Splicing Defects Research Articles

Alternative splicing regulation by tumor suppressing subtransferable candidate 4: a pathway to tumor suppression.

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

Dysregulated RNA splicing induces regeneration failure in alcohol-associated liver disease.

Individuals with progressive liver failure are at a high risk of mortality without liver transplantation. However, our understanding of derailed regenerative responses in failing livers is limited. Here, we performed comprehensive multi-omic profiling of healthy and diseased human livers using bulk and single-nucleus RNA-plus ATAC-seq. We report that hepatic immune milieu alterations in alcohol-associated liver disease (ALD) prevent hepatocytes from transitioning to a proliferative progenitor-like state, trapping them into an unproductive intermediate state. We discovered striking changes in RNA binding protein (RBP) expression, particularly ESRP, PTBP, and SR families, that cause misregulation of developmentally controlled RNA splicing in ALD. Our data pinpoint ESRP2 as a pivotal disease-sensitive RBP and support a causal role of its deficiency in ALD pathogenesis. Notably, splicing defects in ESRP2-targets Tcf4 and Slk , amongst others, directly alter their nuclear localization and activities, disrupting WNT and Hippo signaling pathways, which are critical for normal liver regeneration. We demonstrate that changes in stromal cell populations enrich failing ALD livers with TGF-β, which suppresses ESRP2-driven epithelial splicing program and replaces functional parenchyma with quasi-progenitor-like cells lacking liver-specific functions. This unprecedented account of transcriptional and post-transcriptional dysregulation in ALD suggests that targeting misspliced RNAs could improve recovery and serve as biomarkers for poor ALD outcomes.

Engineered CRISPR-Base Editors as a Permanent Treatment for Familial Dysautonomia.

Familial dysautonomia (FD) is a fatal inherited congenital neuropathy characterized by both progressive neurological symptoms and systemic abnormalities, and patients have a reduced life expectancy. FD is caused by a T-to-C mutation in intron 20 of the Elongator acetyltransferase complex subunit 1 ( ELP1 ) gene, which affects the ELP1 splicing process by causing tissue-specific skipping of exon 20. Here, we developed a CRISPR base editor (BE) approach capable of precisely correct this mutation. Using Cas9 variants and screening multiple gRNAs, we selected an optimized BE combination that was able to promote up to 70% on-target editing in HEK293T cells harboring the ELP1 T-to-C mutation. These editing levels were sufficient to rescue more than 50% of ELP1 transcripts with precise exon 20 inclusion. Moreover, an engineered dual intein-split system was optimized to deliver these constructs in vivo . We packaged these novel constructs in adeno-associated virus (AAV) vectors for further testing in iPSC-derived sympathetic neurons and a mouse model harboring the human ELP1 T6C gene. Our strategy could effectively correct the ELP1 splicing defects in vivo and culminated in phenotypic recovery in human neurons. Minimal off-target editing was observed demonstrating high levels of specificity with these optimized base editors. Therefore, we engineered and validated novel base editor approaches to correct the ELP1 TC6 mutation and ELP1 splicing defect and provided essential proof of concept data towards a permanent treatment for FD.

Modeling of TDP-43 proteinopathy by chronic oxidative stress identifies rapamycin as beneficial in ALS patient-derived 2D and 3D iPSC models

