores several key dysregulated pathways in ALS (RNA processing, proteostasis, metabolism and inflammation) as well as evolving efforts to develop effective therapeutics to restore cellular homeostasis. The authors also detail strategies that are likely to be required to improve clinical studies moving forwards. Key points: Amyotrophic lateral sclerosis (ALS) is a complex clinico-pathological syndrome with multiple pathways contributing to disease pathogenesis that are potentially suitable for therapeutic targeting. RNA-binding protein mislocalisation results in changes to RNA splicing, particularly cryptic exon inclusion, which can be therapeutically targeted by antisense oligonucleotides. Improving protein homeostasis, from folding to transport to degradation, can decrease protein aggregates. Boosting cellular bioenergetics can support neuronal health by correcting dysregulation of metabolic pathways, including mitochondrial dysfunction. Targeting neuronal and/or systemic inflammation has the potential to confer neuroprotection. A combination of therapies is likely to be required to address ALS pathogenesis using a personalized medicine approach. [ABSTRACT FROM AUTHOR]

Schematic regulatory network of the HnRNPR-mediated splicing variant UPF3B-S that promotes HCC cell invasion and migration by exhausting CDH1 mRNA. [Display omitted] • UPF3B-S is elevated in HCC compared to adjacent liver tissues, with higher expression in invasive HCC than noninvasive HCC. • UPF3B-S is a promising prognostic biomarker and predictive metric for the invasive phenotype of HCC. • HnRNPR binds UPF3B pre-mRNA, causing exon 8 skipping and generating truncated splice variant UPF3B-S. • UPF3B-S enhances HCC invasiveness and migration via modulation of the Hippo/YAP/BIRC5 signaling pathway. ·• UPF3B-S degrades CDH1 mRNA by targeting its 3'-UTR, thereby impeding the phosphorylation of LATS1 and YAP1. Abnormal alternative splicing (AS) contributes to aggressive intrahepatic invasion and metastatic spread, leading to the high lethality of hepatocellular carcinoma (HCC). This study aims to investigate the functional implications of UPF3B-S (a truncated oncogenic splice variant) in HCC metastasis. Basescope assay was performed to analyze the expression of UPF3B-S mRNA in tissues and cells. RNA immunoprecipitation, and in vitro and in vivo models were used to explore the role of UPF3B-S and the underlying mechanisms. We show that splicing factor HnRNPR binds to the pre-mRNA of UPF3B via its RRM2 domain to generate an exon 8 exclusion truncated splice variant UPF3B-S. High expression of UPF3B-S is correlated with tumor metastasis and unfavorable overall survival in patients with HCC. The knockdown of UPF3B-S markedly suppresses the invasive and migratory capacities of HCC cells in vitro and in vivo. Mechanistically, UPF3B-S protein targets the 3′-UTR of CDH1 mRNA to enhance the degradation of CDH1 mRNA, which results in the downregulation of E-cadherin and the activation of epithelial–mesenchymal transition. Overexpression of UPF3B-S enhances the dephosphorylation of LATS1 and the nuclear accumulation of YAP1 to trigger the Hippo signaling pathway. Our findings suggest that HnRNPR-induced UPF3B-S promotes HCC invasion and metastasis by exhausting CDH1 mRNA and modulating YAP1-Hippo signaling. UPF3B-S could potentially serve as a promising biomarker for the clinical management of invasive HCC. [ABSTRACT FROM AUTHOR]

Human genetic studies have nominated cadherin-like and PC-esterase domain-containing 1 (CPED1) as a candidate target gene mediating bone mineral density (BMD) and fracture risk heritability. Recent efforts to define the role of CPED1 in bone in mouse and human models have revealed complex alternative splicing and inconsistent results arising from gene targeting, making its function in bone difficult to interpret. To better understand the role of CPED1 in adult bone mass and morphology, we conducted a comprehensive genetic and phenotypic analysis of cped1 in zebrafish, an emerging model for bone and mineral research. We analyzed two different cped1 mutant lines and performed deep phenotyping to characterize more than 200 measures of adult vertebral, craniofacial, and lean tissue morphology. We also examined alternative splicing of zebrafish cped1 and gene expression in various cell/tissue types. Our studies fail to support an essential role of cped1 in adult zebrafish bone. Specifically, homozygous mutants for both cped1 mutant alleles, which are expected to result in loss-of-function and impact all cped1 isoforms, exhibited no significant differences in the measures examined when compared to their respective wildtype controls, suggesting that cped1 does not significantly contribute to these traits. We identified sequence differences in critical residues of the catalytic triad between the zebrafish and mouse orthologs of CPED1, suggesting that differences in key residues, as well as distinct alternative splicing, could underlie different functions of CPED1 orthologs in the two species. Our studies fail to support a requirement of cped1 in zebrafish bone and lean tissue, adding to evidence that variants at 7q31.31 can act independently of CPED1 to influence BMD and fracture risk. [ABSTRACT FROM AUTHOR]

Background: The clinical manifestations of PI4KA-related disorders are characterized by considerable variability, predominantly featuring neurological impairments, gastrointestinal symptoms, and a combined immunodeficiency. The aim of this study was to delineate the novel spectrum of PI4KA variants detected prenatally and to assess their influence on fetal development. Methods: A thorough fetal ultrasound screening was conducted, supplemented by both antenatal and post-abortion magnetic resonance imaging (MRI) studies. Novel PI4KA variants were detected through clinical Whole exon sequencing (WES) and validated by Sanger sequencing. The functional consequences of these variants were evaluated using bioinformatics tools. The effects of the identified variants on splicing were analyzed through minigene splicing assays. Subsequently, both wild-type and mutant PI4KA protein fragments were purified, and their enzymatic activities were quantitatively assessed. Results: Ultrasound imaging, MRI scans revealed a dilated small intestine with an obstruction. Compound heterozygous variants (NM_058004.3: c.2802_2863-40del and c.2819 C > T, p.Ala940Val) were identified in the PI4KA of the affected fetus through clinical trio-WES. Both variants were predicted deleterious. The PI4KA variant c.2802_2863-40del resulted in the production of three distinct mRNA isoforms. The PI4KA variant c.2819 C > T (p.Ala940Val) significantly reduced the enzyme activity. Conclusions: This study extended the mutational spectrum of PI4KA and may provide guidance for genetic counseling. Functional studies confirmed that the identified variant induces alterations in RNA splicing and impairs enzyme activity. [ABSTRACT FROM AUTHOR]

