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Splicing factor 3b subunit 1 (SF3B1) is the largest subunit and core component of the spliceosome. Inhibition of SF3B1 was associated with an increase in broad intron retention (IR) on most transcripts, suggesting that IR can be used as a marker of spliceosome inhibition in chronic lymphocytic leukemia (CLL) cells. Furthermore, we separately analyzed exonic and intronic mapped reads on annotated RNA-sequencing transcripts obtained from B cells (n = 98 CLL patients) and healthy volunteers (n = 9). We measured intron/exon ratio to use that as a surrogate for alternative RNA splicing (ARS) and found that 66% of CLL-B cell transcripts had significant IR elevation compared with normal B cells (NBCs) and that correlated with mRNA downregulation and low expression levels. Transcripts with the highest IR levels belonged to biological pathways associated with gene expression and RNA splicing. A >2-fold increase of active pSF3B1 was observed in CLL-B cells compared with NBCs. Additionally, when the CLL-B cells were treated with macrolides (pladienolide-B), a significant decrease in pSF3B1, but not total SF3B1 protein, was observed. These findings suggest that IR/ARS is increased in CLL, which is associated with SF3B1 phosphorylation and susceptibility to SF3B1 inhibitors. These data provide additional support to the relevance of ARS in carcinogenesis and evidence of pSF3B1 participation in this process.

Pre-mRNA splicing is a crucial regulatory process in eukaryotes, ensuring accurate mRNA maturation and contributing to transcript diversity through alternative splicing (AS). This process is mediated by the spliceosome, a large molecular complex composed of small nuclear RNAs (snRNAs) and proteins. Structural and comparative analysis have showed that splicing factors are highly conserved across eukaryotes, although the extent of their functional conservation remains to be fully elucidated. In Arabidopsis, two homologous genes, PRP43a and PRP43b, encode components involved in spliceosome disassembly. To investigate the roles of these genes, we characterized T-DNA insertion mutants prp43a-1 and prp43b-1. While prp43b-1 homozygous mutants were embryonic lethal, prp43a-1 mutants displayed no significant developmental defects under normal conditions, nor did they exhibit impaired heat tolerance. However, RNA-seq analysis revealed transcriptional changes in prp43a-1 under normal conditions. Gene ontology (GO) analysis indicated an enrichment of stress-related processes among the differentially expressed genes (DEGs), suggesting a role for PRP43a in stress responses. Additionally, differential alternative splicing (DAS) analysis identified numerous PRP43a-dependent intron retention (IR) events, particularly under heat stress, indicating that PRP43a influences AS under environmental conditions. These findings suggest PRP43a as a modulator of splicing and gene expression, with potential roles in stress responses. [ABSTRACT FROM AUTHOR]

Intron retention (IR) constitutes a less explored form of alternative splicing, wherein introns are retained within mature mRNA transcripts. This investigation demonstrates that the cell division cycle (CDC)‐like kinase 2 (CLK2) undergoes liquid–liquid phase separation (LLPS) within nuclear speckles in response to heat shock (HS). The formation of CLK2 condensates depends on the intrinsically disordered region (IDR) located within the N‐terminal amino acids 1‐148. Phosphorylation at residue T343 sustains CLK2 kinase activity and promotes overall autophosphorylation, which inhibits the LLPS activity of the IDR. These CLK2 condensates initiate the reorganization of nuclear speckles, transforming them into larger, rounded structures. Moreover, these condensates facilitate the recruitment of splicing factors into these compartments, restricting their access to mRNA for intron splicing and promoting the IR. The retained introns lead to the sequestration of transcripts within the nucleus. These findings extend to the realm of glioma stem cells (GSCs), where a physiological state mirroring HS stress inhibits T343 autophosphorylation, thereby inducing the formation of CLK2 condensates and subsequent IR. Notably, expressing the CLK2 condensates hampers the maintenance of GSCs. In conclusion, this research unveils a mechanism by which IR is propelled by CLK2 condensates, shedding light on its role in coping with cellular stress. [ABSTRACT FROM AUTHOR]

Background: Peripheral inflammation is often associated with depressive disorders, and immunological biomarkers of depression remain a focus of investigation. Methods: We performed RNA-seq analysis of RNA transcripts of human peripheral blood mononuclear cells from a case-control study including subjects with self-reported depression in the pre-symptomatic state of major depressive disorder and analyzed differentially expressed genes (DEGs) and the frequency of intron retention (IR) using rMATS. Results: Among the statistically significant DEGs identified, the 651 upregulated DEGs were particularly enriched in the term "bacterial infection and phagocytosis", whereas the 820 downregulated DEGs were enriched in the terms "antigen presentation" and "T-cell proliferation and maturation". We also analyzed 158 genes for which the IR was increased (IncIR) and 211 genes for which the IR was decreased (DecIR) in the depressed subjects. Although the Gene Ontology terms associated with IncIR and DecIR were very similar to those of the up- and downregulated genes, respectively, IR genes appeared to be particularly enriched in genes with sensor functions, with a preponderance of the term "ciliary assembly and function". The observation that IR genes specifically interact with innate immunity genes suggests that immune-related genes, as well as cilia-related genes, may be excellent markers of depression. Re-analysis of previously published RNA-seq data from patients with MDD showed that common IR genes, particularly our predicted immune- and cilia-related genes, are commonly detected in populations with different levels of depression, providing validity for using IR to detect depression. Conclusion: Depression was found to be associated with activation of the innate immune response and relative inactivation of T-cell signaling. The DEGs we identified reflect physiological demands that are controlled at the transcriptional level, whereas the IR results reflect a more direct mechanism for monitoring protein homeostasis. Accordingly, an alteration in IR, namely IncIR or DecIR, is a stress response, and intron-retained transcripts are sensors of the physiological state of the cytoplasm. The results demonstrate the potential of relative IR as a biomarker for the immunological stratification of depressed patients and the utility of IR for the discovery of novel pathways involved in recovery from depression. [ABSTRACT FROM AUTHOR]

The plastid-targeted transcription factor Whirly1 (WHY1) has been implicated in chloroplast biogenesis, plastid genome stability, and fungal defense response, which together represent characteristics of interest for the study of autotrophic losses across the angiosperms. While gene loss in the plastid and nuclear genomes has been well studied in mycoheterotrophic plants, the evolution of the molecular mechanisms impacting genome stability is completely unknown. Here, we characterize the evolution of WHY1 in four early transitional mycoheterotrophic orchid species in the genus Corallorhiza by synthesizing the results of phylogenetic, transcriptomic, and comparative genomic analyses with WHY1 genomic sequences sampled from 21 orders of angiosperms. We found an increased number of non-canonical WHY1 isoforms assembled from all but the greenest Corallorhiza species, including intron retention in some isoforms. Within Corallorhiza, phylotranscriptomic analyses revealed the presence of tissue-specific differential expression of WHY1 in only the most photosynthetically capable species and a coincident increase in the number of non-canonical WHY1 isoforms assembled from fully mycoheterotrophic species. Gene- and codon-level tests of WHY1 selective regimes did not infer significant signal of either relaxed selection or episodic diversifying selection in Corallorhiza but did so for relaxed selection in the late-stage full mycoheterotrophic orchids Epipogium aphyllum and Gastrodia elata. Additionally, nucleotide substitutions that most likely impact the function of WHY1, such as nonsense mutations, were only observed in late-stage mycoheterotrophs. We propose that our findings suggest that splicing and expression changes may precede the selective shifts we inferred for late-stage mycoheterotrophic species, which therefore does not support a primary role for WHY1 in the transition to mycoheterotrophy in the Orchidaceae. Taken together, this study provides the most comprehensive view of WHY1 evolution across the angiosperms to date. [ABSTRACT FROM AUTHOR]

