Splicing Machinery Research Articles

An overview of cdc2-like kinase 1 (Clk1) inhibitors and their therapeutic indications.

Over the past decade, Clk1 has been identified as a promising target for the treatment of various diseases, in which deregulated alternative splicing plays a role. First small molecules targeting Clk1 are in clinical trials for the treatment of solid cancer, where variants of oncogenic proteins derived from alternative splicing promote tumor progression. Since many infectious pathogens hi-jack the host cell's splicing machinery to ensure efficient replication, further indications in this area are under investigation, such as Influenza A, HIV-1 virus, and Trypanosoma infections, and more will likely be discovered in the future. In addition, Clk1 was found to contribute to the progression of Alzheimer's disease through causing an imbalance of tau splicing products. Interestingly, homozygous Clk1 knockout mice showed a rather mild phenotype, opposed to what might be expected in view of the profound role of Clk1 in alternative splicing. A major drawback of most Clk1 inhibitors is their insufficient selectivity; in particular, Dyrk kinases and haspin were frequently identified as off-targets, besides the other Clk isoforms. Only few inhibitors were shown to be selective over Dyrk1A and haspin, whereas no Clk1 inhibitor so far achieved selectivity over the Clk4 isoform. In this review, we carefully compiled all Clk1 inhibitors from the scientific literature and summarized their structure-activity relationships (SAR). In addition, we critically discuss the available selectivity data and describe the inhibitor's efficacy in cellular models, if reported. Thus, we provide a comprehensive overview on the current state of Clk1 drug discovery and highlight the most promising chemotypes.

HiFENS: high-throughput FISH detection of endogenous pre-mRNA splicing isoforms.

Splicing factors play an essential role in regulation of alternative pre-mRNA splicing. While much progress has been made in delineating the mechanisms of the splicing machinery, the identity of signal transduction pathways and upstream factors that regulate splicing factor activity is largely unknown. A major challenge in the discovery of upstream regulatory factors of pre-mRNA splicing is the scarcity of functional genomics screening methods to monitor splicing outcomes of endogenous genes. Here, we have developed HiFENS (high throughput FISH detection of endogenous splicing isoforms), a high-throughput imaging assay based on hybridization chain reaction (HCR) and used HiFENS to screen for cellular factors that regulate alternative splicing of endogenous genes. We demonstrate optimized detection with high specificity of endogenous splicing isoforms and multiplexing of probes for accurate detection of splicing outcomes with single cell resolution. As proof-of-principle, we perform an RNAi screen of 702 human kinases and identify potential candidate upstream splicing regulators of the FGFR2 gene. HiFENS should be a useful tool for the unbiased delineation of cellular pathways involved in alternative splicing regulation.

Investigation of the effect of meclofenamic acid on the proteome of LNCaP cells reveals changes in alternative polyadenylation and splicing machinery

Investigation of the effect of meclofenamic acid on the proteome of LNCaP cells reveals changes in alternative polyadenylation and splicing machinery

Regulatory Network of Serine/Arginine-Rich (SR) Proteins: The Molecular Mechanism and Physiological Function in Plants.

Serine/arginine-rich (SR) proteins are a type of splicing factor. They play significant roles in constitutive and alternative pre-mRNA splicing, and are involved in post-splicing activities, such as mRNA nuclear export, nonsense-mediated mRNA decay, mRNA translation, and miRNA biogenesis. In plants, SR proteins function under a complex regulatory network by protein–protein and RNA–protein interactions between SR proteins, other splicing factors, other proteins, or even RNAs. The regulatory networks of SR proteins are complex—they are regulated by the SR proteins themselves, they are phosphorylated and dephosphorylated through interactions with kinase, and they participate in signal transduction pathways, whereby signaling cascades can link the splicing machinery to the exterior environment. In a complex network, SR proteins are involved in plant growth and development, signal transduction, responses to abiotic and biotic stresses, and metabolism. Here, I review the current status of research on plant SR proteins, construct a model of SR proteins function, and ask many questions about SR proteins in plants.