A bidirectional nucleus-mitochondria communication is essential for homeostasis and stress. By acting as critical molecules, the nuclear-encoded lncRNAs (nulncRNAs) have been implicated in the nucleus-to-mitochondria anterograde regulation. However, role of mitochondrial-derived lncRNAs (mtlncRNAs) in the mitochondria-to-nucleus retrograde regulation remains elusive. Here, we identify functional implication of the mtlncRNAs MDL1AS, lncND5 and lncCyt b in retrograde regulation. Mediated by HuR and PNPT1 proteins, the mtlncRNAs undergo a mitochondria-to-nucleus traveling and then regulate a network of nuclear genes. Moreover, as an example of the functional consequence, we showed that the nuclear-translocated lncCyt b cooperates with the splicing factor hnRNPA2B1 to influence several aspects of cell metabolism including glycolysis, possibly through their regulatory effect on the post-transcriptional processing of related nuclear genes. This study advances our knowledge in mitochondrial biology and provides new insights into the role of mtlncRNAs in mitochondria-nucleus communications. Author summary: Due to the essential role of the nucleus-mitochondria crosstalk in homeostasis and stress, identifying and characterizing critical molecules that contribute to the nucleus-to-mitochondria (anterograde) and mitochondria-to-nucleus (retrograde) communication is fundamental to human biology. In mammals, mitochondria are the only organelles that possess their own genetic material (mtDNA), which produces a set of lncRNAs (mitochondrial-derived lncRNAs, mtlncRNAs). Many lncRNAs encoded by nuclear genome (nuclear-encoded lncRNAs, nulncRNAs) have been involved in the regulation of mitochondrial function. Nevertheless, whether and how the mtlncRNAs operate in the retrograde regulation signaling to modulate nuclear events remains largely unclear. Here, we identify that the mtlncRNAs MDL1AS, lncND5 and lncCyt b undergo a mitochondria-to-nucleus traveling and then regulate a network of nuclear genes, and describe an example of how the nuclear-translocated lncCyt b modulates nuclear genes at the post-transcriptional level via its binding partner, the splicing factor hnRNPA2B1, to influence several aspects of cell metabolism including glycolysis, thereby acting as a critical regulatory molecule in retrograde signaling. Our study illustrates that certain mtlncRNAs, on the basis of their nuclear translocation, can operate in the mitochondria-to-nucleus retrograde regulation, advancing knowledge in mitochondrial biology as well as our understanding of implication of mtlncRNAs in the regulation of inter-compartmental crosstalk in cells. [ABSTRACT FROM AUTHOR]

Black men suffer disproportionately from prostate cancer (PCa) compared to men of other races and ethnicities. Comparing the molecular landscape of PCa among Black and White patients has the potential to identify targets for development of new precision medicine interventions. Herein, we conducted transcriptomic analysis of prostate tumors and paired tumor-adjacent normals from self-reported Black and White PCa patients and estimated patient genetic ancestry. Clinical follow-up revealed increased biochemical recurrence (BCR) among Black patients compared to White patients with high-grade PCa. Transcriptomic analysis identified differential alternative RNA splicing events (ARSs) between Black and White PCa patients. Genes undergoing genetic ancestry-concordant ARSs in high-grade or low-grade tumors involved cancer promoting genes. Most genes undergoing genetic ancestry-concordant ARSs did not exhibit differential aggregate gene expression or alternative polyadenylation. A number of the genetic ancestry-concordant ARSs associated with BCR; thus, genetic ancestry-concordant RNA splice variants may represent unique targets for PCa precision oncology. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) in eukaryotic RNA is an epigenetic modification that is critical for RNA metabolism, gene expression regulation, and the development of organisms. Aberrant expression of m6A components appears in a variety of human diseases. RNA m6A modification in Drosophila has proven to be involved in sex determination regulated by Sxl and may affect X chromosome expression through the MSL complex. The dosage-related effects under the condition of genomic imbalance (i.e. aneuploidy) are related to various epigenetic regulatory mechanisms. Here, we investigated the roles of RNA m6A modification in unbalanced genomes using aneuploid Drosophila. The results showed that the expression of m6A components changed significantly under genomic imbalance, and affected the abundance and genome-wide distribution of m6A, which may be related to the developmental abnormalities of aneuploids. The relationships between methylation status and classical dosage effect, dosage compensation, and inverse dosage effect were also studied. In addition, we demonstrated that RNA m6A methylation may affect dosage-dependent gene regulation through dosage-sensitive modifiers, alternative splicing, the MSL complex, and other processes. More interestingly, there seems to be a close relationship between MSL complex and RNA m6A modification. It is found that ectopically overexpressed MSL complex, especially the levels of H4K16Ac through MOF, could influence the expression levels of m6A modification and genomic imbalance may be involved in this interaction. We found that m6A could affect the levels of H4K16Ac through MOF, a component of the MSL complex, and that genomic imbalance may be involved in this interaction. Altogether, our work reveals the dynamic and regulatory role of RNA m6A modification in unbalanced genomes, and may shed new light on the mechanisms of aneuploidy-related developmental abnormalities and diseases. [ABSTRACT FROM AUTHOR]

Proteins that bind to DNA/RNA are typically evolutionarily conserved with multiple regulatory functions in transcription initiation, mRNA translation, stability of RNAs, and RNA splicing. Therefore, dysregulation of DNA/RNA binding proteins such as purine-rich element binding protein alpha (PURα) disrupts signaling transduction and often leads to human diseases including cancer. PURα was initially recognized as a tumor suppressor in acute myeloid leukemia (AML) and prostate cancer (PC). Most recently, several studies have revealed that PURα is dysregulated in multiple cancers, such as breast cancer (BC) and esophageal squamous cell carcinoma (ESCC). The oncogenic or tumor-suppressive functions of PURα are realized via regulating RNA/protein interaction, mRNA translation, formation of stress granules (SGs), and transcriptional regulation of several oncogenes and tumor suppressors. Although DNA/RNA binding proteins are hardly targeted, novel strategies have been applied to identify compounds targeting PURα and have demonstrated promising anti-tumor efficacy in the preclinical study. The present review summarizes the most recently discovered critical roles of PURα in various cancer types, providing an overview of the biomarker and therapeutic target potential of PURα for patients with cancer. [ABSTRACT FROM AUTHOR]

Split inteins are biological mechanisms for the operation of the spatiotemporal control of protein activities. They function through protein trans-splicing, in which their N- and C-terminal fragments are expressed contiguously with two protein halves. The subsequent self-excision upon recognition of the complimentary fragment yields a mature, complete, and functional protein. The conditional regulation of protein splicing through environmental factors or the attachment of regulatory modules can be used to determine when and where a protein will operate, providing potential novel approaches for engineering biology applications. This review will discuss current split intein applications and the mechanistic basis for novel species classification. These considerations can provide guidance in intein and extein engineering through activation strategies, in the design of spatial arrangements, and in taking advantage of unique reaction environments. This can pave the way for the future implementation of novel split intein discoveries and the selection of appropriate intein species and aid in designing novel protein engineering strategies. [ABSTRACT FROM AUTHOR]

Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and extensive ITK mRNA alternative splicing. We further introduce the most comprehensive list to date of proteins involved in splicing and leverage cysteine- and protein-directed activity-based protein profiling with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry. [Display omitted] • Electrophilic probe suppresses T cell activation by targeting a core splicing factor • Inhibition of SF3B1 leads to T cell state-dependent decrease in ITK protein levels • Protein-directed TMT-ABPP is a valuable complementary chemical proteomic approach • Chemical proteomics reveals broad potential for covalent targeting of splicing There is a growing appreciation that alternative splicing can play distinct roles in immune cell pathophysiology. 1,2,3 However, our understanding of the existing opportunities for targeting of splicing in the context of immunopathologies is limited. Here we report a small-molecule covalent electrophile that inhibits T cell activation and induces a T cell state-dependent loss of an important immune kinase, ITK, by targeting a major splicing factor SF3B1. The lack of observed cytotoxicity or gross protein expression changes associated with inhibition of splicing suggests that targeting of splicing could be a viable therapeutic strategy in immune-related disorders. To further investigate this opportunity, we generated the most comprehensive to date list of splicing factors and splicing regulators, which can be targeted with small molecule covalent electrophiles based on peptide-level and protein-level TMT-ABPP experiments with scout fragments. This approach produced a valuable community resource, aimed at facilitating future target follow up and ligand development efforts. Scott et al. perform mechanism-of-action studies of an acrylamide stereoprobe, which lead to the discovery of an unanticipated mechanism of suppression of T cell activation via inhibition of a core splicing factor (SF3B1). Evaluation of splicing factor ligandability using electrophilic scout fragments suggests a broad potential for targeting splicing. [ABSTRACT FROM AUTHOR]

