Splicing Regulatory Factors Research Articles

Investigating the influence of the SIRT6 gene and alternative splicing on canine longevity: an in-depth bioinformatics analysis and experimental confirmation via NGS-based targeted sequencing.

Sirtuin 6 (SIRT6) has many functions, but its most notable contribution lies in the intricate regulation of cell senescence and lifespan. The effect of the SIRT6 gene on body size and longevity in dogs has not been extensively studied, particularly with regard to alternative splicing mechanisms. To address this gap, the present study used a comprehensive approach that integrated bioinformatics analysis, DNA sequence analysis, and next-generation sequencing-based targeted sequencing analyses. Our results show that, according to the reference genomes of different dog breeds, the canine SIRT6 gene exhibits different variants according to the dog breed. Except for the exonic variant g.55,146,051C > T (rs851065050) detected in the Boxer breed, all variants obtained from other genomes were determined to be intronic variants. The g.56,075,604 G > T (rs3343377774) intronic variant previously detected in the Labrador Retriever breed was only detected in the small breed group in our study. As a result of in silico analysis, the g.56,075,604 G > T variant has an exonic splicing enhancer (ESE) site; this variant has created the motif of the binding site for the splicing factor ESE_SRp55. The g.55.146,051C > T variant was associated with a change in the ratio of exonic splicing silencer/ESE binding motifs. This change indicates an increased probability of exon skipping for the mutant allele. Thus, as with the intronic variant g.56,075,604 G > T, the mis-splicing induced by the exonic variant g.55,146,051C > T could potentially be associated with an altered distribution of regulatory splicing factors of the canine SIRT6 gene.

Identification of alternative splicing regulatory patterns and characteristic splicing factors in heart failure using RNA-seq data and machine learning

Heart failure (HF) represents the advanced stage of several cardiovascular disorders. This study aimed to build an alternative splicing regulatory network and identify potential splicing factors involved in HF utilizing RNA-seq data and machine learning algorithms. We performed bioinformatics analysis on RNA-seq datasets containing samples from HF patients and normal individuals to obtain gene expression matrices and identify differently regulated alternative splicing events in HF. By calculating percent spliced-in (PSI) value, we identified 4055 abnormal alternative splicing events of 3142 genes in HF. These genes were significantly enriched in PPAR signaling, regulation of actin cytoskeleton, and muscle contraction. Interestingly, based on abnormal alternative splicing events, two distinct clusters of HF patients with distinct molecular mechanisms and pathways were identified using unsupervised clustering. Additionally, we built a regulatory network consisting of heart failure-related alternative splicing and splicing factors. Subsequently, we identify 203 HF specific pairs between splicing factors and alternative splicing events. Four splicing factors (RBM5, ZRANB2, HnRNPF, and HnRNPA0) were found using LASSO and SVM-RFE algorithms, their expression patterns were confirmed in two other microarray datasets. Our study clarifies involvement of splicing factors and alternative splicing events in HF by thoroughly analyzing RNA-seq data with machine learning methods. The findings may advance our understanding of the regulatory systems underlying biological processes associated with heart failure by providing candidates for further investigation and markers for diagnostic and therapeutic purposes.