Exploring the mechanistic link between SF3B1 mutation and ring sideroblast formation in myelodysplastic syndrome

Acquired sideroblastic anemia, characterized by bone marrow ring sideroblasts (RS), is predominantly associated with myelodysplastic syndrome (MDS). Although somatic mutations in splicing factor 3b subunit 1 (SF3B1), which is involved in the RNA splicing machinery, are frequently found in MDS-RS, the detailed mechanism contributing to RS formation is unknown. To explore the mechanism, we established human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells stably expressing SF3B1K700E. SF3B1K700E expressing cells showed higher proportion of RS than the control cells along with erythroid differentiation, indicating the direct contribution of mutant SF3B1 expression in erythroblasts to RS formation. In SF3B1K700E expressing cells, ABCB7 and ALAS2, known causative genes for congenital sideroblastic anemia, were downregulated. Additionally, mis-splicing of ABCB7 was observed in SF3B1K700E expressing cells. ABCB7-knockdown HUDEP-2 cells revealed an increased frequency of RS formation along with erythroid differentiation, demonstrating the direct molecular link between ABCB7 defects and RS formation. ALAS2 protein levels were obviously decreased in ABCB7-knockdown cells, indicating decreased ALAS2 translation owing to impaired Fe–S cluster export by ABCB7 defects. Finally, RNA-seq analysis of MDS clinical samples demonstrated decreased expression of ABCB7 by the SF3B1 mutation. Our findings contribute to the elucidation of the complex mechanisms of RS formation in MDS-RS.

New insights into pathogen-mediated modulation of host RNA splicing

Alternative splicing (AS) regulation of pre-mRNA has been proven to be one of the fundamental layers of plant immune system. How pathogens disrupt plant AS process to suppress plant immunity by secreted effectors remain poorly understood. In the recent study, Gui et al. revealed that a previously identified effector PSR1 of Phytophthora interferes with host RNA splicing machinery to modulate small RNA biogenesis, leading to compromised plant immunity. The study provided a novel insight into the importance of AS process during pathogen-host interactions.

Spliceosomic dysregulation unveils NOVA1 as a candidate actionable therapeutic target in pancreatic neuroendocrine tumors

Dysregulation of the splicing machinery is emerging as a hallmark in cancer due to its association with multiple dysfunctions in tumor cells. Inappropriate function of this machinery can generate tumor-driving splicing variants and trigger oncogenic actions. However, its role in pancreatic neuroendocrine tumors (PanNETs) is poorly defined. In this study we aimed to characterize the expression pattern of a set of splicing machinery components in PanNETs, and their relationship with aggressiveness features. A qPCR-based array was first deployed to determine the expression levels of components of the major (n = 13) and minor spliceosome (n = 4) and associated splicing factors (n = 27), using a microfluidic technology in 20 PanNETs and non-tumoral adjacent samples. Subsequently, in vivo and in vitro models were applied to explore the pathophysiological role of NOVA1. Expression analysis revealed that a substantial proportion of splicing machinery components was altered in tumors. Notably, key splicing factors were overexpressed in PanNETs samples, wherein their levels correlated with clinical and malignancy features. Using in vivo and in vitro assays, we demonstrate that one of those altered factors, NOVA1, is tightly related to cell proliferation, alters pivotal signaling pathways and interferes with responsiveness to drug treatment in PanNETs, suggesting a role for this factor in the aggressiveness of these tumors and its suitability as therapeutic target. Altogether, our results unveil a severe alteration of the splicing machinery in PanNETs and identify the putative relevance of NOVA1 in tumor development/progression, which could provide novel avenues to develop diagnostic biomarkers and therapeutic tools for this pathology.

Wiskott-Aldrich syndrome protein forms nuclear condensates and regulates alternative splicing

The diverse functions of WASP, the deficiency of which causes Wiskott-Aldrich syndrome (WAS), remain poorly defined. We generated three isogenic WAS models using patient induced pluripotent stem cells and genome editing. These models recapitulated WAS phenotypes and revealed that WASP deficiency causes an upregulation of numerous RNA splicing factors and widespread altered splicing. Loss of WASP binding to splicing factor gene promoters frequently leads to aberrant epigenetic activation. WASP interacts with dozens of nuclear speckle constituents and constrains SRSF2 mobility. Using an optogenetic system, we showed that WASP forms phase-separated condensates that encompasses SRSF2, nascent RNA and active Pol II. The role of WASP in gene body condensates is corroborated by ChIPseq and RIPseq. Together our data reveal that WASP is a nexus regulator of RNA splicing that controls the transcription of splicing factors epigenetically and the dynamics of the splicing machinery through liquid-liquid phase separation.