Thioredoxin z (TRX z) plays a significant role in chloroplast development by regulating the transcription of chloroplast genes. In this study, we identified a pentatricopeptide repeat (PPR) protein, rice albino seedling-lethal (RAS), that interacts with OsTRX z. This interaction was initially discovered by using a yeast two-hybrid (Y2H) screening technique and was further validated through Y2H and bimolecular fluorescence complementation (BiFC) experiments. RAS contains 16 PPR motifs and features a small MutS-related (SMR) domain at its C-terminus. CRISPR/Cas9-generated ras mutants exhibited an albino seedling-lethal phenotype characterized by abnormal chloroplast structures and a significantly reduced chlorophyll content. RAS localizes to the chloroplast and is predominantly expressed in young leaves. Mutations in RAS affect RNA editing at the rpl2, rps14, and ndhA sites, as well as RNA splicing at the rpl2, atpF, and ndhA transcripts within the chloroplast. Furthermore, the expression levels of genes associated with chloroplast formation are altered in the ras mutant. Both OsTRX z and RAS were found to interact with chloroplast signal recognition particle (cpSRP) proteins, indicating that their proper localization within the chloroplast may be dependent on the SRP pathway. Collectively, our findings highlight the critical role of RAS in chloroplast development, as it is involved in RNA processing and the regulation of chloroplast gene expression. [ABSTRACT FROM AUTHOR]

ABSTRACT Arthrogryposis multiplex congenita (AMC) is a heterogeneous disorder associated with 1/3000 to 1/5000 live births. We report a consanguineous family with multiple affected members with AMC and identified a recessive mutation in the highly conserved splice donor site, resulting in the mis‐splicing of the affected exons. SENP7 is a deSUMOylase that is critical for sarcomere assembly and skeletal muscle contraction by regulating the transcriptional program in the skeletal muscle. This is a reported case of an affected family with a noncoding, splice site SENP7 variant, expanding the spectrum of SENP7 as a causative gene in rare cases of lethal AMC. [ABSTRACT FROM AUTHOR]

The cell cycle regulator Aurora-A kinase presents an attractive target for cancer therapies, though its inhibition is also associated with toxic side effects. To gain a more nuanced understanding of Aurora-A function, we applied shotgun proteomics to identify 407 specific protein partners, including several splicing factors. Supporting a role in alternative splicing, we found that Aurora-A localizes to nuclear speckles, the storehouse of splicing proteins. Aurora-A interacts with and phosphorylates splicing factors both in vitro and in vivo, suggesting that it regulates alternative splicing by modulating the activity of these splicing factors. Consistently, Aurora-A inhibition significantly impacts the alternative splicing of 505 genes, with RNA motif analysis revealing an enrichment for Aurora-A interacting splicing factors. Additionally, we observed a significant positive correlation between the splicing events regulated by Aurora-A and those modulated by its interacting splicing factors. An interesting example is represented by CLK1 exon 4, which appears to be regulated by Aurora-A through SRSF3. Collectively, our findings highlight a broad role of Aurora-A in the regulation of alternative splicing. [ABSTRACT FROM AUTHOR]

Farnesoid X receptor a (FXRa, NR1H4) is a bile acidactivated nuclear receptor that regulates the expression of glycolytic and lipogenic target genes by interacting with the 9-cis-retinoic acid receptor a (RXRa, NR2B1). Along with cofactors, the FXRa proteins reported thus far in humans and rodents have been observed to regulate both isoform (a1-4)- and tissue-specific gene expression profiles to integrate energy balance and metabolism. Here, we studied the biological functions of an FXRa naturally occurring spliced exon 5 isoform (FXRase5) lacking the second zinc-binding module of the DNA-binding domain. We demonstrate spliced exon 5 FXRa expression in all FXRa-expressing human and mouse tissues and cells, and that it is unable to bind to its response element or activate FXRa dependent transcription. In parallel, this spliced variant displays differential interaction capacities with its obligate heterodimer partner retinoid X receptor a that may account for silencing of this permissive dimer for signal transduction. Finally, deletion of exon 5 by gene edition in HepG2 cells leads to FXRa loss-of-function, increased expression of LRH1 metabolic sensor and CD36 fatty acid transporter in conjunction with changes in glucose and triglycerides homeostasis. Together, these findings highlight a novel mechanism by which alternative splicing may regulate FXRa gene function to fine-tune adaptive and/or metabolic responses. This finding deepens our understanding on the role of splicing events in hindering FXRa activity to regulate specific transcriptional programs and their contribution in modifying energy metabolism in normal tissues and metabolic diseases. [ABSTRACT FROM AUTHOR]

• SPL84 is an ASO developed for the treatment of people with CF. • SPL84 was tested in a blinded, placebo-controlled phase 1 study in thirty two (32) healthy volunteers (HVs). • There were no serious adverse events (SAEs) in the study, and there were no significant adverse events. • The systemic exposure to SPL84 was low and tended to be dose dependent. • SPL84 was safe and well-tolerated when administered to HVs at doses up to 160 mg, importantly, no significant effect on pulmonary function. Antisense Oligonucleotides (ASOs) are small synthetic nucleic acid molecules able to bind specific sequences within target Ribonucleic Acid (RNA) molecules. SPL84 is an ASO drug developed for treatment of cystic fibrosis (CF) patients carrying the 3849 + 10 kb C->T Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) splicing mutation. The 3849 + 10 kb C->T variant leads to inclusion of cryptic exon harboring stop codon leading to the production of truncated non-functional CFTR proteins. in vitro , SPL84 treatment results in splicing modulation, which leads to an increase of correctly spliced CFTR RNA and higher levels of functional CFTR proteins. SPL84 was tested in a blinded, placebo-controlled phase 1 study in thirty two (32) healthy volunteers (HVs), each received a single dose of either SPL84 or placebo by inhalation. A total of 8 participants were randomized to each of the 4 escalating cohorts in a 3:1 ratio (active: placebo). Safety and tolerability were evaluated by monitoring adverse events (AEs), vital signs, physical exam findings, spirometry, electrocardiograms (ECG), and analyses of safety laboratories. Blood samples were obtained periodically over 24 h for measurement of systemic exposure. There were no significant changes from baseline in vital signs, clinical laboratory values, ECG, physical examination, or pulmonary function. There were no Serious Adverse Events (SAEs) in the study, and there were no significant adverse events. The systemic exposure to SPL84 was low and tended to be dose dependent. The exposure, expressed in terms of area under the curve to infinity (AUC inf), at the no observed adverse effect level (NOAEL) in 9-week toxicological mice study was 7.51 µg/ml*hrs, which is ∼20 times higher than the exposure at the 160 mg dose (444 ng/ml*hrs). SPL84 was safe and well-tolerated when administered as a single inhaled dose to HVs at doses up to 160 mg, with minimal systemic exposure. There were no safety issues observed, no SAEs, no significant related AEs, and, importantly, no significant effect on pulmonary function. The successful completion of the study enabled the initiation of multi-dosing of CF patients in a phase 2 clinical study. [ABSTRACT FROM AUTHOR]