The intron retention (IR) is a phenomenon utilized by cells to allow diverse fates at the same mRNA, leading to a different pattern of synthesis of the same protein. In this study, we analyzed the modulation of phosphoinositide‐specific phospholipase C (PI‐PLC) enzymes by Harpagophytum procumbens extract (HPE) in synoviocytes from joins of osteoarthritis (OA) patients. In some samples, the PI‐PLC γ1 isoform mature mRNA showed the IR and, in these synoviocytes, the HPE treatment increased the phenomenon. Moreover, we highlighted that as a consequence of IR, a lower amount of PI‐PLC γ1 was produced. The decrease of PI‐PLC γ1 was associated with the decrease of metalloprotease‐3 (MMP‐3), and MMP‐13, and ADAMTS‐5 after HPE treatment. The altered expression of MMPs is a hallmark of the onset and progression of OA, thus substances able to decrease their expression are very desirable. The interesting outcomes of this study are that 35% of analyzed synovial tissues showed the IR phenomenon in the PI‐PLC γ1 mRNA and that the HPE treatment increased this phenomenon. For the first time, we found that the decrease of PI‐PLC γ1 protein in synoviocytes interferes with MMP production, thus affecting the pathways involved in the MMP expression. This finding was validated by the silencing of PI‐PLC γ1 in synoviocytes where the IR phenomenon was not present. Our results shed new light on the biochemical mechanisms involved in the degrading enzyme production in the joint of OA patients, suggesting a new therapeutic target and highlighting the importance of personalized medicine. Significant Statement: This manuscript focuses on the modulation of phosphoinositide‐specific phospholipase C (PI‐PLC) γ1 in human primary fibroblast‐like synoviocytes, which leads to the modulation of metalloprotease‐3 (MMP‐3), MMP‐13, and ADAMTS‐5, involved in the degradation of extracellular matrix in degenerative joint diseases. We found that in some synoviocyte samples, the PI‐PLC γ1 mRNA incurs in the intron retention (IR) phenomenon, which results in an unsuccessful translation of the proteins associated with the decrease of MMP enzymes. Moreover, IR is increased after cell treatment with Harpagophytum procumbens extract (HPE), traditionally used to treat osteoarthritis. Considering that HPE results are effective in modulating MMPs only in synoviocyte samples where the IR phenomenon is present, it is necessary to evaluate individual variability in the personalized medicine field. [ABSTRACT FROM AUTHOR]

Eukaryotic genomes contain a tiny subset of 'minor class' introns with unique sequence elements that require their own splicing machinery. These minor introns are present in certain gene families with specific functions, such as voltage-gated Na+ and voltage-gated Ca2+ channels. Removal of minor introns by the minor spliceosome has been proposed as a post-transcriptional regulatory layer, which remains unexplored in the heart. Here, we investigate whether the minor spliceosome regulates electrophysiological properties of cardiomyocytes by knocking down the essential minor spliceosome small nuclear snRNA component U6atac in neonatal rat ventricular myocytes. Loss of U6atac led to robust minor intron retention within Scn5a and Cacna1c, resulting in reduced protein levels of Nav1.5 and Cav1.2 channels. Functional consequences were studied through patch-clamp analysis, and revealed reduced Na+ and L-type Ca2+ currents after loss of U6atac. In conclusion, minor intron splicing modulates voltage-dependent ion channel expression and function in cardiomyocytes. This may be of particular relevance in situations in which minor splicing activity changes, such as in genetic diseases affecting minor spliceosome components, or in acquired diseases in which minor spliceosome components are dysregulated, such as heart failure. [ABSTRACT FROM AUTHOR]

Abstract Intron retention (IR) constitutes a less explored form of alternative splicing, wherein introns are retained within mature mRNA transcripts. This investigation demonstrates that the cell division cycle (CDC)‐like kinase 2 (CLK2) undergoes liquid–liquid phase separation (LLPS) within nuclear speckles in response to heat shock (HS). The formation of CLK2 condensates depends on the intrinsically disordered region (IDR) located within the N‐terminal amino acids 1‐148. Phosphorylation at residue T343 sustains CLK2 kinase activity and promotes overall autophosphorylation, which inhibits the LLPS activity of the IDR. These CLK2 condensates initiate the reorganization of nuclear speckles, transforming them into larger, rounded structures. Moreover, these condensates facilitate the recruitment of splicing factors into these compartments, restricting their access to mRNA for intron splicing and promoting the IR. The retained introns lead to the sequestration of transcripts within the nucleus. These findings extend to the realm of glioma stem cells (GSCs), where a physiological state mirroring HS stress inhibits T343 autophosphorylation, thereby inducing the formation of CLK2 condensates and subsequent IR. Notably, expressing the CLK2 condensates hampers the maintenance of GSCs. In conclusion, this research unveils a mechanism by which IR is propelled by CLK2 condensates, shedding light on its role in coping with cellular stress.

Low level HIV transcription during modern antiretroviral therapy (ART) in persons with HIV is linked to residual inflammation and associated diseases, like cardiovascular disease and cancer. The "block and lock" approach to hold HIV in a state of deep latency may help decrease residual inflammation in a person with HIV on ART and thus improve health. A camptothecin analog topotecan (TPT) was previously implicated as an inhibitor of active HIV replication. Using an in vitro primary T cell model of HIV latency, we demonstrated that (i) TPT reduces HIV transcriptional activity in latently infected cells; (ii) downregulation of HIV RNA by TPT cannot be reversed by latency reversing agents; (iii) several primary and secondary mechanism of action of TPT may be involved in control of HIV replication; (iv) regulation of HIV RNA by TPT is dependent on splicing complexity; (v) increase in proportion of unspliced HIV transcripts was facilitated by intron retention and upregulation of splicing factors, specifically SRSF6, by TPT. Although high TPT dosing (10 μM) was needed to achieve the observed effects, viability of primary CD4+ T cells was not greatly affected. Because toxicity can be observed with TPT in persons with cancer, TPT is unlikely to be used as an anti-HIV agent in clinic, but our study provides proof that camptothetin has "block and lock" activity. Other camptothetin analogs, which are less toxic than TPT, should be designed and tested as HIV "block and lock" agents. IMPORTANCE HIV survives in a state of very low activity, called latency, for long periods in persons with HIV on antiretroviral therapy. This low activity of HIV is linked to residual inflammation and associated diseases, such as heart disease and cancer. New strategies are being explored to further silence the HIV provirus and suppress residual inflammation. This study provides strong evidence that the camptothetin analog, Topotecan, can reduce residual activity of HIV in an experimental model of HIV latency. While Topotecan itself is likely not suitable for use in the clinic due to its toxicity, other camptothetin analogs should be designed and investigated as "block and lock" agents.

• We report a recurrent intronic homozygous c.1023+5G> A desmin mutation in indian patients. • Second report of recessive desminopathy associated with mixed limb girdle CMS – myofibrillar myopathy. • Partial loss of desmin leading to NMJ dysfunction and CMS phenotype. • Leaky splice site with intron retention leads to partial desmin loss. Recessive desminopathies are rare and often present as severe early-onset myopathy. Here we report a milder phenotype in three unrelated patients from southern India (2 M, 1F) aged 16, 21, and 22 years, who presented with childhood-onset, gradually progressive, fatigable limb-girdle weakness, ptosis, speech and swallowing difficulties, without cardiac involvement. Serum creatine kinase was elevated, and repetitive nerve stimulation showed decrement in all. Clinical improvement was noted with pyridostigmine and salbutamol in two patients. All three patients had a homozygous substitution in intron 5: DES(NM_001927.4):c.1023+5G> A , predicted to cause a donor splice site defect. Muscle biopsy with ultrastructural analysis suggested myopathy with myofibrillar disarray, and immunohistochemistry showed partial loss of desmin with some residual staining, while western blot analysis showed reduced desmin. RT-PCR of patient muscle RNA revealed two transcripts: a reduced normal desmin transcript and a larger abnormal transcript suggesting leaky splicing at the intron 5 donor site. Sequencing of the PCR products confirmed the inclusion of intron 5 in the longer transcript, predicted to cause a premature stop codon. Thus, we provide evidence for a leaky splice site causing partial loss of desmin associated with a unique phenotypic presentation of a milder form of desmin-related recessive myopathy overlapping with congenital myasthenic syndrome. [ABSTRACT FROM AUTHOR]

Temozolomide (TMZ) resistance remains the major obstacle in the treatment of glioblastoma (GBM). Lactylation is a novel post‐translational modification that is involved in various tumors. However, whether lactylation plays a role in GBM TMZ resistance remains unclear. Here it is found that histone H3K9 lactylation (H3K9la) confers TMZ resistance in GBM via LUC7L2‐mediated intron 7 retention of MLH1. Mechanistically, lactylation is upregulated in recurrent GBM tissues and TMZ‐resistant cells, and is mainly concentrated in histone H3K9. Combined multi‐omics analysis, including CUT&Tag, SLAM‐seq, and RNA‐seq, reveals that H3K9 lactylation is significantly enriched in the LUC7L2 promoter and activates LUC7L2 transcription to promote its expression. LUC7L2 mediates intron 7 retention of MLH1 to reduce MLH1 expression, and thereby inhibit mismatch repair (MMR), ultimately leading to GBM TMZ resistance. Of note, it is identified that a clinical anti‐epileptic drug, stiripentol, which can cross the blood–brain barrier and inhibit lactate dehydrogenase A/B (LDHA/B) activity, acts as a lactylation inhibitor and renders GBM cells more sensitive to TMZ in vitro and in vivo. These findings not only shed light on the mechanism of lactylation in GBM TMZ resistance but also provide a potential combined therapeutic strategy for clinical GBM treatment. [ABSTRACT FROM AUTHOR]