P388: PREFERENTIAL SYNERGISTIC EFFECT OF INDISULAM IN COMBINATION WITH VENETOCLAX AND AZACYTIDINE IN AML CELLS WITH SF3B1 MUTATIONS.

Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with poor prognosis characterized by a high rate of relapse after conventional combination chemotherapy, highlighting the need for improved therapeutic interventions. Genes involved in pre-mRNA splicing, namely SF3B1, U2AF1 and SRSF2, are frequently mutated in AML.Recent studies suggest that pharmacological targeting of the splicing machinery results in preferential lethality of splicing mutant AML cells, potentially providing a novel strategy for treatment of this disease. Existing anti- cancer sulfonamides have been shown to interfere with splicing and induce preferential killing of splicing mutant AML cell lines. A particular sulfonamide drug, indisulam, was previously studied in cancer clinical trials where it was found to be safe but demonstrated limited efficacy, most likely because neither the mechanism of action nor potential biomarkers of response were known. Aims: We investigated, in this study, the efficacy of indisulam, either alone or in combination with ABT-199 (venetoclax) and azacitidine, in cultured primary human AML cells. Methods: Bone marrow cells were collected from adults newly diagnosed with AML, non-treated. Cells were cultured and incubated for 48 and 72 hours with indisulam at a range of nanomolar concentrations. Additionally, cells were incubated with ABT-199 (10nM) and azacitidine (1μM), alone or in combination with indisulam. Cell viability and cell death were measured. For mutation profiling, a targeted myeloid gene panel was used. Results: We found that treatment with indisulam resulted in lethality of primary AML cells harboring mutations in SF3B1, but had minor or no effect on AML cells with other mutations including mutations in splicing factors U2AF1 and SRSF2. In SF3B1-mutated cells, a clear synergistic effect was further observed when indisulam was combined with ABT-199 and azacitidine. The correlation between efficacy of indisulam and synergy with ABT-199 and azacitidine in AML cells with different mutations will be shown and discussed. Summary/Conclusion: Our results indicate that indisulam is preferentially active and synergistic with venetoclax and azacitidine in SF3B1-mutated cells from newly diagnosed, non-treated AML patients.

Selective ablation of 3' RNA ends and processive RTs facilitate direct cDNA sequencing of full-length host cell and viral transcripts.

Alternative splicing (AS) is necessary for viral proliferation in host cells and a critical regulatory component of viral gene expression. Conventional RNA-seq approaches provide incomplete coverage of AS due to their short read lengths and are susceptible to biases and artifacts introduced in prevailing library preparation methodologies. Moreover, viral splicing studies are often conducted separately from host cell transcriptome analysis, precluding an assessment of the viral manipulation of host splicing machinery. To address current limitations, we developed a quantitative full-length direct cDNA sequencing strategy to simultaneously profile viral and host cell transcripts. This nanopore-based approach couples processive reverse transcriptases with a novel one-step chemical ablation of 3′ RNA ends (termed CASPR), which decreases ribosomal RNA reads and enriches polyadenylated coding sequences. We extensively validate our approach using synthetic reference transcripts and show that CASPR doubles the breadth of coverage per transcript and increases detection of long transcripts (>4 kb), while being functionally equivalent to PolyA+ selection for transcript quantification. We used our approach to interrogate host cell and HIV-1 transcript dynamics during viral reactivation and identified novel putative HIV-1 host factors containing exon skipping or novel intron retentions and delineated the HIV-1 transcriptional state associated with these differentially regulated host factors.

Transfer RNA processing- from a structural and disease perspective.