Easyab Unravels Oncokinase Driven Regulation of Splicing Factors in AML

Acute myeloid leukemia (AML) is the most commonly diagnosed leukemia in adults. Gene mutations involved in various cellular functions are known to cause AML. A large proportion of AML is driven by aberrant activation of kinases and signal transducers such as FLT3, cKIT, PTPN11, JAK2, KRAS, NRAS, and to a lesser extent, JAK1, JAK3, and CSF3R. Treatment for AML has remained the same in the past five decades. Recent developments led to the availability of targeted therapies and enabled the practice of additional drugs like tyrosine kinase inhibitors. However, drug resistance and tolerance mechanism remain a challenge to finding a cure for the disease. Though we can identify and target kinase mutations individually, we have yet to learn how these mutations, like FLT3-ITD, change the cell's intrinsic signalling environment and cooperate with other oncogenes. Understanding these signalling behaviors is crucial for effective treatment outcomes and avoiding the development of drug resistance and tolerance mechanisms. Protein post-translational modifications (PTM) such as phosphorylation, ubiquitination, methylation, control the activity, fate, and spatial location of the proteins, thereby controlling processes such as signal transduction. Mass spectrometry-based proteomics offers unbiased profiling of PTMs. However, comprehensive analyses of PTMs require conventional antibody-based enrichment approaches and large starting materials, laborious, low throughput, cost prohibitive, and time-consuming, thus limiting the application to cell line-based studies. It also limits application in exploring unbiased-global cell signaling in patient samples. To overcome these limitations, we developed a loss-less sample preparation method named ‘EasyAb’. We show that our short, high throughput and low-cost approach enables capturing PTM peptides from very small inputs. As proof of principle, we enriched tyrosine-phosphorylated peptides from Molm13 and MV4-11 cell lines expressing FLT3-ITD kinase. Enrichment resulted in the ultra-deep identification of 4,416 tyrosine phosphosites, double the number of sites found in the conventional protocols, and took only one day of processing. About 40 % of the tyrosine phosphorylated proteins enriched are membrane proteins, including seven tyrosine phosphorylation sites on FLT3 kinase, of which FLT3 pY401 is a novel site discovered. To test the applicability of the EasyAb protocol in patient samples, we performed phosphotyrosine profiling of AML samples expressing FLT3 WT (n=9) and FLT3-ITD (n=5) mutation. EasyAb enrichment for phosphotyrosine peptides resulted in identifying 6,339 phosphotyrosine sites, of which 2300 sites were quantified. Comparing FLT3WT and ITD samples revealed more than 500 phosphosites significantly regulated. The significantly changing sites include several known signalling molecules such as STAT5, SYK, BTK, GAB2, GRB2, and PTPN11. The data also revealed differential tyrosine phosphorylation of proteins involved in various cellular functions such as autophagy, apoptosis, cell cycle, DNA damage repair proteins, phosphatases, and kinases. Interestingly, we also uncovered a class of proteins belonging to mRNA processing and splicing pathways distinctly tyrosine phosphorylated in FLT3-ITD samples. It is known that splicing factor mutations drive leukemia. Conversely, little is known about the regulatory mechanism of splicing factors by singling in cancer. To further investigate FLT3-ITD signalling regulation of splicing factors, we treated Molm13 and MV4-11 cells with Quizartinib and profiled tyrosine phosphorylation. Inhibition of FLT3-ITD lead to downregulation in tyrosine phosphorylation of several splicing factors. We have previously reported that splicing factor phosphorylation by JAK2 kinase plays a crucial role in Jak inhibitor persistence phenotype. Importantly, FLT3-ITD-mediated tyrosine phosphorylation-based regulation of splicing factors is unknown, and this process could be crucial for FLT3-ITD-mediated tumorigenesis. The functional significance of this novel regulatory mechanism is being studied. In conclusion, we have developed an effective tool for studying post-translational modifications that require antibody-based enrichment (tyrosine phosphorylation, ubiquitination, methylation, acetylation, and sumoylation) relatively quickly in primary patient samples.

MEN1 is a regulator of alternative splicing and prevents R-loop-induced genome instability through suppression of RNA polymerase II elongation.

The fidelity of alternative splicing (AS) patterns is essential for growth development and cell fate determination. However, the scope of the molecular switches that regulate AS remains largely unexplored. Here we show that MEN1 is a previously unknown splicing regulatory factor. MEN1 deletion resulted in reprogramming of AS patterns in mouse lung tissue and human lung cancer cells, suggesting that MEN1 has a general function in regulating alternative precursor mRNA splicing. MEN1 altered exon skipping and the abundance of mRNA splicing isoforms of certain genes with suboptimal splice sites. Chromatin immunoprecipitation and chromosome walking assays revealed that MEN1 favored the accumulation of RNA polymerase II (Pol II) in regions encoding variant exons. Our data suggest that MEN1 regulates AS by slowing the Pol II elongation rate and that defects in these processes trigger R-loop formation, DNA damage accumulation and genome instability. Furthermore, we identified 28 MEN1-regulated exon-skipping events in lung cancer cells that were closely correlated with survival in patients with lung adenocarcinoma, and MEN1 deficiency sensitized lung cancer cells to splicing inhibitors. Collectively, these findings led to the identification of a novel biological role for menin in maintaining AS homeostasis and link this role to the regulation of cancer cell behavior.

The role of alternative splicing in lung cancer.