Objective: The objective of this study was to pinpoint pathogenic genes and assess the mutagenic pathogenicity in two pediatric patients with hereditary spherocytosis. Methods: We utilized whole-exome sequencing (WES) for individual analysis (case 1) and family-based trio analysis (case 2). The significance of the intronic mutation was validated through a Minigene splicing assay and supported by subsequent in vitro experiments. Results: Both probands received a diagnosis of hereditary spherocytosis. WES identified a novel ANK1 c.1504-9G>A mutation in both patients, causing the retention of seven nucleotides at the 5' end of intron 13, as substantiated by the Minigene assay. This variant results in a premature stop codon and the production of a truncated protein. In vitro studies indicated a reduced expression of the ANK1 gene. Conclusion: The novel ANK1 c.1504-9G>A variant is established as the causative factor for hereditary spherocytosis, with the c.1504-9G site functioning as a splicing receptor. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae (baker´s yeast) has yielded relevant in-sights into some of the basic mechanisms of organismal aging. Among these are genomic instability, oxidative stress, caloric restriction and mitochondrial dysfunction. Several genes are known to have an impact on the aging process, with corresponding mutants exhibiting short- or long-lived phenotypes. Re-search dedicated to unraveling the underlying cellular mechanisms can sup-port the identification of conserved mechanisms of aging in other species. One of the hitherto less studied fields in yeast aging is how the organism regulates its gene expression at the transcriptional level. To our knowledge, we present the first investigation into alternative splicing, particularly intron retention, during replicative aging of S. cerevisiae. This was achieved by utiliz-ing the IRFinder algorithm on a previously published RNA-seq data set by Janssens et al. (2015). In the present work, 44 differentially retained introns in 43 genes were identified during replicative aging. We found that genes with altered intron retention do not display significant changes in overall transcript levels. It was possible to functionally assign distinct groups of these genes to the cellular processes of mRNA processing and export (e.g., YRA1) in early and middle-aged yeast, and protein ubiquitination (e.g., UBC5) in older cells. In summary, our work uncovers a previously unexplored layer of the transcrip-tional program of yeast aging and, more generally, expands the knowledge on the occurrence of alternative splicing in baker´s yeast.

Alternative splicing (AS) is a crucial mechanism for regulating gene expression and isoform diversity in eukaryotes. However, the analysis and visualization of AS events from RNA sequencing data remains challenging. Most tools require a certain level of computer literacy and the available means of visualizing AS events, such as coverage and sashimi plots, have limitations and can be misleading. To address these issues, we present SpliceWiz , an R package with an interactive Shiny interface that allows easy and efficient AS analysis and visualization at scale. A novel normalization algorithm is implemented to aggregate splicing levels within sample groups, thereby allowing group differences in splicing levels to be accurately visualized. The tool also offers downstream gene ontology enrichment analysis, highlighting ASEs belonging to functional pathways of interest. SpliceWiz is optimized for speed and efficiency and introduces a new file format for coverage data storage that is more efficient than BigWig. Alignment files are processed orders of magnitude faster than other R-based AS analysis tools and on par with command-line tools. Overall, SpliceWiz streamlines AS analysis, enabling reliable identification of functionally relevant AS events for further characterization. SpliceWiz is a Bioconductor package and is also available on GitHub (https://github.com/alexchwong/SpliceWiz). [ABSTRACT FROM AUTHOR]

Trichophyton rubrum is the primary causative agent of dermatophytosis worldwide. This fungus colonizes keratinized tissues and uses keratin as a nutritional source during infection. In T. rubrum–host interactions, sensing a hostile environment triggers the adaptation of its metabolic machinery to ensure its survival. The glyoxylate cycle has emerged as an alternative metabolic pathway when glucose availability is limited; this enables the conversion of simple carbon compounds into glucose via gluconeogenesis. In this study, we investigated the impact of stuA deletion on the response of glyoxylate cycle enzymes during fungal growth under varying culture conditions in conjunction with post-transcriptional regulation through alternative splicing of the genes encoding these enzymes. We revealed that the ΔstuA mutant downregulated the malate synthase and isocitrate lyase genes in a keratin-containing medium or when co-cultured with human keratinocytes. Alternative splicing of an isocitrate lyase gene yielded a new isoform. Enzymatic activity assays showed specific instances where isocitrate lyase and malate synthase activities were affected in the mutant strain compared to the wild type strain. Taken together, our results indicate a relevant balance in transcriptional regulation that has distinct effects on the enzymatic activities of malate synthase and isocitrate lyase. [ABSTRACT FROM AUTHOR]

Splicing factor 3b subunit 1 (SF3B1) is the largest subunit and core component of the spliceosome. Inhibition of SF3B1 was associated with an increase in broad intron retention (IR) on most transcripts, suggesting that IR can be used as a marker of spliceosome inhibition in chronic lymphocytic leukemia (CLL) cells. Furthermore, we separately analyzed exonic and intronic mapped reads on annotated RNA-sequencing transcripts obtained from B cells (n = 98 CLL patients) and healthy volunteers (n = 9). We measured intron/exon ratio to use that as a surrogate for alternative RNA splicing (ARS) and found that 66% of CLL-B cell transcripts had significant IR elevation compared with normal B cells (NBCs) and that correlated with mRNA downregulation and low expression levels. Transcripts with the highest IR levels belonged to biological pathways associated with gene expression and RNA splicing. A >2-fold increase of active pSF3B1 was observed in CLL-B cells compared with NBCs. Additionally, when the CLL-B cells were treated with macrolides (pladienolide-B), a significant decrease in pSF3B1, but not total SF3B1 protein, was observed. These findings suggest that IR/ARS is increased in CLL, which is associated with SF3B1 phosphorylation and susceptibility to SF3B1 inhibitors. These data provide additional support to the relevance of ARS in carcinogenesis and evidence of pSF3B1 participation in this process.

The plastid-targeted transcription factor Whirly1 (WHY1) has been implicated in chloroplast biogenesis, plastid genome stability, and fungal defense response, which together represent characteristics of interest for the study of autotrophic losses across the angiosperms. While gene loss in the plastid and nuclear genomes has been well studied in mycoheterotrophic plants, the evolution of the molecular mechanisms impacting genome stability is completely unknown. Here, we characterize the evolution of WHY1 in four early transitional mycoheterotrophic orchid species in the genus Corallorhiza by synthesizing the results of phylogenetic, transcriptomic, and comparative genomic analyses with WHY1 genomic sequences sampled from 21 orders of angiosperms. We found an increased number of non-canonical WHY1 isoforms assembled from all but the greenest Corallorhiza species, including intron retention in some isoforms. Within Corallorhiza, phylotranscriptomic analyses revealed the presence of tissue-specific differential expression of WHY1 in only the most photosynthetically capable species and a coincident increase in the number of non-canonical WHY1 isoforms assembled from fully mycoheterotrophic species. Gene- and codon-level tests of WHY1 selective regimes did not infer significant signal of either relaxed selection or episodic diversifying selection in Corallorhiza but did so for relaxed selection in the late-stage full mycoheterotrophic orchids Epipogium aphyllum and Gastrodia elata. Additionally, nucleotide substitutions that most likely impact the function of WHY1, such as nonsense mutations, were only observed in late-stage mycoheterotrophs. We propose that our findings suggest that splicing and expression changes may precede the selective shifts we inferred for late-stage mycoheterotrophic species, which therefore does not support a primary role for WHY1 in the transition to mycoheterotrophy in the Orchidaceae. Taken together, this study provides the most comprehensive view of WHY1 evolution across the angiosperms to date.

Abstract Temozolomide (TMZ) resistance remains the major obstacle in the treatment of glioblastoma (GBM). Lactylation is a novel post‐translational modification that is involved in various tumors. However, whether lactylation plays a role in GBM TMZ resistance remains unclear. Here it is found that histone H3K9 lactylation (H3K9la) confers TMZ resistance in GBM via LUC7L2‐mediated intron 7 retention of MLH1. Mechanistically, lactylation is upregulated in recurrent GBM tissues and TMZ‐resistant cells, and is mainly concentrated in histone H3K9. Combined multi‐omics analysis, including CUT&Tag, SLAM‐seq, and RNA‐seq, reveals that H3K9 lactylation is significantly enriched in the LUC7L2 promoter and activates LUC7L2 transcription to promote its expression. LUC7L2 mediates intron 7 retention of MLH1 to reduce MLH1 expression, and thereby inhibit mismatch repair (MMR), ultimately leading to GBM TMZ resistance. Of note, it is identified that a clinical anti‐epileptic drug, stiripentol, which can cross the blood–brain barrier and inhibit lactate dehydrogenase A/B (LDHA/B) activity, acts as a lactylation inhibitor and renders GBM cells more sensitive to TMZ in vitro and in vivo. These findings not only shed light on the mechanism of lactylation in GBM TMZ resistance but also provide a potential combined therapeutic strategy for clinical GBM treatment.