Transfer RNAs (tRNAs) are highly structured non-coding RNAs which play key roles in translation and cellular homeostasis. tRNAs are initially transcribed as precursor molecules and mature by tightly controlled, multistep processes that involve the removal of flanking and intervening sequences, over 100 base modifications, addition of non-templated nucleotides and aminoacylation. These molecular events are intertwined with the nucleocytoplasmic shuttling of tRNAs to make them available at translating ribosomes. Defects in tRNA processing are linked to the development of neurodegenerative disorders. Here, we summarize structural aspects of tRNA processing steps with a special emphasis on intron-containing tRNA splicing involving tRNA splicing endonuclease and ligase. Their role in neurological pathologies will be discussed. Identification of novel RNA substrates of the tRNA splicing machinery has uncovered functions unrelated to tRNA processing. Future structural and biochemical studies will unravel their mechanistic underpinnings and deepen our understanding of neurological diseases.

Abstract 986: RBM10 loss in thyroid cancer leads to aberrant splicing of cytoskeletal and extracellular matrix mRNAs and increased metastatic fitness

Abstract NGS studies implicate dysregulation of the splicing machinery in the development of diverse cancers. The X-chromosome RBM10 gene encodes an RNA-binding protein that modulates transcriptome-wide cassette exon splicing. Truncation and missense RBM10 mutations are enriched (11%) in non-anaplastic thyroid cancers of patients who died of metastatic disease. MSK-MET, an integrated pan-cancer cohort of tumor genomic and clinical outcome data showed RBM10 alterations associate with metastatic burden in thyroid cancer. Within the MSK-IMPACT thyroid cancer cohort, 44% of RBM10 alterations co-occur with RAS mutations. We developed a murine Rbm10 floxed allele, which results in a non-functional transcript. Thyroid-specific Rbm10 inactivation through Tpo-Cre did not induce a phenotype. When crossed with FR-HrasG12V mice, which upon recombination generate endogenous expression of HrasG12V, Hras/Rbm10 mice developed thyroid cancers, ~ 20% of which were metastatic to lung. Cell lines derived from these tumors showed high penetrance of lung metastases after tail vein or orthotopic implantation into the thyroid. We identified splicing targets of RBM10 by high depth RNAseq of 5 isogenic human thyroid cancer cell lines (2 RBM10-null with dox-induced RBM10; 3 RBM10 WT with RBM10 shRNA). The common abnormalities associated with RBM10 loss were exon inclusion events. Ingenuity Pathway Analysis of global transcriptomes in RBM10 isogenic human thyroid cancer cell lines showed enrichment in pro-migratory, aberrant integrin and RHO/RAC signaling expression signatures. Enriched GO analysis confined to genes subject to aberrant splicing by RBM10 loss extended these findings, with the top terms being cell adhesion, cytoskeleton, cadherin and integrin binding. RBM10 loss was associated with increased cell migration velocity in vitro as measured by time lapse imaging. Key cytoskeletal and extracellular matrix genes subject to exon inclusion events included vinculin (VCL), tenascin C (TNC), CD44, fibronectin (FN1) and tropomyosin 1 (TPM1). Isoform-specific knockdown of the VCL splice inclusion cassette exon that leads to illegitimate expression of metavinculin (mVCL) reduced cell migration, whereas isoform-specific knockdown of TNC and CD44 exon inclusion events reduced cell invasiveness in vitro. Rho GTPases transduce signals from the ECM to integrin receptors to regulate cell adhesion, migration, and invasiveness. Consistent with this, RBM10 overexpression in RBM10 null cells reduced RAC1-GTP levels. Finally, RBM10 re-expression in RBM10 null cells reversed metastatic competency in vivo. In conclusion, RBM10 loss alters the ratio of cassette exon inclusion events in a subset of transcripts that regulate interactions between the ECM and the cytoskeleton, leading to RHO/RAC activation and governing a process favoring increased cell movement and metastatic competence. Citation Format: Gnana P. Krishnamoorthy, Anthony Glover, Brian Untch, Mahesh Saqcena, Dina Vukel, Katherine Berman, Omar Abdel-Wahab, Robert K. Bradley, Jeffrey A. Knauf, James A. Fagin. RBM10 loss in thyroid cancer leads to aberrant splicing of cytoskeletal and extracellular matrix mRNAs and increased metastatic fitness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 986.