Aberrant alternative splicing (AS) events are frequently observed in lung cancer, which can be attributed to aberrant gene AS, alterations in splicing regulatory factors, or changes in splicing regulatory mechanisms. Consequently, the dysregulation of alternative RNA splicing is the fundamental cause of lung cancer. In this review, we have summarized the pivotal role of AS in the development, progression, invasion, metastasis, angiogenesis, and drug resistance of lung cancer. Ultimately, this review emphasizes the potential of AS as biomarkers in lung cancer prognosis and diagnosis, and introduces some applications of AS isoform in the treatment of lung cancer. The comprehension of the AS may provide a glimmer of hope for the eradication of lung cancer.

Adipose tissue plasticity in pheochromocytoma patients suggests a role of the splicing machinery in human adipose browning

Adipose tissue from pheochromocytoma patients acquires brown fat features, making it a valuable model for studying the mechanisms that control thermogenic adipose plasticity in humans. Transcriptomic analyses revealed a massive downregulation of splicing machinery components and splicing regulatory factors in browned adipose tissue from patients, with upregulation of a few genes encoding RNA-binding proteins potentially involved in splicing regulation. These changes were also observed in cell culture models of human brown adipocyte differentiation, confirming a potential involvement of splicing in the cell-autonomous control of adipose browning. The coordinated changes in splicing are associated with a profound modification in the expression levels of splicing-driven transcript isoforms for genes involved in the specialized metabolism of brown adipocytes and those encoding master transcriptional regulators of adipose browning. Splicing control appears to be a relevant component of the coordinated gene expression changes that allow human adipose tissue to acquire a brown phenotype.

Regulation of Pre-mRNA Splicing: Indispensable Role of Post-Translational Modifications of Splicing Factors

Pre-mRNA splicing is a process used by eukaryotic cells to generate messenger RNAs that can be translated into proteins. During splicing, the non-coding regions of the RNAs (introns) are removed from pre-mRNAs and the coding regions (exons) are joined together, resulting in mature mRNAs. The particular steps of splicing are executed by the multimegadalton complex called a spliceosome. This complex is composed of small nuclear ribonucleoproteins, various splicing factors, and other regulatory and auxiliary proteins. In recent years, various post-translational modifications of splicing factors have been shown to contribute significantly to regulation of processes involved in pre-mRNA splicing. In this review, we provide an overview of the most important post-translational modifications of splicing factors that are indispensable for their normal function during pre-mRNA splicing (i.e., phosphorylation, acetylation, methylation, ubiquitination and sumoylation). Moreover, we also discuss how the defects in regulation of splicing factors are related to the development of cancer.

Myocardin regulates exon usage in smooth muscle cells through induction of splicing regulatory factors

Differentiation of smooth muscle cells (SMCs) depends on serum response factor (SRF) and its co-activator myocardin (MYOCD). The role of MYOCD for the SMC program of gene transcription is well established. In contrast, the role of MYOCD in control of SMC-specific alternative exon usage, including exon splicing, has not been explored. In the current work we identified four splicing factors (MBNL1, RBPMS, RBPMS2, and RBFOX2) that correlate with MYOCD across human SMC tissues. Forced expression of MYOCD family members in human coronary artery SMCs in vitro upregulated expression of these splicing factors. For global profiling of transcript diversity, we performed RNA-sequencing after MYOCD transduction. We analyzed alternative transcripts with three different methods. Exon-based analysis identified 1637 features with differential exon usage. For example, usage of 3´ exons in MYLK that encode telokin increased relative to 5´ exons, as did the 17 kDa telokin to 130 kDa MYLK protein ratio. Dedicated event-based analysis identified 239 MYOCD-driven splicing events. Events involving MBNL1, MCAM, and ACTN1 were among the most prominent, and this was confirmed using variant-specific PCR analyses. In support of a role for RBPMS and RBFOX2 in MYOCD-driven splicing we found enrichment of their binding motifs around differentially spliced exons. Moreover, knockdown of either RBPMS or RBFOX2 antagonized splicing events stimulated by MYOCD, including those involving ACTN1, VCL, and MBNL1. Supporting an in vivo role of MYOCD-SRF-driven splicing, we demonstrate altered Rbpms expression and splicing in inducible and SMC-specific Srf knockout mice. We conclude that MYOCD-SRF, in part via RBPMS and RBFOX2, induce a program of differential exon usage and alternative splicing as part of the broader program of SMC differentiation.

Dysregulated minor intron splicing in cancer.