Aging and senescence are characterized by pervasive transcriptional dysfunction, including increased expression of transposons and introns. Our aim was to elucidate mechanisms behind this increased expression. Most transposons are found within genes and introns, with a large minority being close to genes. This raises the possibility that transcriptional readthrough and intron retention are responsible for age-related changes in transposon expression rather than expression of autonomous transposons. To test this, we compiled public RNA-seq datasets from aged human fibroblasts, replicative and drug-induced senescence in human cells, and RNA-seq from aging mice and senescent mouse cells. Indeed, our reanalysis revealed a correlation between transposons expression, intron retention, and transcriptional readthrough across samples and within samples. Both intron retention and readthrough increased with aging or cellular senescence and these transcriptional defects were more pronounced in human samples as compared to those of mice. In support of a causal connection between readthrough and transposon expression, analysis of models showing induced transcriptional readthrough confirmed that they also show elevated transposon expression. Taken together, our data suggest that elevated transposon reads during aging seen in various RNA-seq dataset are concomitant with multiple transcriptional defects. Intron retention and transcriptional readthrough are the most likely explanation for the expression of transposable elements that lack a functional promoter.

Abstract Background Amelogenesis imperfecta (AI) is a developmental enamel defect affecting the structure of enamel, esthetic appearance, and the tooth masticatory function. Gene mutations are reported to be relevant to AI. However, the mechanism underlying AI caused by different mutations is still unclear. This study aimed to reveal the molecular pathogenesis in AI families with 2 novel pre-mRNA splicing mutations. Methods Two Chinese families with AI were recruited. Whole-exome sequencing and Sanger sequencing were performed to identify mutations in candidate genes. Minigene splicing assays were performed to analyze the mutation effects on mRNA splicing alteration. Furthermore, three-dimensional structures of mutant proteins were predicted by AlphaFold2 to evaluate the detrimental effect. Results The affected enamel in family 1 was thin, rough, and stained, which was diagnosed as hypoplastic-hypomature AI. Genomic analysis revealed a novel splicing mutation (NM_001142.2: c.570 + 1G > A) in the intron 6 of amelogenin (AMELX) gene in family 1, resulting in a partial intron 6 retention effect. The proband in family 2 exhibited a typical hypoplastic AI, and the splicing mutation (NM_031889.2: c.123 + 4 A > G) in the intron 4 of enamelin (ENAM) gene was observed in the proband and her father. This mutation led to exon 4 skipping. The predicted structures showed that there were obvious differences in the mutation proteins compared with wild type, leading to impaired function of mutant proteins. Conclusions In this study, we identified two new splicing mutations in AMELX and ENAM genes, which cause hypoplastic-hypomature and hypoplastic AI, respectively. These results expand the spectrum of genes causing AI and broaden our understanding of molecular genetic pathology of enamel formation.

Alterations in RNA splicing are associated with different malignancies, including leukemia, lymphoma, and solid tumors. The RNA splicing modulators such as FD-895 and pladienolide B have been investigated in different malignancies to target/modulate spliceosome for therapeutic purpose. Different cell lines were screened using an RNA splicing modulator to test in vitro cytotoxicity and the ability to modulate RNA splicing capability via induction of intron retention (using RT-PCR and qPCR). The Cignal Finder Reporter Array evaluated [pathways affected by the splice modulators in HeLa cells. Further, the candidates associated with the pathways were validated at protein level using western blot assay, and gene-gene interaction studies were carried out using GeneMANIA. We show that FD-895 and pladienolide B induces higher apoptosis levels than conventional chemotherapy in different solid tumors. In addition, both agents modulate Wnt signaling pathways and mRNA splicing. Specifically, FD-895 and pladienolide B significantly downregulates Wnt signaling pathway-associated transcripts (GSK3β and LRP5) and both transcript and proteins including LEF1, CCND1, LRP6, and pLRP6 at the transcript, total protein, and protein phosphorylation's levels. These results indicate FD-895 and pladienolide B inhibit Wnt signaling by decreasing LRP6 phosphorylation and modulating mRNA splicing through induction of intron retention in solid tumors.

Summary: Background: Tau is a microtubule-binding protein encoded by the MAPT gene. Tau is essential for several physiological functions and associated with pathological processes, including Alzheimer's disease (AD). Six tau isoforms are typically described in the central nervous system, but current research paints a more diverse landscape and a more nuanced balance between isoforms. Recent work has described tau isoforms generated by intron 11 and intron 12 retention. This work adds to that evidence, proving the existence of MAPT transcripts retaining intron 3. Our aim is to demonstrate the existence of mature MAPT RNA species that retain intron 3 in human brain samples and to study its correlation with Alzheimer's disease across different regions. Methods: Initial evidence of intron-3-retaining MAPT species come from in silico analysis of RNA-seq databases. We further demonstrate the existence of these mature RNA species in a human neuroepithelioma cell line and human brain samples by quantitative PCR. We also use digital droplet PCR to demonstrate the existence of RNA species that retain either intron 3, intron 12 or both introns. Findings: Intron-3-retaining species are even more prominently present that intron-12-retaining ones. We show the presence of MAPT transcripts that retain both introns 3 and 12. These intron-retaining species are diminished in brain samples of patients with Alzheimer's disease with respect to individuals without dementia. Conversely, relative abundance of intron-3- or intron-12-retaining MAPT species with respect to double-retaining species as well as their percentage of expression with respect to total MAPT are increased in patients with Alzheimer's disease, especially in hippocampal samples. Among these TIR-MAPT species, TIR3+12 double truncation allows better classification potential of Alzheimer's disease samples. Moreover, we find a significant increase in intron-3- or intron-12-retaining species and its relative abundance with respect to double-retaining MAPT species in cerebellum in contrast to frontal lateral cortex and hippocampus in individuals with no signs of dementia. Interpretation: Intron retention constitutes a potential mechanism to generate Tau isoforms whose mature RNA expression levels correlate with Alzheimer's pathology showing its potential as a biomarker associated to the disease. Funding: This research was funded by the Spanish Ministry of Science, Innovation and Universities: PGC2018-096177-B-I00 (J.A.); Spanish Ministry of Science and Innovation (MCIN): PID2020-113204GB-I00 (F.H.) and PID2021-123859OB-100 from MCIN/AEI/10.13039/501100011033/FEDER, UE (J.A.). It was also supported by CSIC through an intramural grant (201920E104) (J.A.) and the Centre for Networked Biomedical Research on Neurodegenerative Diseases (J.A.). The Centro de Biología Molecular Severo Ochoa (CBMSO) is a Severo Ochoa Center of Excellence (MICIN, award CEX2021-001154-S).

Simple Summary: Alternative splicing generates transcripts that can promote tumorigenesis, but intron retention (IR), a form of alternative splicing, remains understudied. We investigated the role of IR in tumorigenesis and its potential clinical applications at the pan-cancer level. We identified IR events from The Cancer Genome Atlas (TCGA) that differ between tumor and normal samples, as well as IR events associated with patient survival. These IR events may modulate the expression of host genes, including those that play a role in cancer biology. For example, we experimentally validated a novel IR event within the STN1 5′UTR. The excellent performance of IR-based diagnostic and prognostic models and their biological importance in cancer suggest that IR is a potential biomarker and drug target. Alternative splicing can produce transcripts that affect cancer development and thus shows potential for cancer diagnosis and treatment. However, intron retention (IR), a type of alternative splicing, has been studied less in cancer biology research. Here, we generated a pan-cancer IR landscape for more than 10,000 samples across 33 cancer types from The Cancer Genome Atlas (TCGA). We characterized differentially retained introns between tumor and normal samples and identified retained introns associated with survival. We discovered 988 differentially retained introns in 14 cancers, some of which demonstrated diagnostic potential in multiple cancer types. We also inferred a large number of prognosis-related introns in 33 cancer types, and the associated genes included well-known cancer hallmarks such as angiogenesis, metastasis, and DNA mutations. Notably, we discovered a novel intron retention inside the 5′UTR of STN1 that is associated with the survival of lung cancer patients. The retained intron reduces translation efficiency by producing upstream open reading frames (uORFs) and thereby inhibits colony formation and cell migration of lung cancer cells. Besides, the IR-based prognostic model achieved good stratification in certain cancers, as illustrated in acute myeloid leukemia. Taken together, we performed a comprehensive IR survey at a pan-cancer level, and the results implied that IR has the potential to be diagnostic and prognostic cancer biomarkers, as well as new drug targets. [ABSTRACT FROM AUTHOR]