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterized neuropathologically by TDP-43 proteinopathy with loss of TDP-43 nuclear splicing activity and formation of cytoplasmic TDP-43 aggregates. The lack of suitable experimental models of TDP-43 proteinopathy has hampered the discovery of effective therapies. We already showed that chronic and mild oxidative insult by sodium arsenite (ARS) triggered TDP-43 cytoplasmic aggregation and stress granules (SGs) formation in ALS patient-derived fibroblasts and motor neurons differentiated from induced pluripotent stem cells (iPSC-MNs). However, whether this insult induces a reduction of TDP-43 splicing activity in the nucleus, thus recapitulating both gain and loss of function pathomechanisms, still remains to be determined.In this study we first showed that chronic ARS in human neuroblastoma cells triggered TDP-43 cytoplasmic mislocalization, SGs formation and defective splicing of TDP-43 target genes UNC13A and POLDIP3 as functional readouts of TDP-43 proteinopathy. Additionally, a dysregulation of autophagy and senescence markers was observed in this condition. In a preliminary drug screening approach with autophagy-promoting drugs, namely rapamycin, lithium carbonate and metformin, only rapamycin prevented ARS-induced loss of TDP-43 splicing activity. We then demonstrated that, in addition to TDP-43 cytoplasmic aggregation, chronic ARS triggered TDP-43 loss of splicing activity also in ALS patient-derived primary fibroblasts and iPSC-MNs and that rapamycin was beneficial to reduce these TDP-43 pathological features. By switching to a neuro-glial 3D in vitro model, we observed that treatment of ALS iPSC-brain organoids with chronic ARS also induced a defective TDP-43 splicing activity which was prevented by rapamycin.Collectively, we established different human cell models of TDP-43 proteinopathy which recapitulate TDP-43 gain and loss of function, prevented by rapamycin administration. Human neuroblastoma cells and patient-derived fibroblasts and 2D- and 3D-iPSC models exposed to chronic oxidative stress represent therefore suitable in vitro platforms for future drug screening approaches in ALS.

A defective splicing machinery promotes senescence through MDM4 alternative splicing.

Defects in the splicing machinery are implicated in various diseases, including cancer. We observed a general reduction in the expression of spliceosome components and splicing regulators in human cell lines undergoing replicative, stress-induced, and telomere uncapping-induced senescence. Supporting the view that defective splicing contributes to senescence, splicing inhibitors herboxidiene, and pladienolide B induced senescence in normal and cancer cell lines. Furthermore, depleting individual spliceosome components also promoted senescence. All senescence types were associated with an alternative splicing transition from the MDM4-FL variant to MDM4-S. The MDM4 splicing shift was reproduced when splicing was inhibited, and spliceosome components were depleted. While decreasing the level of endogenous MDM4 promoted senescence and cell survival independently of the MDM4-S expression status, cell survival was also improved by increasing MDM4-S. Overall, our work establishes that splicing defects modulate the alternative splicing of MDM4 to promote senescence and cell survival.

Psr1 phosphatase regulates pre-mRNA splicing through spliceosomal B complex factor Snu66.

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

The MTR4/hnRNPK complex surveils aberrant polyadenylated RNAs with multiple exons

RNA surveillance systems degrade aberrant RNAs that result from defective transcriptional termination, splicing, and polyadenylation. Defective RNAs in the nucleus are recognized by RNA-binding proteins and MTR4, and are degraded by the RNA exosome complex. Here, we detect aberrant RNAs in MTR4-depleted cells using long-read direct RNA sequencing and 3′ sequencing. MTR4 destabilizes intronic polyadenylated transcripts generated by transcriptional read-through over one or more exons, termed 3′ eXtended Transcripts (3XTs). MTR4 also associates with hnRNPK, which recognizes 3XTs with multiple exons. Moreover, the aberrant protein translated from KCTD13 3XT is a target of the hnRNPK-MTR4-RNA exosome pathway and forms aberrant condensates, which we name KCTD13 3eXtended Transcript-derived protein (KeXT) bodies. Our results suggest that RNA surveillance in human cells inhibits the formation of condensates of a defective polyadenylated transcript-derived protein.