Abstract 2692: Identification of functional single nucleotide polymorphisms in cryptic exon 3 of the androgen receptor gene

Abstract Castration resistant prostate cancer (CRPC) involves the upregulation of androgen receptor variants (AR-Vs), of which AR-V7 is clinically the most relevant. By lacking the ligand-binding domain, AR-V7 is constitutively active and may therefore bypass AR signaling inhibitors. Treating CRPC involves improved AR signaling inhibitors or taxane-based chemotherapy. However, the optimal treating sequence may differ from patient to patient. The presence of AR-V7 in the nucleus of circulating PCa cells, which is associated with AR targeted therapy resistance, is used as a therapy-guiding biomarker. This stratification has a high predictive power and specificity, but its sensitivity is unsatisfying and can only be done by highly specialized laboratories. A PCR/sequencing based test would improve the sensitivity of this biomarker, but it requires a better understanding of AR-V7’s functional regulation. Therefore, we aim here to analyze single nucleotide polymorphisms (SNPs) in the 3’ untranslated region (UTR) of AR-V7 that might have an impact on AR-V7’s expression and/or functional activity. The LD trait tool of NIH was used to search for trait association with AR cryptic exon (CE3) SNPs. Four RNA binding motif databases (oRNAment, ATtRACT, RBP map, and RBPDB) and SpliceAid v2 were used for evaluating SNPs in their capacity to alter motifs. By CRISPR/CAS9 mediated non-homologous end joining (NHEJ) these predictions were assessed experimentally using qPCR and Westen Blot. SNP rs5918762 was selected as it is in linkage disequilibrium with several PCa tagSNPs (rs5919393, rs5919432). rs5918762T/C is characterized by an unequal minor allele frequency (MAF) distribution among different populations. Using an in silico approach, this SNP is predicted to destroy/create a binding site for the splicing machinery involved protein SRSF9. Disrupting this SNP region via NHEJ resulted respectively in an increased and decreased expression of AR-FL and AR-V7 mRNA in 22RV1 cells (C allele). Additionally, analysis of the SU2C dataset (CRPC patients) correlated SRSF9 expression with AR-V7 expression. Subsequently, SRSF9 knock out (KO) resulted in reduced AR-V7 mRNA expression. Our data suggests that SNP rs5918762T/C is located in a region important for CE3 inclusion during AR splicing, which might involve SRSF9. Further validation of these results is in need and encompasses splicing assays by an AR-V7 mini-gene assay, protein-RNA interaction assays and an analysis of a lack of effect in cell lines expressing the T allele. Citation Format: Jasper Van Goubergen, Florian Handle, Marcus V. Cronauer, Frédéric R. Santer. Identification of functional single nucleotide polymorphisms in cryptic exon 3 of the androgen receptor gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2692.

Phytophthora effector PSR1 hijacks the host pre-mRNA splicing machinery to modulate small RNA biogenesis and plant immunity.

Phytophthora effector PSR1 suppresses small RNA (sRNA)-mediated immunity in plants, but the underlying mechanism remains unknown. Here, we show that Phytophthora suppressor of RNA silencing 1 (PSR1) contributes to the pathogenicity of Phytophthora sojae and specifically binds to three conserved C-terminal domains of the eukaryotic PSR1-Interacting Protein 1 (PINP1). PINP1 encodes PRP16, a core pre-mRNA splicing factor that unwinds RNA duplexes and binds to primary microRNA transcripts and general RNAs. Intriguingly, PSR1 decreased both RNA helicase and RNA-binding activity of PINP1, thereby dampening sRNA biogenesis and RNA metabolism. The PSR1-PINP1 interaction caused global changes in alternative splicing (AS). A total of 5,135 genes simultaneously exhibited mis-splicing in both PSR1-overexpressing and PINP1-silenced plants. AS upregulated many mRNA transcripts that had their introns retained. The high occurrence of intron retention in AS-induced transcripts significantly promoted Phytophthora pathogen infection in Nicotiana benthamiana, and this might be caused by the production of truncated proteins. Taken together, our findings reveal a key role for PINP1 in regulating sRNA biogenesis and plant immunity.