Substantial evidence highlights divergences in immune responses between men and women. Women are more susceptible to autoimmunity, whereas men suffer from the more severe presentation of autoimmune disorders. The molecular mechanism of this sexual dimorphism remains elusive. Herein, we conducted a comprehensive analysis of sex differences in whole-blood gene expression focusing on alternative splicing (AS) events in systemic lupus erythematosus (SLE), which is a prototype sex-biased disease. This study included 79 SLE patients with active disease and 58 matched healthy controls who underwent whole-blood RNA sequencing. Sex differences in splicing events were widespread, existent in both SLE and a healthy state. However, we observed distinct gene sets and molecular pathways targeted by sex-dependent AS in SLE patients as compared to healthy subjects, as well as a notable sex dissimilarity in intron retention events. Sexually differential spliced genes specific to SLE patients were enriched for dynamic cellular processes including chromatin remodeling, stress and inflammatory responses. Remarkably, the extent of sexual differences in AS in the SLE patients and healthy individuals exceeded those in gene expression. Overall, this study reveals an unprecedent variation in sex-dependent splicing events in SLE and the healthy state, with potential implications for understanding the molecular basis of sexual dimorphism in autoimmunity. [ABSTRACT FROM AUTHOR]

Background: Amelogenesis imperfecta (AI) is a developmental enamel defect affecting the structure of enamel, esthetic appearance, and the tooth masticatory function. Gene mutations are reported to be relevant to AI. However, the mechanism underlying AI caused by different mutations is still unclear. This study aimed to reveal the molecular pathogenesis in AI families with 2 novel pre-mRNA splicing mutations. Methods: Two Chinese families with AI were recruited. Whole-exome sequencing and Sanger sequencing were performed to identify mutations in candidate genes. Minigene splicing assays were performed to analyze the mutation effects on mRNA splicing alteration. Furthermore, three-dimensional structures of mutant proteins were predicted by AlphaFold2 to evaluate the detrimental effect. Results: The affected enamel in family 1 was thin, rough, and stained, which was diagnosed as hypoplastic-hypomature AI. Genomic analysis revealed a novel splicing mutation (NM_001142.2: c.570 + 1G > A) in the intron 6 of amelogenin (AMELX) gene in family 1, resulting in a partial intron 6 retention effect. The proband in family 2 exhibited a typical hypoplastic AI, and the splicing mutation (NM_031889.2: c.123 + 4 A > G) in the intron 4 of enamelin (ENAM) gene was observed in the proband and her father. This mutation led to exon 4 skipping. The predicted structures showed that there were obvious differences in the mutation proteins compared with wild type, leading to impaired function of mutant proteins. Conclusions: In this study, we identified two new splicing mutations in AMELX and ENAM genes, which cause hypoplastic-hypomature and hypoplastic AI, respectively. These results expand the spectrum of genes causing AI and broaden our understanding of molecular genetic pathology of enamel formation. [ABSTRACT FROM AUTHOR]

Summary: The regulatory roles of RNA splicing in plant immunity are emerging but still largely obscure. We reported previously that Phytophthora pathogen effector Avr3c targets a soybean protein SKRP (serine/lysine/arginine‐rich protein) to impair soybean basal immunity by regulating host pre‐mRNA alternative splicing, while the biochemical nature of SKRP remains unknown.Here, by using Arabidopsis as a model, we studied the mechanism of SKRP in regulating pre‐mRNA splicing and plant immunity.AtSKRP confers impaired plant immunity against Phytophthora capsici and associates with spliceosome component PRP8 and splicing factor SR45, which positively and negatively regulate plant immunity, respectively. Enhanced crosslinking and immunoprecipitation followed by high‐throughput sequencing (eCLIP‐seq) showed AtSKRP is a novel RNA‐binding protein that targets exon 3′ end of unspliced RNA. Such position‐specific binding of SKRP is associated with its activity in suppressing intron retention, including at positive immune regulatory genes UBP25 and RAR1. In addition, we found AtSKRP self‐interact and forms oligomer, and these properties are associated with its function in plant immunity.Overall, our findings reveal that the immune repressor SKRP is a spliceosome‐associated protein that targets exon 3′ end to regulate pre‐mRNA splicing in Arabidopsis. [ABSTRACT FROM AUTHOR]

The monocarboxylate transporter 4 (MCT4; Slc16a3) is expressed in the central nervous system, notably by astrocytes. It is implicated in lactate release and the regulation of glycolytic flux. Whether its expression varies during normal and/or pathological aging is unclear. As the presence of its mature transcript in the brain of young and old mice was determined, an unexpectedly longer RT-PCR fragment was detected in the mouse frontal cortex and hippocampus at 12 vs. 3 months of age. Cultured astrocytes expressed the expected 516 base pair (bp) fragment but treatment with IL-1β to mimic inflammation as can occur during aging led to the additional expression of a 928 bp fragment like that seen in aged mice. In contrast, cultured pericytes (a component of the blood–brain barrier) only exhibited the 516 bp fragment. Intriguingly, cultured endothelial cells constitutively expressed both fragments. When RT-PCR was performed on brain subregions of an Alzheimer mouse model (APPswePS1dE9), no fragment was detected at 3 months, while only the 928 bp fragment was present at 12 months. Sequencing of MCT4 RT-PCR products revealed the presence of a remaining intron between exon 2 and 3, giving rise to the longer fragment detected by RT-PCR. These results unravel the existence of intron retention for the MCT4 gene in the central nervous system. Such alternative splicing appears to increase with age in the brain and might be prominent in neurodegenerative diseases such as Alzheimer's disease. Hence, further studies in vitro and in vivo of intron 2 retention in the Slc16a3 gene transcript are required for adequate characterization concerning the biological roles of Slc16a3 isoforms in the context of aging and Alzheimer's disease pathology. [ABSTRACT FROM AUTHOR]

Human transcriptome can undergo RNA mis-splicing due to spliceopathies contributing to the increasing number of genetic diseases including muscular dystrophy (MD), Alzheimer disease (AD), Huntington disease (HD), myelodysplastic syndromes (MDS). Intron retention (IR) is a major inducer of spliceopathies where two or more introns remain in the final mature mRNA and account for many intronic expansion diseases. Potential removal of such introns for therapeutic purposes can be feasible when utilizing bioinformatics, catalytic RNAs, and nano-drug delivery systems. Overcoming delivery challenges of catalytic RNAs was discussed in this review as a future perspective highlighting the significance of utilizing synthetic biology in addition to high throughput deep sequencing and computational approaches for the treatment of mis-spliced transcripts.

OsPLS4 encodes a β-ketoacyl carrier protein reductase (KAR). The role of OsPLS4 in rice sheath blight (Rhizoctonia solani) remains unclear. Our preliminary studies showed that premature leaf senescence mutants (pls4) were highly susceptive to sheath blight in the early stage of rice development. To explore the role of this gene in the development of rice sheath blight, the transcriptome profiles of the rice pls4 mutant and wild type were compared by RNA-seq. The results revealed 2,569 differentially expressed genes (DEGs). The down-regulated genes were significantly enriched in the defense response-related biological processes. These down-regulated genes included the chitinase genes and WRKY genes, which were significantly changed in pls4 mutants. Furthermore, 467 genes induced significant alternative splicing (AS) events. Among them, intron retention (IR) affected gene expression levels and functions of the vitamin B6 (VB6) metabolism pathway related to sheath blight. This result suggests that IR plays an important role in the sheath blight resistance of mutant pls4. Together, these results indicate that pls4 could be involved in the biological process of sheath blight via DEGs and the fine-tuning of IR. The present study provides a molecular basis for further investigation of the resistance of rice to sheath blight. [ABSTRACT FROM AUTHOR]

The decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1 kb). Splicing reporter studies have shown that decoy splice sites are essential for activity, suggesting that decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The decoy model predicts that antisense oligonucleotides may be able to block decoy splice sites in endogenous pre-mRNA, thereby reducing IR and increasing productive gene expression. Indeed, we now demonstrate that targeting a decoy 5' splice site in the O-GlcNAc transferase (OGT) gene reduced IR from ∼80% to ∼20% in primary human erythroblasts, accompanied by increases in spliced OGT RNA and OGT protein expression. The remaining OGT IR was refractory to antisense treatment and might be mediated by independent mechanism(s). In contrast, other retained introns were strongly dependent on decoy function, since antisense targeting of decoy 5' splice sites greatly reduced (SNRNP70) or nearly eliminated (SF3B1) IR in two widely expressed splicing factors, and also greatly reduced IR in transcripts encoding the erythroid-specific structural protein, α-spectrin (SPTA1). These results show that modulating decoy exon function can dramatically alter IR and suggest that dynamic regulation of decoy exons could be a mechanism to fine-tune gene expression post-transcriptionally in many cell types.