A systematic review and meta-analysis of GFAP gene variants in Alexander disease

Alexander disease (ALXDRD) is a rare neurodegenerative disorder of astrocytes resulting from pathogenic variants in the GFAP gene. The genotype-phenotype correlation remains elusive due to the variable expressivity of clinical manifestations. In an attempt to clarify the effects of GFAP variants in ALXDRD, numerous studies were collected and analyzed. In particular, we systematically searched for GFAP variants associated with ALXDRD and collected information on the location within the gene and protein, prediction of deleteriousness/pathogenicity, occurrence, sex and country of origin of patients, DNA source, genetic testing, and clinical signs. To identify possible associations, statistical analyses and meta-analyses were applied, thus revealing a higher than expected percentage of adult patients with ALXDRD. Furthermore, substitution of Arginine, the most frequently altered residue among the 550 predominantly missense causative GFAP variants collected, were mostly de novo and more prevalent in early-onset forms of ALXDRD. The effect of defective splicing in modifying the impact of GFAP variants on the age of onset of ALXDRD was also postulated after evaluating the distribution of the corresponding deleterious predictive values. In conclusion, not only previously unrecognized genotype-phenotype correlations were revealed in ALXDRD, but also subtle mechanisms could explain the variable manifestations of the ALXDRD clinical phenotype.

Mitochondrial Splicing Efficiency Is Lower in Holoparasites Than in Free-Living Plants.

Mitochondria play a crucial role in eukaryotic organisms, housing their own genome with genes vital for oxidative phosphorylation. Coordination between nuclear and mitochondrial genomes is pivotal for organelle gene expression. Splicing, editing and processing of mitochondrial transcripts are regulated by nuclear-encoded factors. Splicing efficiency (SEf) of the many group II introns present in plant mitochondrial genes is critical for mitochondrial function since a splicing defect or splicing deficiency can severely impact plant growth and development. This study investigates SEf in free-living and holoparasitic plants, focusing on 25 group II introns from 15 angiosperm species. Our comparative analyses reveal distinctive splicing patterns with holoparasites exhibiting significantly lower SEf, potentially linked to their unique evolutionary trajectory. Given the preponderance of horizontal gene transfer (HGT) in parasitic plants, we investigated the effect of HGT on SEf, such as the presence of foreign introns or foreign nuclear-encoded splicing factors. Contrary to expectations, the SEf reductions do not correlate with HGT events, suggesting that other factors are at play, such as the loss of photosynthesis or the transition to a holoparasitic lifestyle. The findings of this study broaden our understanding of the molecular evolution in parasitic plants and shed light on the multifaceted factors influencing organelle gene expression.

7694 Characterizing NF1 Germline Pathogenic Variants In A Cohort Of Patients With Pheochromocytomas And Neurofibromatosis Type 1

Abstract Disclosure: T. Hristova: None. S. Parisien-La Salle: None. S. Lapointe: None. D. Tremblay: None. N. Dumas: None. M. Labidi: None. Z. El Haffaf: None. I. Bourdeau: None. Introduction: Pheochromocytomas and paragangliomas (PPGLs) are the tumors with the highest heritability in adult patients, with identification of a germline mutation in more than 30% of cases. Up to 20 susceptibility genes for PPGLs are known, including NF1. Neurofibromatosis type 1 (NF1) is an inherited disease affecting approximately 1 in 3000 people that predisposes to the development of tumors, such as pheochromocytomas (PHEO). Up to recently, the diagnosis of NF1 was mainly based on clinical manifestations and NF1 genetic characterization was not systematically performed. Objectives: To characterize germline pathogenic variants in the NF1 gene in patients with NF1 developing and PHEO. Methods: We reviewed the charts of patients with a pathology-proven diagnosis of PHEOs that were investigated at Centre hospitalier de l’Université de Montréal (CHUM) between 2000 and May 2023. Genetic analysis included gene sequencing by Sanger method or multigene sequencing by NGS with a panel (Invitae, CA) Results: In our cohort of 220 PHEOs, 15 patients (6.8%) (Males: 6, Females: 9) had a diagnosis of NF1. Mean age at diagnosis of PHEO in NF1 patients was 48 ± 13.6 years, contrasting to the mean age of 38,7 ± 15.2 years in patients carrying germline mutations in non-NF1 genes, most likely reflecting lack of systematic biochemically screening of PHEO in NF1. Urinary metanephrines were elevated in 9/15 patients. 2/15 (15.4%) NF1 patients had bilateral PHEO and 1/15 (7,7%) metastatic disease. Mean tumour diameter was 4.64cm (min-max 1.5 – 12.5 cm). Eight out of 15 NF1 patients underwent NF1 genetic analysis. Heterozygous NF1 germline pathogenic variants were found in all of them: 3 deletions (c.7379delG, c.5844_5845delAA and one encompassing the entire gene), 3 premature stop codons leading to truncated proteins or loss of function (c.7549C>T, c.3916C>T, c.1246C>T), and 2 splicing defects (c.4269+1 G>A, c.1885G>A). Conclusions: We report a large cohort of patients with NF1 and PHEO. The older age at diagnosis of PHEO in NF1 is expected based on lack of systematic biochemical screening for PHEO in NF1 clinical guidelines. We report 8 germline NF1 mutations associated with PHEOs. Further studies are required to fully understand the impact of genetic pathogenic variants in this population and to unravel a possible genotype-phenotype correlation. Presentation: 6/2/2024