A Chemical Biology Perspective to Therapeutic Regulation of RNA Splicing in Spinal Muscular Atrophy (SMA).

Manipulation of RNA splicing machinery has emerged as a drug modality. Here, we illustrate the potential of this novel paradigm to correct aberrant splicing events focused on the recent therapeutic advances in spinal muscular atrophy (SMA). SMA is an incurable neuromuscular disorder and at present the primary genetic cause of early infant death. This Review summarizes the exciting journey from the first reported SMA cases to the currently approved splicing-switching treatments, i.e., antisense oligonucleotides and small-molecule modifiers. We emphasize both chemical structures and molecular bases for recognition. We briefly discuss the advantages and disadvantages of these treatments and include the remaining challenges and future directions. Finally, we also predict that these success stories will contribute to further therapies for human diseases by RNA-splicing control.

Meta-Analysis Suggests That Intron Retention Can Affect Quantification of Transposable Elements from RNA-Seq Data.

Simple SummaryTransposable elements (TEs) are repetitive sequences comprising more than one third of the human genome with the original ability to change their location within the genome. Owing to their repetitive nature, the quantification of TEs results often challenging. RNA-seq is a useful tool for genome-wide TEs quantification, nevertheless it also presents technical issues, including low reads mappability and erroneous quantification derived from the transcription of TEs fragments embedded in canonical transcripts. Fragments derived from TEs are found within the introns of most genes, which led to the hypothesis that intron retention (IR) can affect the unbiased quantification of TEs expression. Performing meta-analysis of public RNA-seq datasets, here we observe that IR can indeed impact the quantification of TEs by increasing the number of reads mapped on intronic TE copies. Our work highlights a correlation between IR and TEs expression measurement by RNA-seq that should be taken into account to achieve reliable TEs quantification, especially in samples characterized by extensive IR, because differential IR might be confused with differential TEs expression.Transposable elements (TEs), also known as “jumping genes”, are repetitive sequences with the capability of changing their location within the genome. They are key players in many different biological processes in health and disease. Therefore, a reliable quantification of their expression as transcriptional units is crucial to distinguish between their independent expression and the transcription of their sequences as part of canonical transcripts. TEs quantification faces difficulties of different types, the most important one being low reads mappability due to their repetitive nature preventing an unambiguous mapping of reads originating from their sequences. A large fraction of TEs fragments localizes within introns, which led to the hypothesis that intron retention (IR) can be an additional source of bias, potentially affecting accurate TEs quantification. IR occurs when introns, normally removed from the mature transcript by the splicing machinery, are maintained in mature transcripts. IR is a widespread mechanism affecting many different genes with cell type-specific patterns. We hypothesized that, in an RNA-seq experiment, reads derived from retained introns can introduce a bias in the detection of overlapping, independent TEs RNA expression. In this study we performed meta-analysis using public RNA-seq data from lymphoblastoid cell lines and show that IR can impact TEs quantification using established tools with default parameters. Reads mapped on intronic TEs were indeed associated to the expression of TEs and influence their correct quantification as independent transcriptional units. We confirmed these results using additional independent datasets, demonstrating that this bias does not appear in samples where IR is not present and that differential TEs expression does not impact on IR quantification. We concluded that IR causes the over-quantification of intronic TEs and differential IR might be confused with differential TEs expression. Our results should be taken into account for a correct quantification of TEs expression from RNA-seq data, especially in samples in which IR is abundant.