Abstract Background Alternative splicing is a widespread regulatory phenomenon that enables a single gene to produce multiple transcripts. Among the different types of alternative splicing, intron retention is one of the least explored despite its high prevalence in both plants and animals. The recent discovery that the majority of splicing is co-transcriptional has led to the finding that chromatin state affects alternative splicing. Therefore, it is plausible that transcription factors can regulate splicing outcomes. Results We provide evidence for the hypothesis that transcription factors are involved in the regulation of intron retention by studying regions of open chromatin in retained and excised introns. Using deep learning models designed to distinguish between regions of open chromatin in retained introns and non-retained introns, we identified motifs enriched in IR events with significant hits to known human transcription factors. Our model predicts that the majority of transcription factors that affect intron retention come from the zinc finger family. We demonstrate the validity of these predictions using ChIP-seq data for multiple zinc finger transcription factors and find strong over-representation for their peaks in intron retention events. Conclusions This work opens up opportunities for further studies that elucidate the mechanisms by which transcription factors affect intron retention and other forms of splicing. Availability Source code available at https://github.com/fahadahaf/chromir

Background & Aims: Hepatoblastoma (HB) is a common pediatric malignant liver tumor that is characterized by a low level of genetic mutations. Alternative splicing (AS) has been shown to be closely associated with cancer progression, especially in tumors with a low mutational burden. However, the role of AS in HB remains unknown. Methods: Transcriptome sequencing was performed on 5 pairs of HB tissues and matched non-tumor tissues to delineate the AS landscape in HB. AS events were validated in 92 samples from 46 patients. RNA pull-down and RNA immunoprecipitation assays were carried out to identify splicing factors that regulate the AS of small nucleolar RNA host genes (SNHG). Patient-derived organoids (PDOs) were established to investigate the role of the splicing factor polyadenylate-binding nuclear protein 1 (PABPN1). Results: This study uncovered aberrant alternative splicing in HB, including lncRNAs from SNHG family that undergo intron retention in HB. Further investigations revealed that PABPN1, a significantly upregulated RNA binding protein, interacts with splicing machinery in HB, inducing the intron retention of these SNHG RNAs and the downregulation of intronic small nucleolar RNAs (snoRNAs). Functionally, PABPN1 acts as an oncofetal splicing regulator in HB by promoting cell proliferation and DNA damage repair via inducing the intron retention of SNHG19. Knock-down of PABPN1 increases the cisplatin sensitivity of HB PDOs. Conclusions: Our findings revealed the role of intron retention in regulating snoRNA expression in hepatoblastoma, explained detailed regulatory mechanism between PABPN1 and the intron retention of SNHG RNAs, and provided insight into the development of new HB treatment options.

Abstract Background There is growing interest in retained introns in a variety of disease contexts including cancer and aging. Many software tools have been developed to detect retained introns from short RNA-seq reads, but reliable detection is complicated by overlapping genes and transcripts as well as the presence of unprocessed or partially processed RNAs. Results We compared introns detected by 8 tools using short RNA-seq reads with introns observed in long RNA-seq reads from the same biological specimens. We found significant disagreement among tools (Fleiss’ $$\kappa = 0.113$$ κ = 0.113 ) such that 47.7% of all detected intron retentions were not called by more than one tool. We also observed poor performance of all tools, with none achieving an F1-score greater than 0.26, and qualitatively different behaviors between general-purpose alternative splicing detection tools and tools confined to retained intron detection. Conclusions Short-read tools detect intron retention with poor recall and precision, calling into question the completeness and validity of a large percentage of putatively retained introns called by commonly used methods.

Summary: Alternative splicing (AS) is a critical means by which plants respond to changes in the environment, but few splicing factors contributing to AS have been reported and functionally characterized in rice (Oryza sativa L.). Here, we explored the function and molecular mechanism of the spliceosome‐associated protein OsFKBP20‐1b during AS.We determined the AS landscape of wild‐type and osfkbp20‐1b knockout plants upon abscisic acid (ABA) treatment by transcriptome deep sequencing. To capture the dynamics of translating intron‐containing mRNAs, we blocked transcription with cordycepin and performed polysome profiling. We also analyzed whether OsFKBP20‐1b and the splicing factors OsSR34 and OsSR45 function together in AS using protoplast transfection assays.We show that OsFKBP20‐1b interacts with OsSR34 and regulates its stability, suggesting a role as a chaperone‐like protein in the spliceosome. OsFKBP20‐1b facilitates the splicing of mRNAs with retained introns after ABA treatment; some of these mRNAs are translatable and encode functional transcriptional regulators of stress‐responsive genes. In addition, interacting proteins, OsSR34 and OsSR45, regulate the splicing of the same retained introns as OsFKBP20‐1b after ABA treatment.Our findings reveal that spliceosome‐associated immunophilin functions in alternative RNA splicing in rice by positively regulating the splicing of retained introns to limit ABA response. [ABSTRACT FROM AUTHOR]

Sacred lotus (Nelumbo nucifera) is a typical aquatic plant, belonging to basal eudicot plant, which is ideal for genome and genetic evolutionary study. Understanding lotus gene diversity is important for the study of molecular genetics and breeding. In this research, public RNA-seq data and the annotated reference genome were used to identify the genes in lotus. A total of 26,819 consensus and 1,081 novel genes were identified. Meanwhile, a comprehensive analysis of gene alternative splicing events was conducted, and a total of 19,983 "internal" alternative splicing (AS) events and 14,070 "complete" AS events were detected in 5,878 and 5,881 multi-exon expression genes, respectively. Observations made from the AS events show the predominance of intron retention (IR) subtype of AS events representing 33%. IR is followed by alternative acceptor (AltA), alternative donor (AltD) and exon skipping (ES), highlighting the universality of the intron definition model in plants. In addition, functional annotations of the gene with AS indicated its relationship to a number of biological processes such as cellular process and metabolic process, showing the key role for alternative splicing in influencing the growth and development of lotus. The results contribute to a better understanding of the current gene diversity in lotus, and provide an abundant resource for future functional genome analysis in lotus. [ABSTRACT FROM AUTHOR]

Peptidyl‐prolyl isomerase‐like 1 (PPIL1) is associated with the human spliceosome complex. However, its function in pre‐mRNA splicing remains unclear. In this study, we show that Arabidopsis thaliana CYCLOPHILIN 18‐2 (AtCYP18‐2), a PPIL1 homolog, plays an essential role in heat tolerance by regulating pre‐mRNA splicing. Under heat stress conditions, AtCYP18‐2 expression was upregulated in mature plants and GFP‐tagged AtCYP18‐2 redistributed to nuclear and cytoplasmic puncta. We determined that AtCYP18‐2 interacts with several spliceosome complex BACT components in nuclear puncta and is primarily associated with the small nuclear RNAs U5 and U6 in response to heat stress. The AtCYP18‐2 loss‐of‐function allele cyp18‐2 engineered by CRISPR/Cas9‐mediated gene editing exhibited a hypersensitive phenotype to heat stress relative to the wild type. Moreover, global transcriptome profiling showed that the cyp18‐2 mutation affects alternative splicing of heat stress–responsive genes under heat stress conditions, particularly intron retention (IR). The abundance of most intron‐containing transcripts of a subset of genes essential for thermotolerance decreased in cyp18‐2 compared to the wild type. Furthermore, the intron‐containing transcripts of two heat stress‐related genes, HEAT SHOCK PROTEIN 101 (HSP101) and HEAT SHOCK FACTOR A2 (HSFA2), produced functional proteins. HSP101‐IR‐GFP localization was responsive to heat stress, and HSFA2‐III‐IR interacted with HSF1 and HSP90.1 in plant cells. Our findings reveal that CYP18‐2 functions as a splicing factor within the BACT spliceosome complex and is crucial for ensuring the production of adequate levels of alternatively spliced transcripts to enhance thermotolerance. [ABSTRACT FROM AUTHOR]