471P Interfering with CUG toxic repeats using AAV.U7snRNAs rescue myotonia and splicing defects in myotonic dystrophy type 1

471P Interfering with CUG toxic repeats using AAV.U7snRNAs rescue myotonia and splicing defects in myotonic dystrophy type 1

Exonic splice variant discovery using in vitro models of inherited retinal disease

Correct identification of the molecular consequences of pathogenic genetic variants is essential to the development of allele-specific therapies. However, such molecular effects may remain ambiguous following genetic sequence analysis alone. Here, we identify exonic codon-altering variants that are also predicted to disrupt normal RNA splicing in the context of inherited retinal disease. NR2E3 c.932G>A (p.Arg311Gln) is a variant commonly associated with enhanced S cone syndrome. Previous studies using mutagenized cDNA constructs have shown that the arginine to glutamine substitution at position 311 of NR2E3 does not meaningfully diminish function of the rod-specific transcription factor. Using retinal organoids, we explored the molecular consequences of NR2E3 c.932G>A when expressed endogenously during human rod photoreceptor cell development. Retinal organoids carrying the NR2E3 c.932G>A allele expressed a transcript containing a 186-nucleotide deletion of exon 6 within the ligand binding domain. This short transcript was not detected in control organoids or control human donor retina samples. A minigene containing exons 5 and 6 of NR2E3 showed sufficiency of the c.932G>A variant to cause the observed splicing defect. These results support the hypothesis that the pathogenic NR2E3 c.932G>A variant leads to photoreceptor disease by causing a splice defect and not through an amino acid substitution as previously supposed. They also explain the relatively mild effect of Arg311Gln on NR2E3 function invitro. We also used in silico prediction tools to show that similar changes are likely to affect other inherited retinal disease variants in genes such as CEP290, ABCA4, and BEST1.

The role of the U1 snRNP complex in the regulation of gene expression: recent reports

U1 snRNP (U1 small nuclear ribonucleoprotein) is a nuclear ribonucleoprotein complex involved mainly in pre-mRNA splicing, which is a key regulatory process in the eukaryotic gene expression pathway, but also in the process of preventing premature transcription termination (telescripting). U1 snRNP interacts directly with RNA polymerase II, thereby influencing the synthesis and maturation of transcripts in the cell nucleus, including the formation of the 3' end of mRNA and polyadenylation. At the level of cell physiology, it regulates the functioning of mitochondria and energy metabolism. The core of the U1 snRNP complex is U1 snRNA, encoded by many copies of genes that differ in sequence and expression level, and the expression of some of them leads to the formation of defective products. According to current reports, U1 snRNA can be used for therapeutic purposes to regulate gene expression and improve mRNA splicing defects, which are the cause of many diseases. Here we present selected recent discoveries and achievements related to U1 snRNP.