The discovery of tumor-derived neoantigens which elicit an immune response through major histocompatibility complex (MHC-I/II) binding has led to significant advancements in immunotherapy. While many neoantigens have been discovered through the identification of non-synonymous mutations, the rate of these is low in some cancers, including head and neck squamous cell carcinoma. Therefore, the identification of neoantigens through additional means, such as aberrant splicing, is necessary. To achieve this, we developed the splice isoform neoantigen evaluator (SINE) pipeline. Our tool documents peptides present on spliced or inserted genomic regions of interest using Patient Harmonic-mean Best Rank scores, calculating the MHC-I/II binding affinity across the complete human leukocyte antigen landscape. Here, we found 125 potentially immunogenic events and 9 principal binders in a cohort of head and neck cancer patients where the corresponding wild-type peptides display no MHC-I/II affinity. Further, in a melanoma cohort of patients treated with anti-PD1 therapy, the expression of immunogenic splicing events identified by SINE predicted response, potentially indicating the existence of immune editing in these tumors. Overall, we demonstrate SINE's ability to identify clinically relevant immunogenic neojunctions, thus acting as a useful tool for researchers seeking to understand the neoantigen landscape from aberrant splicing in cancer. [ABSTRACT FROM AUTHOR]

Alternative splicing plays a fundamental role in gene expression and protein complexity. Aberrant splicing impairs cell homeostasis and is closely associated with aging and cellular senescence. Significant changes to alternative splicing, including dysregulated splicing events and the abnormal expression of splicing factors, have been detected during the aging process or in age-related disorders. Here, we highlight the possibility of suppressing aging and cellular senescence by controlling alternative splicing. In this review, we will summarize the latest research progress on alternative splicing in aging and cellular senescence, discuss the roles and regulatory mechanisms of alternative splicing during aging, and then excavate existing and potential approaches to anti-aging by controlling alternative splicing. Novel therapeutic breakthroughs concerning aging and senescence entail a further understanding of regulating alternative splicing mechanically and accurately. [ABSTRACT FROM AUTHOR]

Genetic analysis is important for modern plant molecular biology, and in this regard, the existence of specific mutants is crucial. While genome editing technologies, particularly CRISPR-Cas9, have revolutionized plant molecular biology by enabling precise gene disruption, knockout methods are ineffective for lethal genes, necessitating alternatives like gene knockdown. This study demonstrates the practical generation of a hypomorphic mutant allele, alongside severe null mutant alleles, via the targeting of mRNA splicing sites using CRISPR-Cas9. The Arabidopsis HIGH PLOIDY 2 (HPY2) encodes a yeast NSE2 ortholog, part of the conserved eukaryotic SMC5/6 complex, with SUMO E3 ligase activity essential for cell cycle progression and plant development. Loss-of-function HPY2 mutants exhibit severe dwarfism and seedling lethality, making functional analysis challenging. To overcome these limitations, we created HPY2 knockdown mutants as novel tools to investigate gene function. Of the three mutant alleles, the hpy2-cr1 and hpy2-cr2 mutants resembled the existing severe hpy2-1 allele, both harboring a single base pair insertion in one exon, causing significant root shortening and seedling lethality. In contrast, the hypomorphic mutant hpy2-cr3, which has a five bp deletion at an intron-exon junction, showed relatively longer root growth and survived until the reproductive stage. RT-PCR analysis of hpy2-cr3 revealed atypical mRNAs producing truncated polypeptides that retained some HPY2 function, explaining the milder phenotype. These results establish the successful generation of novel hypomorphic mutant alleles critical for studying the lethal gene HPY2, and demonstrate the usefulness of CRISPR-Cas9 for producing viable hypomorphic mutants for investigating complex genetic interactions. [ABSTRACT FROM AUTHOR]

Phosphatase and tensin homologue (PTEN) is the main antagonist of the phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR signalling pathway and mutated in 10%–20% of individuals with autism spectrum disorder (ASD) exhibiting macrocephaly. Hyperactive mTOR signalling is responsible for some aspects during PTEN-ASD progression, e.g. neuronal hypertrophy and -excitability, but PI3K/mTOR-independent processes have additionally been described. There is emerging evidence that PTEN regulates gene transcription, spliceosome formation and pre-mRNA splicing independently of PI3K/mTOR. Altered splicing is a hallmark of brains from individuals with idiopathic and PTEN-ASD, however, molecular mechanisms are yet to be identified. We performed RNA-sequencing (RNA-Seq), followed by analysis of altered transcript splicing in Pten -deficient primary cortical mouse neurons, which we compared with published data from PTEN -deficient human neuronal stem cells. This analysis identified that transcripts were globally mis-spliced in a developmentally regulated fashion and cluster in synaptic and gene expression regulatory processes. Strikingly, splicing defects following Pten -deficiency represent a significant number of other known ASD-susceptibility genes. Furthermore, we show that exons with strong 3′ splice sites are more frequently mis-spliced under Pten -deficient conditions. Our study indicates that PTEN-ASD is a multifactorial condition involving the dysregulation of other known ASD-susceptibility genes. [ABSTRACT FROM AUTHOR]

Elucidating the gene regulatory mechanisms underlying the gut–brain axis is critical for uncovering novel gut–brain interaction pathways and developing therapeutic strategies for gut bacteria-associated neurological disorders. Most studies have primarily investigated how gut bacteria modulate host epigenetics and gene expression; their impact on host alternative splicing, particularly in the brain, remains largely unexplored. Here, we investigated the effects of the gut-associated probiotic Lacidofil® on alternative splicing across 10 regions of the rat brain using published RNA-sequencing data. The Lacidofil® altogether altered 2941 differential splicing events, predominantly, skipped exon (SE) and mutually exclusive exon (MXE) events. Protein–protein interactions and a KEGG analysis of differentially spliced genes (DSGs) revealed consistent enrichment in the spliceosome and vesicle transport complexes, as well as in pathways related to neurodegenerative diseases, synaptic function and plasticity, and substance addiction across brain regions. Using the PsyGeNET platform, we found that DSGs from the locus coeruleus (LConly), medial preoptic area (mPOA), and ventral dentate gyrus (venDG) were enriched in depression-associated or schizophrenia-associated genes. Notably, we highlight the App gene, where Lacidofil® precisely regulated the splicing of two exons causally involved in amyloid β protein-based neurodegenerative diseases. Although the splicing factors exhibited both splicing plasticity and expression plasticity in response to Lacidofil®, the overlap between DSGs and differentially expressed genes (DEGs) in most brain regions was rather low. Our study provides novel mechanistic insight into how gut probiotics might influence brain function through the modulation of RNA splicing. [ABSTRACT FROM AUTHOR]

Background 17α-hydroxylase/17,20-lyase deficiency (17-OHD) is a rare subtype of congenital adrenal hyperplasia caused by homozygous or compound heterozygous pathogenic variants in the CYP17A1 gene. Purpose This study aimed to identify and characterize pathogenic variants in individuals with 17-OHD and to classify and validate the pathogenicity of novel variants. Methods Variants were identified via targeted long-read sequencing (TLRS) of the entire CYP17A1 gene in enrolled 17-OHD patients. The American College of Medical Genetics and Genomics (ACMG) guidelines were employed to assess the pathogenicity of novel variants. Minigene splicing assays were utilized to determine the impact of variants on RNA splicing. Results This study encompassed 26 patients with 17-OHD, detecting 2 trans pathogenic variants per patient using the TLRS method. A total of 20 pathogenic variants in the CYP17A1 were identified, with variant c.985_987delinsAA being the most frequent (28/52 alleles), followed by variant c.1459_1467del (4/52 alleles). Five novel variants including c.280T > C, c.470T > A, c.636_637del, c.866A > G, and c.1095del, were classified as pathogenic/likely pathogenic ones according to ACMG criteria. The minigene splicing assays revealed c.866A > G in exon 5 causes a frameshift due to a 104 base pair deletion, while c.470T > A generates 2 transcripts, with the vast majority spliced like the wild-type and a small fraction lacking 35 base pairs in the 5′ flank of exon 3. Conclusion The TLRS can determine the cis/trans orientation of 2 distant variants. Five novel pathogenic variants were reported, broadening the spectrum of the CYP17A1 pathogenic variants. The variant c.866A > G, located deep in the exon, affects gene function through mechanisms of aberrant splicing. [ABSTRACT FROM AUTHOR]