Enhanced Disease Susceptibility 1 (EDS1), a key component of microbe‐triggered immunity and effector‐triggered immunity in most higher plants, forms functional heterodimeric complexes with its homologs Phytoalexin Deficient 4 (PAD4) or Senescence‐associated Gene 101 (SAG101). Here, the crystal structure of VvEDS1Nterm, the N‐terminal domain of EDS1 from Vitis vinifera, is reported, representing the first structure of an EDS1 entity beyond the model plant Arabidopsis thaliana. VvEDS1Nterm has an α/β‐hydrolase fold, is similar to the N‐terminal domain of A. thaliana EDS1 and forms stable homodimers in solution as well as in crystals. These VvEDS1Nterm homodimers are spatially incompatible with heterodimers with PAD4 or SAG101, they explain why VvEDS1Nterm does not interact with V. vinifera PAD4 according to gel filtration, and they serve as a guide to develop a plausible, albeit experimentally not verified model of full‐length EDS1. VvEDS1Nterm is a splicing variant comprising two of three exons of the VvEDS1 gene. It originates from a naturally occurring mRNA, in which the first of two introns was removed while the second one containing a stop codon close to the exon/intron border was retained. This is a potential case of intron retention and the first report of this phenomenon in the context of EDS1. Its biological significance has not yet been clarified, nor has the question if a VvEDS1Nterm protein with a specific function can occur under physiological conditions. PDB Code(s): 8B28; [ABSTRACT FROM AUTHOR]

ABSTRACT Nonsense-mediated decay (NMD) is a conserved mRNA quality control process that eliminates transcripts bearing a premature termination codon. In addition to its role in removing erroneous transcripts, NMD is involved in post-transcriptional regulation of gene expression via programmed intron retention in metazoans. The apicomplexan parasite Plasmodium falciparum shows relatively high levels of intron retention, but it is unclear whether these variant transcripts are functional targets of NMD. In this study, we use CRISPR-Cas9 to disrupt and epitope-tag the P. falciparum orthologs of two core NMD components: PfUPF1 (PF3D7_1005500) and PfUPF2 (PF3D7_0925800). We localize both PfUPF1 and PfUPF2 to puncta within the parasite cytoplasm and show that these proteins interact with each other and other mRNA-binding proteins. Using RNA-seq, we find that although these core NMD orthologs are expressed and interact in P. falciparum, they are not required for degradation of nonsense transcripts. Furthermore, our work suggests that the majority of intron retention in P. falciparum has no functional role and that NMD is not required for parasite growth ex vivo. IMPORTANCE In many organisms, the process of destroying nonsense transcripts is dependent on a small set of highly conserved proteins. We show that in the malaria parasite, these proteins do not impact the abundance of nonsense transcripts. Furthermore, we demonstrate efficient CRISPR-Cas9 editing of the malaria parasite using commercial Cas9 nuclease and synthetic guide RNA, streamlining genomic modifications in this genetically intractable organism.

Post-transcriptional regulation of RNA stability is a key step in gene expression control. We describe a regulatory program, mediated by the RNA binding protein TARBP2, that controls RNA stability in the nucleus. TARBP2 binding to pre-mRNAs results in increased intron retention, subsequently leading to targeted degradation of TARBP2-bound transcripts. This is mediated by TARBP2 recruitment of the m6A RNA methylation machinery to its target transcripts, where deposition of m6A marks influences the recruitment of splicing regulators, inhibiting efficient splicing. Interactions between TARBP2 and the nucleoprotein TPR then promote degradation of these TARBP2-bound transcripts by the nuclear exosome. Additionally, analysis of clinical gene expression datasets revealed a functional role for TARBP2 in lung cancer. Using xenograft mouse models, we find that TARBP2 affects tumor growth in the lung and that this is dependent on TARBP2-mediated destabilization of ABCA3 and FOXN3. Finally, we establish ZNF143 as an upstream regulator of TARBP2 expression.

Abstract Background Dyskerin is a nuclear protein involved in H/ACA box snoRNA-guided uridine modification of RNA. In humans, its defective function is associated with cancer development and induces specific post-transcriptional alterations of gene expression. In this study, we seek to unbiasedly identify mRNAs regulated by dyskerin in human breast cancer-derived cells. Results We find that dyskerin depletion affects the expression and the association with polysomes of selected mRNA isoforms characterized by the retention of H/ACA box snoRNA-containing introns. These snoRNA retaining transcripts (snoRTs) are bound by dyskerin in the cytoplasm in the form of shorter 3′ snoRT fragments. We then characterize the whole cytoplasmic dyskerin RNA interactome and find both H/ACA box snoRTs and protein-coding transcripts which may be targeted by the snoRTs’ guide properties. Since a fraction of these protein-coding transcripts is involved in the nuclear hormone receptor binding, we test to see if this specific activity is affected by dyskerin. Obtained results indicate that dyskerin dysregulation may alter the dependence on nuclear hormone receptor ligands in breast cancer cells. These results are paralleled by consistent observations on the outcome of primary breast cancer patients stratified according to their tumor hormonal status. Accordingly, experiments in nude mice show that the reduction of dyskerin levels in estrogen-dependent cells favors xenograft development in the absence of estrogen supplementation. Conclusions Our work suggests a cytoplasmic function for dyskerin which could affect mRNA post-transcriptional networks relevant for nuclear hormone receptor functions.

STXBP1 variants are one of the most common genetic causes of neurodevelopmental disorders and epilepsy, wherein STXBP1-related disorders are characterized by neurodevelopmental abnormalities in 95% and seizures in 89% of affected patients. However, the spectrums of both genotype and phenotype are quite wide and diverse, with a high baseline variability even for recurrent STXBP1 variants. Until now, no clear genotype-phenotype correlations have been established and multiple disease mechanisms have been proposed for STXBP1-related disorders. Without an ascertained disease cause for many cases of STXBP1 variants, it is challenging to manage this disease in an effective manner and current symptom-based treatments are focused on seizure control only, which has a minimal impact on global development. A novel STXBP1 canonical splice variant, NM_001032221.4:c.578+2T>C, was reported in this study, together with detailed documentation of disease manifestations and treatment management. Further RNA expression analysis revealed abnormal intron retention and possible production of truncated STXBP1 proteins as a likely pathogenic mechanism. More importantly, the landscape of previously understudied STXBP1 splice variants and functional investigations was assessed for the first time to provide a context for the discussion of the complicated genotype-phenotype relationship of STXBP1-related disorders. Future cases of this disorder and a deeper mechanism-based understanding of its pathogenic cause are required for precision medicine and better disease management. [ABSTRACT FROM AUTHOR]

Alternative splicing (AS) is a key post-transcriptional modification that helps in increasing protein diversity. Almost 90% of the protein-coding genes in humans are known to undergo AS and code for different transcripts. Some transcripts are associated with diseases such as breast cancer, lung cancer and glioblastoma. Hence, these transcripts can serve as novel therapeutic and prognostic targets for drug discovery. Herein, we have developed a pipeline, Finding Alternative Splicing Events (FASE), as the R package that includes modules to determine the structure and concentration of transcripts using differential AS. To predict the correct structure of expressed transcripts in given conditions, FASE combines the AS events with the information of exons, introns and junctions using graph theory. The estimated concentration of predicted transcripts is reported as the relative expression in terms of log2CPM. Using FASE, we were able to identify several unique transcripts of EMILIN1 and SLK genes in the TCGA-BRCA data, which were validated using RT-PCR. The experimental study demonstrated consistent results, which signify the high accuracy and precision of the developed methods. In conclusion, the developed pipeline, FASE, can efficiently predict novel transcripts that are missed in general transcript-level differential expression analysis. It can be applied selectively from a single gene to simple or complex genome even in multiple experimental conditions for the identification of differential AS-based biomarkers, prognostic targets and novel therapeutics. [ABSTRACT FROM AUTHOR]

An intron with an IR ratio of 0.5 means that this intron is retained in 50% of the transcripts. Keywords: detained intron; ferns; genome; intron retention; post-transcriptional regulation; Salviniales EN detained intron ferns genome intron retention post-transcriptional regulation Salviniales 1490 1494 5 02/06/23 20230301 NES 230301 Intron retention (IR), a form of alternative splicing, plays a key role in post-transcriptional gene regulation. Our RNA-seq coverage plots clearly showed that there were minimal number of reads (if not none) that span intron-exon junctions (Figs 2b, S1), and all the introns in these 17 genes had low IR ratios across the three time points. [Extracted from the article]