PTEN controls alternative splicing of autism spectrum disorder-associated transcripts in primary neurons.

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

Lipopeptide-mediated Cas9 RNP delivery: A promising broad therapeutic strategy for safely removing deep-intronic variants in ABCA4

Deep-intronic (DI) variants represent approximately 10-12% of disease-causing genetic defects in ABCA4-associated Stargardt disease (STGD1). Although many of these DI variants are amenable to antisense oligonucleotide-based splicing modulation therapy, no treatment is currently available. These molecules are mostly variant-specific, limiting their applicability to a broader patient population. In this study, we investigated the therapeutic potential of the CRISPR-Cas9 system combined with the amphipathic lipopeptide C18:1-LAH5 for intracellular delivery to correct splicing defects caused by different DI variants within the same intron. The combination of these components facilitated efficient editing of two target introns (introns 30 and 36) of ABCA4 in which several recurrent DI variants are found. The partial removal of these introns did not affect ABCA4 splicing or its expression levels when assessed in two different human cellular models: fibroblasts and induced pluripotent stem cell-derived photoreceptor precursor cells (PPCs). Furthermore, the DNA editing in STGD1 patient-derived PPCs led to a ∼50% reduction of the pseudoexon-containing transcripts resulting from the c.4539+2001G>A variant in intron 30. Overall, we provide proof-of-concept evidence of the use of C18:1-LAH5 as a delivery system for therapeutic genome editing for ABCA4-associated DI variants, offering new opportunities for clinical translation.

Rare occurrence of cryptic 5' splice sites by downstream 3' splice site/exon boundary mutations in a heavy-ion-induced egy1-4 allele of Arabidopsis thaliana.

Pre-mRNA splicing is a fundamental process in eukaryotic gene expression, and the mechanism of intron definition, involving the recognition of the canonical GU (5'-splice site) and AG (3'-splice site) dinucleotides by splicing factors, has been postulated for most cases of splicing initiation in plants. Splice site mutations have played crucial roles in unraveling the mechanism of pre-mRNA splicing in planta. Typically, splice site mutations abolish splicing events or activate one or more cryptic splice sites surrounding the mutated region. In this report, we investigated the splicing pattern of the EGY1 gene in an Ar-ion-induced egy1-4 allele of Arabidopsis thaliana. egy1-4 has an AG-to-AC mutation in the 3'-end of intron 3, along with 4-bp substitutions and a 5-bp deletion in adjacent exon 4. RT-PCR, cDNA cloning, and amplicon sequencing analyses of EGY1 revealed that while most wild-type EGY1 mRNAs had a single splicing pattern, egy1-4 mRNAs had multiple splicing defects. Almost half of EGY1 transcripts showed 'intron retention' at intron 3, while the other half exhibited activation of 3' cryptic splice sites either upstream or downstream of the original 3'-splice site. Unexpectedly, around 8% of EGY1 transcripts in egy1-4 exhibited activation of cryptic 5'-splice sites positioned upstream of the authentic 5'-splice site of intron 3. Whole genome resequencing of egy1-4 indicated that it has no other known impactful mutations. These results may provide a rare, but real case of activation of cryptic 5'-splice sites by downstream 3'-splice site/exon mutations in planta.