X-linked hypophosphatemic rickets (XLH), the most common form of hereditary rickets, is characterized by renal phosphate wasting and abnormal vitamin D metabolism due to elevated circulating levels of the phosphatonin fibroblast growth factor 23 (FGF23). Dominant inactivating variants of the phosphate regulating endopeptidase homolog, X-linked (PHEX), gene are present in patients with XLH, and more than half of affected patients carry de novo variants. We report on 3 families in whom affected members had highly unusual PHEX pathogenic variants. In 1 family we identified a previously described deep intronic PHEX variant (c.1768 + 173A>G) in the proband and her affected son. This variant is also near a previously reported PHEX variant (c.1768 + 177_1768 + 180dupGTAA) and is predicted to affect splicing by SpliceAI (delta score: 0.95) through creation of a new donor splice site. In a second proband we identified 2 pathogenic de novo and novel PHEX variants, c.2083delT (p.Ser695Profs*45) and c.2085delC (p.Tyr696Thrfs*44), that were present on different alleles, consistent with mosaicism for 3 PHEX alleles. The third proband also carried 2 PHEX variants (c.755 T>C [p.Phe252Ser] and c.759G>A [p.Met253Ile]), but in this case both variants were present on the same PHEX allele. These studies expand the molecular catalog of pathogenic PHEX variants in XLH and emphasize the importance of deep intronic sequencing and comprehensive family studies. Conventional approaches to genetic diagnosis may not be adequate to identify or characterize the disease-causing variants in the PHEX gene in some patients with likely XLH. [ABSTRACT FROM AUTHOR]

TAR DNA-binding protein 43 (TDP-43) has emerged as a critical player in neurodegenerative disorders, with its dysfunction implicated in a wide spectrum of diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and Alzheimer's disease (AD). This comprehensive review explores the multifaceted roles of TDP-43 in both physiological and pathological contexts. We delve into TDP-43's crucial functions in RNA metabolism, including splicing regulation, mRNA stability, and miRNA biogenesis. Particular emphasis is placed on recent discoveries regarding TDP-43's involvement in DNA interactions and chromatin dynamics, highlighting its broader impact on gene expression and genome stability. The review also examines the complex pathogenesis of TDP-43-related disorders, discussing the protein's propensity for aggregation, its effects on mitochondrial function, and its non-cell autonomous impacts on glial cells. We provide an in-depth analysis of TDP-43 pathology across various neurodegenerative conditions, from well-established associations in ALS and FTLD to emerging roles in diseases such as Huntington's disease and Niemann-Pick C disease. The potential of TDP-43 as a therapeutic target is explored, with a focus on recent developments in targeting cryptic exon inclusion and other TDP-43-mediated processes. This review synthesizes current knowledge on TDP-43 biology and pathology, offering insights into the protein's central role in neurodegeneration and highlighting promising avenues for future research and therapeutic interventions. [ABSTRACT FROM AUTHOR]

Background: Posttranslational modifications (PTMs) play critical roles in hepatocellular carcinoma (HCC). However, the locations of PTM-modified sites across protein secondary structures and regulatory patterns in HCC remain largely uncharacterized. Methods: Total proteome and nine PTMs (phosphorylation, acetylation, crotonylation, ubiquitination, lactylation, N-glycosylation, succinylation, malonylation, and β-hydroxybutyrylation) in tumor sections and paired normal adjacent tissues derived from 18 HCC patients were systematically profiled by 4D-Label free proteomics analysis combined with PTM-based peptide enrichment. Results: We detected robust preferences in locations of intrinsically disordered protein regions (IDRs) with phosphorylated sites and other site biases to locate in folded regions. Integrative analyses revealed that phosphorylated and multiple acylated-modified sites are enriched in proteins containing RRM1 domain, and RNA splicing is the key feature of this subset of proteins, as indicated by phosphorylation and acylation of splicing factor NCL at multiple residues. We confirmed that NCL-S67, K398, and K646 cooperate to regulate RNA processing. Conclusion: Together, this proteome profiling represents a comprehensive study detailing regulatory patterns based on multiple PTMs of HCC. [ABSTRACT FROM AUTHOR]

Innate immune signaling is essential for clearing pathogens and damaged cells and must be tightly regulated to avoid excessive inflammation or autoimmunity. Here, we found that the alternative splicing of exons derived from transposable elements is a key mechanism controlling immune signaling in human cells. By analyzing long-read transcriptome datasets, we identified numerous transposon exonization events predicted to generate functional protein variants of immune genes, including the type I interferon receptor IFNAR2. We demonstrated that the transposon-derived isoform of IFNAR2 is more highly expressed than the canonical isoform in almost all tissues and functions as a decoy receptor that potently inhibits interferon signaling, including in cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our findings uncover a primate-specific axis controlling interferon signaling and show how a transposon exonization event can be co-opted for immune regulation. [Display omitted] • Long-read transcriptomes enable improved characterization of transposon exonization • Exonization generates many robustly expressed alternative isoforms of immune genes • IFNAR2-S is a primate-specific isoform of IFNAR2 that functions as a decoy receptor • Dysregulation of IFNAR2 splicing is associated with human immune disease Transposable element exonization can yield functional protein isoforms as seen for primate-specific IFNAR2. [ABSTRACT FROM AUTHOR]

Alternative splicing enhances protein diversity in different ways, including through exonization of transposable elements (TEs). Recent transcriptomic analyses identified thousands of unannotated spliced transcripts with exonizing TEs, but their contribution to the proteome and biological relevance remains unclear. Here, we use transcriptome assembly, ribosome profiling, and proteomics to describe a population of 1,227 unannotated TE exonizing isoforms generated by mRNA splicing and recurrent in human populations. Despite being shorter and lowly expressed, these isoforms are shared between individuals and efficiently translated. Functional analyses show stable expression, specific cellular localization, and, in some cases, modified functions. Exonized TEs are rich in ancient genes, whereas the involved splice sites are recent and can be evolutionarily conserved. In addition, exonized TEs contribute to the secondary structure of the emerging isoforms, supporting their functional relevance. We conclude that TE-spliced isoforms represent a diversity reservoir of functional proteins on which natural selection can act. [Display omitted] • Transposable element (TE) exonization expands the diversity of the human proteome • TE-spliced isoforms can have distinct functions compared with canonical isoforms • Exonized TEs can be evolutionarily conserved and add young sequences to ancient genes • Exonized TEs contribute to the overall secondary structure of proteins Transposable element exonization by unannotated splicing events produces stable protein isoforms with acquired functions that are subject to evolutionary selection. [ABSTRACT FROM AUTHOR]