Alternative splicing plays a vital role in regulating pollen allergen (Ole e 1) P19963 protein in Gossypium arboreum

Alternative splicing regulation (AS) is an important post-transcriptional gene regulatory mechanism for increasing the complexity and diversity of transcriptomes and subsequently proteomes. In this study, we performed transcriptomic analysis of Gossypium arboreum to explore the process of alternative splicing regulation in pollen allergen (Ole e 1) P19963 protein. The P19963 causes skin allergies because of its acidic properties. The P19963 protein was observed to undergo intron retention at seedling stage (IR); a type of alternative splicing regulation events when intron is not spliced out after the action of splicing machinery. Experimental validation using RT-PCR and sanger sequencing also proved retention of intronic region in the mature mRNA transcript of P19963 protein. We observed that even if this gene was expressing at later stages of plant development i.e., pollen producing stage, but due to alternative splicing it became disrupted at the seedling stage. The non-functionality of protein can be beneficial or harmful but in our case as the gene is having IR and becomes non-functional, it is beneficial as the pollen allergies and skin allergies can be minimized at this stage or not required at all.

Bap1/SMN axis in Dpp4+ skeletal muscle mesenchymal cells regulates the neuromuscular system.

The survival of motor neuron (SMN) protein is a major component of the pre-mRNA splicing machinery and is required for RNA metabolism. Although SMN has been considered a fundamental gene for the central nervous system, due to its relationship with neuromuscular diseases, such as spinal muscular atrophy, recent studies have also revealed the requirement of SMN in non-neuronal cells in the peripheral regions. Here, we report that the fibro-adipogenic progenitor subpopulation expressing Dpp4 (Dpp4+ FAPs) is required for the neuromuscular system. Furthermore, we also reveal that BRCA1-associated protein-1 (Bap1) is crucial for the stabilization of SMN in FAPs by preventing its ubiquitination-dependent degradation. Inactivation of Bap1 in FAPs decreased SMN levels and accompanied degeneration of the neuromuscular junction, leading to loss of motor neurons and muscle atrophy. Overexpression of the ubiquitination-resistant SMN variant, SMNK186R, in Bap1-null FAPs completely prevented neuromuscular degeneration. In addition, transplantation of Dpp4+ FAPs, but not Dpp4– FAPs, completely rescued neuromuscular defects. Our data reveal the crucial role of Bap1-mediated SMN stabilization in Dpp4+ FAPs for the neuromuscular system and provide the possibility of cell-based therapeutics to treat neuromuscular diseases.

RNA splicing is a key mediator of tumour cell plasticity and a therapeutic vulnerability in colorectal cancer

Tumour cell plasticity is a major barrier to the efficacy of targeted cancer therapies but the mechanisms that mediate it are poorly understood. Here, we identify dysregulated RNA splicing as a key driver of tumour cell dedifferentiation in colorectal cancer (CRC). We find that Apc-deficient CRC cells have dysregulated RNA splicing machinery and exhibit global rewiring of RNA splicing. We show that the splicing factor SRSF1 controls the plasticity of tumour cells by controlling Kras splicing and is required for CRC invasion in a mouse model of carcinogenesis. SRSF1 expression maintains stemness in human CRC organoids and correlates with cancer stem cell marker expression in human tumours. Crucially, partial genetic downregulation of Srsf1 does not detrimentally affect normal tissue homeostasis, demonstrating that tumour cell plasticity can be differentially targeted. Thus, our findings link dysregulation of the RNA splicing machinery and control of tumour cell plasticity.

Alternative splicing as a key player in the fine-tuning of the immunity response in Arabidopsis.

Plants, like animals, are constantly exposed to abiotic and biotic stresses, which often inhibit plant growth and development, and cause tissue damage, disease, and even plant death. Efficient and timely response to stress requires appropriate co‐ and posttranscriptional reprogramming of gene expression. Alternative pre‐mRNA splicing provides an important layer of this regulation by controlling the level of factors involved in stress response and generating additional protein isoforms with specific features. Recent high‐throughput studies have revealed that several defence genes undergo alternative splicing that is often affected by pathogen infection. Despite extensive work, the exact mechanisms underlying these relationships are still unclear, but the contribution of alternative protein isoforms to the defence response and the role of regulatory factors, including components of the splicing machinery, have been established. Modulation of gene expression in response to stress includes alternative splicing, chromatin remodelling, histone modifications, and nucleosome occupancy. How these processes affect plant immunity is mostly unknown, but these facets open new regulatory possibilities. Here we provide an overview of the current state of knowledge and recent findings regarding the growing importance of alternative splicing in plant response to biotic stress.