Eukaryotic genomes contain a tiny subset of 'minor class' introns with unique sequence elements that require their own splicing machinery. These minor introns are present in certain gene families with specific functions, such as voltage-gated Na+ and voltage-gated Ca2+ channels. Removal of minor introns by the minor spliceosome has been proposed as a post-transcriptional regulatory layer, which remains unexplored in the heart. Here, we investigate whether the minor spliceosome regulates electrophysiological properties of cardiomyocytes by knocking down the essential minor spliceosome small nuclear snRNA component U6atac in neonatal rat ventricular myocytes. Loss of U6atac led to robust minor intron retention within Scn5a and Cacna1c, resulting in reduced protein levels of Nav1.5 and Cav1.2 channels. Functional consequences were studied through patch-clamp analysis, and revealed reduced Na+ and L-type Ca2+ currents after loss of U6atac. In conclusion, minor intron splicing modulates voltage-dependent ion channel expression and function in cardiomyocytes. This may be of particular relevance in situations in which minor splicing activity changes, such as in genetic diseases affecting minor spliceosome components, or in acquired diseases in which minor spliceosome components are dysregulated, such as heart failure. [ABSTRACT FROM AUTHOR]

Alternative splicing (AS) and intron retention (IR) implicated in multiple pathophysiological processes, have rarely been reported in systemic sclerosis (SSc). We integrated bulk RNA-seq and 4D label-free mass spectrometry to perform a multi-omics analysis of AS and IR in SSc skin tissue and fibroblasts. RMATS and iREAD were used to identify AS and IR, which were validated by real-time PCR. Spearman correlation and the LASSO method were employed to assess correlations among clinical features, introns, splicing factors (regulators of AS) and proteins. AS profiles showed distinct alterations in SSc skin tissue, with the most pronounced changes occurring in IR. AS and IR were associated with total modified Rodnan skin score (mRSS) and local skin score. Upon TGF-β stimulation, fibroblasts exhibited significant alterations in IR profiles, affecting genes related to fibroblast proliferation and collagen fibril organization. A comprehensive integrated analysis of introns, exons, and proteome profiles revealed that IR exerted a negative impact on protein expression, with certain changes being under intronic control. RT-PCR confirmed the presence of intron and exon-derived sequences of CTTN, OGA, MED16 and PHYKPL. Additionally, notable changes were observed in the regulatory network of splicing factors in SSc skin tissues. These factors are also involved in fibrosis pathways and correlated with clinical features. Totally, abnormal AS, IR profiles and splicing factors were identified in SSc, altered IRs and splicing factors participated in fibrosis-related pathways. IR exerted a negative impact on protein expression in TGF-β-stimulated fibroblasts. Clarification of the IR mechanisms will provide new insights into the pathophysiology of SSc. A genome-wide analysis of alternative splicing (AS) and intron retention (IR) was conducted across systemic sclerosis skin (SSc = 48, HC = 33) and TGF-β stimulated fibroblasts based on bulk RNA-seq and 4D label-free mass spectrometry. AS and IR profiles were identified and functional analysis was conducted through Gene Ontology enrichment of gene with differential expressed AS and IR. The correlations between AS/IR and clinical features were assessed by spearman correlation and t -test. LASSO method was employed to assess the correlation between splicing factors and IR. IR profiles were integrated with proteome and transcriptome analysis. Specific IR and protein were validated by RT-PCR and Western blot. [Display omitted] • Alterations of AS/IR were identified across SSc skin and TGF-β-stimulated fibroblasts, related to skin score and ECM score. • Splicing factors were dysregulated and related to IR regulation in the process of skin fibrosis. • IR had a negative impact on protein and were implicated in the processes associated with fibrosis. [ABSTRACT FROM AUTHOR]

Substantial evidence highlights divergences in immune responses between men and women. Women are more susceptible to autoimmunity, whereas men suffer from the more severe presentation of autoimmune disorders. The molecular mechanism of this sexual dimorphism remains elusive. Herein, we conducted a comprehensive analysis of sex differences in whole-blood gene expression focusing on alternative splicing (AS) events in systemic lupus erythematosus (SLE), which is a prototype sex-biased disease. This study included 79 SLE patients with active disease and 58 matched healthy controls who underwent whole-blood RNA sequencing. Sex differences in splicing events were widespread, existent in both SLE and a healthy state. However, we observed distinct gene sets and molecular pathways targeted by sex-dependent AS in SLE patients as compared to healthy subjects, as well as a notable sex dissimilarity in intron retention events. Sexually differential spliced genes specific to SLE patients were enriched for dynamic cellular processes including chromatin remodeling, stress and inflammatory responses. Remarkably, the extent of sexual differences in AS in the SLE patients and healthy individuals exceeded those in gene expression. Overall, this study reveals an unprecedent variation in sex-dependent splicing events in SLE and the healthy state, with potential implications for understanding the molecular basis of sexual dimorphism in autoimmunity.

Intron Retention (IR) is an alternative splicing mode through which introns are retained in mature RNAs rather than being spliced in most cases. IR has been gaining increasing attention in recent years because of its recognized association with gene expression regulation and complex diseases. Continuous efforts have been dedicated to the development of IR detection methods. These methods differ in their metrics to quantify retention propensity, performance to detect IR events, functional enrichment of detected IRs, and computational speed. A systematic experimental comparison would be valuable to the selection and use of existing methods. In this work, we conduct an experimental comparison of existing IR detection methods. Considering the unavailability of a gold standard dataset of intron retention, we compare the IR detection performance on simulation datasets. Then, we compare the IR detection results with real RNA-Seq data. We also describe the use of differential analysis methods to identify disease-associated IRs and compare differential IRs along with their Gene Ontology enrichment, which is illustrated on an Alzheimer’s disease RNA-Seq dataset. We discuss key principles and features of existing approaches and outline their differences. This systematic analysis provides helpful guidance for interrogating transcriptomic data from the point of view of IR.

During terminal erythropoiesis, the splicing machinery in differentiating erythroblasts executes a robust intron retention (IR) program that impacts expression of hundreds of genes. We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ∼50% of its transcripts in late erythroblasts as a nuclear isoform that retains intron 4. RNA-seq analysis of nonsense-mediated decay (NMD)-inhibited cells revealed previously undescribed splice junctions, rare or not detected in normal cells, that connect constitutive exons 4 and 5 to highly conserved cryptic cassette exons within the intron. Minigene splicing reporter assays showed that these cassettes promote IR. Genome-wide analysis of splice junction reads demonstrated that cryptic noncoding cassettes are much more common in large (>1 kb) retained introns than they are in small retained introns or in nonretained introns. Functional assays showed that heterologous cassettes can promote retention of intron 4 in the SF3B1 splicing reporter. Although many of these cryptic exons were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that these exons function as decoys that engage the intron-terminal splice sites, thereby blocking cross-intron interactions required for excision. Developmental regulation of decoy function underlies a major component of the erythroblast IR program.

Eukaryotic genomes contain a tiny subset of 'minor class' introns with unique sequence elements that require their own splicing machinery. These minor introns are present in certain gene families with specific functions, such as voltage-gated Na+ and voltage-gated Ca2+ channels. Removal of minor introns by the minor spliceosome has been proposed as a post-transcriptional regulatory layer, which remains unexplored in the heart. Here, we investigate whether the minor spliceosome regulates electrophysiological properties of cardiomyocytes by knocking down the essential minor spliceosome small nuclear snRNA component U6atac in neonatal rat ventricular myocytes. Loss of U6atac led to robust minor intron retention within Scn5a and Cacna1c, resulting in reduced protein levels of Nav1.5 and Cav1.2 channels. Functional consequences were studied through patch-clamp analysis, and revealed reduced Na+ and L-type Ca2+ currents after loss of U6atac. In conclusion, minor intron splicing modulates voltage-dependent ion channel expression and function in cardiomyocytes. This may be of particular relevance in situations in which minor splicing activity changes, such as in genetic diseases affecting minor spliceosome components, or in acquired diseases in which minor spliceosome components are dysregulated, such as heart failure. [ABSTRACT FROM AUTHOR]

PURPL is a p53-induced lncRNA that suppresses basal p53 levels. Here, we investigated PURPL upon p53 activation in liver cancer cells, where it is expressed at significantly higher levels than other cell types. Using isoform sequencing, we discovered novel PURPL transcripts that have a retained intron and/or previously unannotated exons. To determine PURPL function upon p53 activation, we performed transcriptome sequencing (RNA-Seq) after depleting PURPL using CRISPR interference (CRISPRi), followed by Nutlin treatment to induce p53. Strikingly, although loss of PURPL in untreated cells altered the expression of only 7 genes, loss of PURPL resulted in altered expression of ~ 800 genes upon p53 activation, revealing a context-dependent function of PURPL. Pathway analysis suggested that PURPL is important for finetuning the expression of specific genes required for mitosis. Consistent with these results, we observed a significant decrease in the percentage of mitotic cells upon PURPL depletion. Collectively, these data identify novel transcripts from the PURPL locus and suggest that PURPL delicately moderates the expression of mitotic genes in the context of p53 activation to control cell cycle arrest. [ABSTRACT FROM AUTHOR]