Species-specific interferon responses are shaped by the virus-host arms race. The human interferon-induced transmembrane protein (IFITM) family consists of three antiviral IFITM genes that arose by gene duplication. These genes restrict virus entry and are key players in antiviral interferon responses. The unique IFITM repertoires in different species influence their resistance to viral infections, but the role of IFITMs in shaping the enhanced antiviral immunity of reservoir bat species is unclear. Here, we identified an IFITM gene in Chinese rufous horseshoe bat, a natural host of severe acute respiratory syndrome (SARS)-related coronaviruses, that is alternatively spliced to produce two IFITM isoforms in native cells as shown by transcriptomics. These bat IFITMs have conserved structures in vitro as demonstrated by circular dichroism spectroscopy, yet they exhibit distinct antiviral specificities against influenza A virus, Nipah virus and coronaviruses including SARS-CoV, SARS-CoV-2 and MERS-CoV. In parallel with human IFITM1-3, bat IFITM isoforms localize to distinct sites of virus entry which influences their antiviral potency. Further bioinformatic analysis of IFITM repertoires in 206 mammals reveals that alternative splicing is a recurring strategy for IFITM diversification, albeit less widely adopted than gene duplication. These findings demonstrate that alternative splicing is a key strategy for evolutionary diversification in the IFITM family. Our study also highlights an example of convergent evolution where species-specific selection pressures led to expansion of the IFITM family through multiple means, underscoring the importance of IFITM diversity as a component of innate immunity. Author summary: Zoonotic transmission occurs when viruses 'jump' from animals into human. This may lead to viral outbreaks such as the COVID-19 pandemic, posing a significant threat to public health. Bats are the origin of many zoonotic viruses as their unique immunity may allow them to carry viruses without developing disease. Interferon-induced transmembrane proteins (IFITMs) are important antiviral proteins that have been shown to influence the pathogenesis of viral infections. It is currently unclear whether IFITMs also contribute to the high viral tolerance of bats, so characterization of bat IFITMs is needed to identify factors that predispose species to act as viral reservoirs. Here, we find that the Chinese rufous horseshoe bat, a natural host of SARS-related coronaviruses, adopts a distinct strategy known as alternative splicing to functionally diversify their IFITM family. We also demonstrate that alternative splicing is a recurring strategy in the evolution of IFITMs and is evident in at least 75 mammalian species. Our study therefore provides novel insights into how epidemiologically significant species could take advantage of different evolutionary strategies to enhance their resistance to viruses. [ABSTRACT FROM AUTHOR]

Breast cancer (BCa) is a highly prevalent pathological condition (̴30% in women) with limited and subtype-dependent prognosis and therapeutic options. Therefore, BCa management might benefit from the identification of novel molecular elements with clinical potential. Since splicing process is gaining a great relevance in cancer, this work analysed the expression of multiple Spliceosome Components (SCs = 17) and Splicing Factors (SFs = 26) and found a drastic dysregulation in BCa (n = 69) vs. control (negative biopsies; n = 50) samples. Among all the components analysed, we highlight the upregulation of ESRP1 and down-regulation of PRPF8 and NOVA1 in BCa vs. control samples. Indeed, ESRP1 was specially overexpressed in triple-negative BCa (TNBCa) and associated with worse prognosis (i.e., higher BCa grade and lower overall survival), suggesting an association of ESRP1 with BCa aggressiveness. On the other hand, PRPF8 expression was generally downregulated in BCa with no associations to clinical characteristics, while NOVA1 expression was lower in TNBCa patients and highly aggressive tumours. Consistently, NOVA1 overexpression in vitro reduced functional parameters of aggressiveness in ER-/PR- cell lines (MDA-MB-231 and BT-549) but not in ER+/PR+ cells (MCF7), suggesting a critical role of NOVA1 in subtype-specific BCa. Finally, the in vitro pharmacological inhibition of splicing machinery using pladienolide B decreased aggressiveness features in all the BCa cell lines, showing a subtype-independent inhibitory potential, but being relatively innocuous in normal-like breast cells. These results demonstrate the profound dysregulation of the splicing machinery in BCa and their potential as source of promising diagnosis/prognosis markers, as well as valuable therapeutic targets for BCa. [ABSTRACT FROM AUTHOR]

Mutations that affect RNA splicing significantly impact human diversity and disease. Here we present a method using transformers, a type of machine learning model, to detect splicing from raw 45,000-nucleotide sequences. We generate embeddings with residual neural networks and apply hard attention to select splice site candidates, enabling efficient training on long sequences. Our method surpasses the leading tool, SpliceAI, in detecting splice sites in GENCODE and ENSEMBL annotations. Using extensive RNA sequencing data from an Icelandic cohort of 17,848 individuals and the Genotype-Tissue Expression (GTEx) project, our method demonstrates superior performance in detecting splice junctions compared to SpliceAI-10k (PR-AUC = 0.834 vs. PR-AUC = 0.820) and is more effective at identifying disease-related splice variants in ClinVar (PR-AUC = 0.997 vs. PR-AUC = 0.996). These advancements hold promise for improving genetic research and clinical diagnostics, potentially leading to better understanding and treatment of splicing-related diseases. A transformer-based method efficiently detects RNA splicing from 45,000-nucleotide sequences by applying hard attention to select splice site candidates, outperforming SpliceAI in identifying splice sites and disease-related variants. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is linked to the reduction of certain nucleoporins in neurons. Increased nuclear localization of charged multivesicular body protein 7 (CHMP7), a protein involved in nuclear pore surveillance, has been identified as a key factor damaging nuclear pores and disrupting transport. Using CRISPR-based microRaft, followed by gRNA identification (CRaft-ID), we discovered 55 RNA-binding proteins (RBPs) that influence CHMP7 localization, including SmD1, a survival of motor neuron (SMN) complex component. Immunoprecipitation-mass spectrometry (IP-MS) and enhanced crosslinking and immunoprecipitation (CLIP) analyses revealed CHMP7's interactions with SmD1, small nuclear RNAs, and splicing factor mRNAs in motor neurons (MNs). ALS induced pluripotent stem cell (iPSC)-MNs show reduced SmD1 expression, and inhibiting SmD1/SMN complex increased CHMP7 nuclear localization. Crucially, overexpressing SmD1 in ALS iPSC-MNs restored CHMP7's cytoplasmic localization and corrected STMN2 splicing. Our findings suggest that early ALS pathogenesis is driven by SMN complex dysregulation. [Display omitted] • CRaft-ID identifies key regulators of CHMP7 nuclear localization • CHMP7 interacts with snRNP assembly factors, SM proteins, and Gemin proteins • CHMP7 binds snRNAs and mRNAs that encode essential splicing factors • Inhibition of SMN complex triggers CHMP7 nuclear entry, affecting TDP-43 function Al-Azzam et al. reveal that SmD1, a member of the SMN complex, regulates CHMP7 translocation. Reduced SmD1 expression in sporadic ALS leads to CHMP7 nuclear translocation, while overexpression prevents it, highlighting SmD1's role in ALS pathogenesis. [ABSTRACT FROM AUTHOR]