Alternative splicing dysregulation across tissue and therapeutic approaches in a mouse model of myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1), the leading cause of adult-onset muscular dystrophy, is caused by a CTG repeat expansion. Expression of the repeat causes widespread alternative splicing (AS) defects and downstream pathogenesis including significant skeletal muscle impacts. The HSALR mouse model plays a significant role in therapeutic development. This mouse model features a transgene composed of approximately 220 interrupted CTG repeats, which results in skeletal muscle pathology that mirrors DM1. To better understand this model and the growing number of therapeutic approaches developed with it, we performed a meta-analysis of publicly available RNA sequencing data for AS changes across three widely examined skeletal muscles: quadriceps, gastrocnemius, and tibialis anterior. Our analysis demonstrated that transgene expression correlated with the extent of splicing dysregulation across these muscles from gastrocnemius (highest), quadriceps, to tibialis anterior (lowest). We identified 95 splicing events consistently dysregulated across all examined datasets. Comparison of splicing rescue across seven therapeutic approaches showed a range of rescue across the 95 splicing events from the three muscle groups. This analysis contributes to our understanding of the HSALR model and the growing number of therapeutic approaches currently in preclinical development for DM1.

Splicing control by PHF5A is crucial for melanoma cell survival.

Abnormalities in alternative splicing are a hallmark of cancer formation. In this study, we investigated the role of the splicing factor PHD finger protein 5A (PHF5A) in melanoma. Malignant melanoma is the deadliest form of skin cancer, and patients with a high PHF5A expression show poor overall survival. Our data revealed that an siRNA-mediated downregulation of PHF5A in different melanoma cell lines leads to massive splicing defects of different tumour-relevant genes. The loss of PHF5A results in an increased rate of apoptosis by triggering Fas- and unfolded protein response (UPR)-mediated apoptosis pathways in melanoma cells. These findings are tumour-specific because we did not observe this regulation in fibroblasts. Our study identifies a crucial role of PHF5A as driver for melanoma malignancy and the described underlying splicing network provides an interesting basis for the development of new therapeutic targets for this aggressive form of skin cancer.

KHSRP ameliorates acute liver failure by regulating pre-mRNA splicing through its interaction with SF3B1

Acute liver failure (ALF) is characterized by the rapidly progressive deterioration of hepatic function, which, without effective medical intervention, results in high mortality and morbidity. Here, using proteomic and transcriptomic analyses in murine ALF models, we found that the expression of multiple splicing factors was downregulated in ALF. Notably, we found that KH-type splicing regulatory protein (KHSRP) has a protective effect in ALF. Knockdown of KHSRP resulted in dramatic splicing defects, such as intron retention, and led to the exacerbation of liver injury in ALF. Moreover, we demonstrated that KHSRP directly interacts with splicing factor 3b subunit 1 (SF3B1) and enhances the binding of SF3B1 to the intronic branch sites, thereby promoting pre-mRNA splicing. Using splicing inhibitors, we found that Khsrp protects against ALF by regulating pre-mRNA splicing in vivo. Overall, our findings demonstrate that KHSRP is an important splicing activator and promotes the expression of genes associated with ALF progression by interacting with SF3B1; thus, KHSRP could be a possible target for therapeutic intervention in ALF.

Disruption of nuclear speckle integrity dysregulates RNA splicing in C9ORF72-FTD/ALS

Expansion of an intronic (GGGGCC)n repeat within the C9ORF72 gene is the most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (C9-FTD/ALS), characterized with aberrant repeat RNA foci and noncanonical translation-produced dipeptide repeat (DPR) protein inclusions. Here, we elucidate that the (GGGGCC)n repeat RNA co-localizes with nuclear speckles and alters their phase separation properties and granule dynamics. Moreover, the essential nuclear speckle scaffold protein SRRM2 is sequestered into the poly-GR cytoplasmic inclusions in the C9-FTD/ALS mouse model and patient postmortem tissues, exacerbating the nuclear speckle dysfunction. Impaired nuclear speckle integrity induces global exon skipping and intron retention in human iPSC-derived neurons and causes neuronal toxicity. Similar alternative splicing changes can be found in C9-FTD/ALS patient postmortem tissues. This work identified novel molecular mechanisms of global RNA splicing defects caused by impaired nuclear speckle function in C9-FTD/ALS and revealed novel potential biomarkers or therapeutic targets.