Introduction: RNA splicing is a crucial posttranscriptional process that governs gene expression, and defects in alternative splicing contribute to various diseases, including cancer. Tumor suppressing subtransferable candidate 4 (TSSC4) is a known tumor suppressor and has been identified as part of the U5 small nuclear ribonucleoprotein (snRNP), which is involved in tri-snRNP biogenesis. However, the precise role of TSSC4 in regulating alternative splicing and its impact on tumor growth remain unclear. Methods: To explore the link between splicing modulation and tumor suppression driven by TSSC4, we conducted transcriptome sequencing (RNA-seq) on TSSC4-knockout and wild-type HeLa cells. Additionally, we analyzed alternative splicing and gene expression in various cancer cell lines, including TSSC4-knockout A549 cells and TSSC4-knockdown PANC-1, MDA-MB-231, and MCF-7 cells. Splicing patterns and gene expression profiles were compared between TSSC4-deficient and control cells. Results: Our RNA-seq analysis revealed that TSSC4 deficiency in HeLa cells results in widespread alterations in splicing patterns and gene expression. Specifically, the loss of TSSC4 led to abnormal alternative splicing events and dysregulation of tumor-associated genes, including several oncogenes. This effect was confirmed across multiple cancer cell lines, highlighting a consistent role of TSSC4 in splicing regulation. Discussion: These findings demonstrate that TSSC4 plays a crucial role in regulating RNA splicing, particularly in controlling the splicing of many oncogenes. Our results reveal a novel mechanism by which TSSC4 mediates tumor suppression through the modulation of alternative splicing, which could provide implications for understanding TSSC4's role in cancer biology. [ABSTRACT FROM AUTHOR]

Regulated precursor messenger RNA (pre‐mRNA) splicing modulates gene expression and promotes alternative splicing. The process is regulated by modifications of spliceosomal proteins and small nuclear RNAs (snRNAs). Here, we show that the protein phosphatase Psr1, known for its plasma membrane localisation and function in general stress response in Saccharomyces cerevisiae, also plays a regulatory role in pre‐mRNA splicing. Independently of its presence at the plasma membrane, Psr1 binds and dephosphorylates the core splicing factor Snu66. The enzyme is not an integral component of the spliceosome. Psr1 deletion in yeast, or tethering of its catalytic mutant to Snu66, results in splicing defects of introns with non‐canonical 5′ splice sites (ss). While the Psr1 binding site on Snu66 is distinct from the Hub1 interaction domains (HIND), Hub1 displaces Psr1 from Snu66. Thus, Psr1 phosphatase plays a regulatory role in pre‐mRNA splicing by modulating Snu66 functions. [ABSTRACT FROM AUTHOR]

Taybi-Linder syndrome (TALS) is a rare autosomal recessive disorder characterized by severe microcephaly with abnormal gyral pattern, severe growth retardation and bone abnormalities. It is caused by pathogenic variants in the RNU4ATAC gene. Its transcript, the small nuclear RNA U4atac, is involved in the excision of ~850 minor introns. Here, we report a patient presenting with TALS features but no pathogenic variants were found in RNU4ATAC, instead the homozygous RTTN c.2953A>G variant was detected by whole-exome sequencing. After deciphering the impact of the variant on the RTTN protein function at centrosome in engineered RTTN-depleted RPE1 cells and patient fibroblasts, we analysed neuronal stem cells (NSC) derived from CRISPR/Cas9-edited induced pluripotent stem cells and revealed major cell cycle and mitotic abnormalities, leading to aneuploidy, cell cycle arrest and cell death. In cortical organoids, we discovered an additional function of RTTN in the self-organisation of NSC into neural rosettes, by observing delayed apico-basal polarization of NSC. Altogether, these defects resulted to a marked delay of rosette formation in RTTN-mutated organoids, thus impeding their overall growth and shedding light on mechanisms leading to microcephaly. Author summary: Primary microcephaly is defined as a severe reduction of the brain size that occurs prenatally. Variants in about 50 genes have been associated to primary microcephaly, and most of them encode proteins that regulate cell cycle, notably by participating to centrosome biogenesis. Intriguingly, some other genes involved in the process of minor splicing, such as RNU4ATAC, are also related to primary microcephaly without clear understanding of the underlying pathophysiological mechanisms. In our previous work, we discovered that alterations of minor splicing result into dysfunction of the centrosome/cilium complex. Here, we further feed this link between minor splicing and centrosome/primary cilium by reporting the particular case of a patient who presents with all features of the rare RNU4ATAC-associated syndrome, called the Taybi-Linder syndrome, and yet, is homozygous for the only recurrent pathogenic variant in the centrosomal RTTN gene. Hence, to decipher the underlying cellular mechanisms, we generated unique human neuronal cellular models–iPSC-derived neuronal stem cells (NSC) and cortical organoids–and unveiled the combination of events that contribute to the depletion of the NSC pool and explain RTTN-associated microcephaly. Our work gives thus precious hints for the understanding of the Taybi-Linder syndrome physiopathology. [ABSTRACT FROM AUTHOR]

AbstractObjectiveDatasourceStudy SelectionsResultsConclusionTo synthesize the current understanding of RNA-based regulatory mechanisms, focusing on how RNA splicing and non-coding RNAs shape immune responses and airway remodeling in asthma, with the aim of exploring their potential as therapeutic targets for asthma treatment.Recent advances and emerging research in molecular biology and immunology related to RNA splicing, non-coding RNAs (lncRNAs, circRNAs), and N6-methyladenosine (m6A) RNA methylation in asthma pathogenesis.The review incorporates studies highlighting the roles of alternative RNA splicing, non-coding RNAs (lncRNAs and circRNAs), and RNA methylation (m6A) in regulating immune and inflammatory pathways involved in asthma.RNA splicing events, non-coding RNAs, and m6A RNA methylation are critical in modulating immune dysregulation, airway remodeling, and inflammation in asthma. These mechanisms influence key inflammatory pathways, mRNA stability, and the overall immune landscape of the disease.RNA splicing and non-coding RNAs represent promising areas of research for understanding asthma’s immune pathology and hold potential as novel therapeutic targets for more effective treatment strategies. [ABSTRACT FROM AUTHOR]

SUMMARY: In plants, pre‐mRNA alternative splicing has been demonstrated to be a crucial tier that regulates gene expression in response to salt stress. However, the underlying mechanisms remain elusive. Here, we studied the roles of DIGEORGE‐SYNDROME CRITICAL REGION 14‐like (AtDGCR14L) in regulating pre‐mRNA splicing and salt stress tolerance. We discovered that Arabidopsis AtDGCR14L is required for maintaining plant salt stress tolerance and the constitutively spliced and active isoforms of important stress‐ and/or abscisic acid (ABA)‐responsive genes. We also identified the interaction between AtDGCR14L and splicing factor U1‐70k, which needs a highly conserved three amino acid (TWG) motif in DGCR14. Different from wild‐type AtDGCR14L, the overexpression of the TWG‐substituted AtDGCR14L mutant did not change salt stress tolerance or pre‐mRNA splicing of stress/ABA‐responsive genes. Additionally, SWITCH3A (SWI3A) is a core subunit of the SWI/SUCROSE NONFERMENTING (SWI/SNF) chromatin‐remodeling complexes. We found that SWI3A, whose splicing depends on AtDGCR14L, actively enhances salt stress tolerance. These results revealed that AtDGCR14L may play an essential role in crosstalk between plant salt‐stress response and pre‐mRNA splicing mechanisms. We also unveiled the potential role of SWI3A in controlling salt stress tolerance. The TWG motif in the intrinsically disordered region of AtDGCR14L is highly conserved and crucial for DGCR14 functions. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is a prevalent yet complex reproductive endocrine disorder affecting 11–13% of women worldwide. Its main symptoms include elevated androgen levels, irregular menstrual cycles, and long-term metabolic and offspring health implications. Despite the disease's multifaceted nature involving genetic, epigenetic, and environmental factors, the role of alternative splicing in ovarian granulosa cells remains relatively unexplored. This study aims to investigate the transcriptional and alternative splicing characteristics of granulosa cells in PCOS patients and to elucidate the potential functional consequences of these changes. Analysis of previous published transcriptome sequencing data identified 491 upregulated and 401 downregulated genes in granulosa cells of PCOS patients, significantly involved in immune-related processes. Additionally, 1250 differential splicing events, predominantly involving exon skipping and affecting 947 genes, were detected. These genes with alternative splicing patterns were found to be enriched in endoplasmic reticulum stress and protein post-translational modification processes, suggesting their role in PCOS pathology. Moreover, the study highlighted that the utilization of different splice isoforms of the YAP1 gene may impact its interaction in the Hippo signaling pathway, influencing the pathogenesis of PCOS. These findings underscore substantial alterations in alternative splicing in granulosa cells of PCOS patients, providing a novel viewpoint for comprehending the molecular underpinnings of PCOS and suggesting potential avenues for therapeutic intervention. [ABSTRACT FROM AUTHOR]

RNA-modifying enzymes have recently garnered considerable attention due to their relevance in cancer biology, identifying them as potential targets for novel therapeutic intervention. THUMPD3 was recently identified as an RNA methyltransferase catalysing N2-methylguanosine (m2G) within certain tRNAs. In this study, we unveil a novel role for THUMPD3 in lung cancer cells. Depletion of the enzyme from lung cancer cells significantly impairs their fitness, negatively impacting key cellular processes such as proliferation and migration. Notably, exogenous expression of THUMPD3 in normal lung fibroblasts stimulates their proliferation rate. Additionally, transcriptome-wide analyses reveal that depletion of THUMPD3 from lung cancer cells induces substantial changes in the expression of cell surface proteins, including those comprising the extracellular matrix (ECM). We further demonstrate that THUMPD3 maintains expression of an extra-domain B (EDB) containing pro-tumour isoform of Fibronectin-1 mRNA, encoding FN1, an important ECM protein. Crucially, depletion of THUMPD3 promotes an alternative splicing event that removes the EDB-encoding exon from Fibronectin-1. This is consistent with THUMPD3 depletion reducing cellular proliferation and migration. Moreover, depletion of THUMPD3 selectively and preferentially affects the alternative splicing of ECM and cell adhesion molecule encoding transcripts, as well as those encoding neurodevelopmental proteins. Overall, these findings highlight THUMPD3 as an important player in regulating cancer-relevant alternative splicing and they provide a rationale for further investigations into THUMPD3 as a candidate target in anti-cancer therapy. [ABSTRACT FROM AUTHOR]

Introduction: The main obstacle to achieving an HIV-1 cure is the proviral reservoir. To promote equity in HIV cure strategies, it is crucial to study the viral reservoir of the predominant HIV-1 subtype C in both women and men. Therefore, we investigated the dynamics of the (intact) viral reservoir in relation to plasma viral load (VL), CD4+ T cell count, and immune activation before and during 96 weeks of successful antiretroviral therapy (ART). Methods: Eighty-two participants (62% female) newly initiating ART in a rural clinic in South Africa were included in the study. Blood samples were collected at baseline, week 48, and week 96, and CD4 count was determined. Plasma was used for VL and immune marker analyses, while isolated peripheral blood mononuclear cells (PBMCs) were used for the quantification of cellular multiple spliced HIV-1 RNA (msRNA) and the intact proviral DNA assay. For the longitudinal analyses on ART, we selected only those participants who durably suppressed their VL to <200 copies/mL during 48 (n=65) and/or 96 (n=60) weeks of treatment. Results: At ART initiation, the median CD4 count was 234 cells/mm3 and VL was 68,897 copies/mL. Interestingly, at baseline the number of defective proviruses was significantly correlated with VL (p<0.0001), msRNA (p<0.0001), CD4 count (p=0.0008), CXCL10 (p=0.0003) and TNF-α (p=0.0394). During successful ART, a significant decrease of both the intact and defective proviral reservoir was observed (p<0.0001). The decrease of the intact proviral reservoir was more profound compared to the defective fraction after 96 weeks of therapy. In addition, a significant decrease in cellular msRNA and IL-6, IL-7, TNF-α, sCD14, sCD163, CCL2, CXCL10, and CRP was detected. Discussion: This study underscores the significant relationship observed prior to therapy initiation between the number of defective proviruses, viral transcription/production and their association with immune response indicators such as CD4 count, CXCL10, and TNF-α. Furthermore, the observation of a less pronounced decrease of the defective proviral DNA highlights the importance of addressing both intact and defective proviruses in therapeutic strategies to enhance clinical outcomes for people with HIV-1. Together, these findings suggest a significant role of the defective proviruses in HIV-related disease progression. [ABSTRACT FROM AUTHOR]

The KEAP1-NRF2 system plays a crucial role in responding to oxidative and electrophilic stress. Its dysregulation can cause the overexpression of downstream genes, a known cancer hallmark. Understanding and detecting abnormal KEAP1-NRF2 activity is essential for understanding disease mechanisms and identifying therapeutic targets. This study presents an approach that analyzes splicing patterns by a naive Bayes-based classifier to identify constitutive activation of the KEAP1-NRF2 system, focusing on the higher presence of abnormal splicing junctions as a subproduct of overexpression of downstream genes. Our splicing-based classifier demonstrated robust performance, reliably identifying activation of the KEAP1-NRF2 pathway across extensive datasets, including The Cancer Genome Atlas and the Sequence Read Archive. This shows the classifier's potential to analyze hundreds of thousands of transcriptomes, highlighting its utility in broad-scale genomic studies and provides a new perspective on utilizing splicing aberrations caused by overexpression as diagnostic markers, offering potential improvements in diagnosis and treatment strategies. [ABSTRACT FROM AUTHOR]

Cellular growth and organismal development are remarkably complex processes that require the nutrient-responsive kinase mechanistic target of rapamycin complex 1 (mTORC1). Anticipating that important mTORC1 functions remained to be identified, we employed genetic and bioinformatic screening in C. elegans to uncover mechanisms of mTORC1 action. Here, we show that during larval growth, nutrients induce an extensive reprogramming of gene expression and alternative mRNA splicing by acting through mTORC1. mTORC1 regulates mRNA splicing and the production of protein-coding mRNA isoforms largely independently of its target p70 S6 kinase (S6K) by increasing the activity of the serine/arginine-rich (SR) protein RSP-6 (SRSF3/7) and other splicing factors. mTORC1-mediated mRNA splicing regulation is critical for growth; mediates nutrient control of mechanisms that include energy, nucleotide, amino acid, and other metabolic pathways; and may be conserved in humans. Although mTORC1 inhibition delays aging, mTORC1-induced mRNA splicing promotes longevity, suggesting that when mTORC1 is inhibited, enhancement of this splicing might provide additional anti-aging benefits. [Display omitted] • During C. elegans growth, mTORC1 broadly controls mRNA splicing and expression • mTORC1 increases the expression and activity of SR proteins and other splicing factors • mTORC1 controls growth-related metabolic pathways by regulating mRNA splicing • Regulating metabolic genes through mRNA splicing is a conserved mTORC1 function Ogawa et al. show in Caenorhabditis elegans that the essential growth regulator mTORC1 extensively reprograms gene expression and alternative mRNA splicing. This splicing regulation is critical for growth, orchestrates growth-associated metabolism, and appears to be conserved. Maintaining mTORC1-induced splicing may be beneficial for longevity under conditions of mTORC1 inhibition. [ABSTRACT FROM AUTHOR]