Pre‐mRNA splicing is now widely recognized as a cotranscriptional and post‐transcriptional mechanism essential for regulating gene expression and modifying gene product function. Mutations in genes encoding core spliceosomal proteins and accessory regulatory splicing factors are now considered among the most recurrent genetic abnormalities in patients with cancer, particularly hematologic malignancies. These include mutations in the major (U2‐type) and minor (U12‐type) spliceosomes, which remove >99% and ~0.35% of introns, respectively. Growing evidence indicates that aberrant splicing of evolutionarily conserved U12‐type minor introns plays a crucial role in cancer as the minor spliceosome component, ZRSR2, is subject to recurrent, leukemia‐associated mutations, and intronic mutations have been shown to disrupt the splicing of minor introns. Here, we review the importance of minor intron regulation, the molecular effects of the minor (U12‐type) spliceosomal mutations and cis‐regulatory regions, and the development of minor intron studies for better understanding of cancer biology.

An oligonucleotide/oligosaccharide-binding-fold protein enhances the alternative splicing event producing thylakoid membrane-bound ascorbate peroxidase in Nicotiana tabacum.

The stromal and thylakoid membrane-bound ascorbate peroxidase isoforms are produced by the alternative splicing event of the 3′-terminal region of the APXII gene in spinach (Spinacia oleracea) and tobacco (Nicotiana tabacum), but not in Arabidopsis (Arabidopsis thaliana). However, all alternative splicing variants were detected in APXII gene-transformed Arabidopsis, indicating the occurrence of its regulatory mechanisms in Arabidopsis. The efficiency of this alternative splicing event in producing thylakoid membrane-bound ascorbate peroxidase mRNA is regulated by a splicing regulatory cis element, but trans splicing regulatory factor(s) for alternative splicing remain unclear. To identify this factor, we conducted a forward genetic screen using Arabidopsis in combination with a luciferase reporter system to evaluate the alternative splicing efficiency of thylakoid membrane-bound ascorbate peroxidase mRNA production. We isolated 9 mutant lines that showed low efficiency of the AS in producing thylakoid membrane-bound ascorbate peroxidase mRNA compared with that in the control plants. From one mutant [APXII alternative splicing inhibition (apsi1)], the causal gene responsible for the phenotype, AT5G38890 (oligonucleotide/oligosaccharide-binding-fold protein, APSI1), was identified. The levels of thylakoid membrane-bound ascorbate peroxidase mRNA from the transformed APXII gene decreased and increased in APSI1 knockout and APSI1-overexpressing plants, respectively. APSI1 was localized to the nucleus and specifically bound to the splicing regulatory cis element sequence. Tobacco plants that disrupted the closest homologs of APSI1 showed low levels of endogenous thylakoid membrane-bound ascorbate peroxidase mRNA. These results indicate that APSI1 is an enhancing component of the alternative splicing event of APXII.

Exon junction complex-associated multi-adapter RNPS1 nucleates splicing regulatory complexes to maintain transcriptome surveillance.

The exon junction complex (EJC) is an RNA-binding multi-protein complex with critical functions in post-transcriptional gene regulation. It is deposited on the mRNA during splicing and regulates diverse processes including pre-mRNA splicing and nonsense-mediated mRNA decay (NMD) via various interacting proteins. The peripheral EJC-binding protein RNPS1 was reported to serve two insufficiently characterized functions: suppressing mis-splicing of cryptic splice sites and activating NMD in the cytoplasm. The analysis of transcriptome-wide effects of EJC and RNPS1 knockdowns in different human cell lines supports the conclusion that RNPS1 can moderately influence NMD activity, but is not a globally essential NMD factor. However, numerous aberrant splicing events strongly suggest that the main function of RNPS1 is splicing regulation. Rescue analyses revealed that the RRM and C-terminal domain of RNPS1 both contribute partially to regulate RNPS1-dependent splicing events. We defined the RNPS1 core interactome using complementary immunoprecipitations and proximity labeling, which identified interactions with splicing-regulatory factors that are dependent on the C-terminus or the RRM domain of RNPS1. Thus, RNPS1 emerges as a multifunctional splicing regulator that promotes correct and efficient splicing of different vulnerable splicing events via the formation of diverse splicing-promoting complexes.

Serine Arginine Splicing Factor 7 (SRSF7) is a critical regulator of innate immune activation in macrophages

Abstract Macrophages are immune cells that receive signals from pathogens and respond by reprogramming gene expression to activate an innate immune response. We currently have a poor understanding of how this response is fine-tuned at a post-transcriptional level. To address this, we turned to recent proteomics studies that identified mRNA processing as a pathway enriched for differentially phosphorylated proteins in macrophages infected with the important human pathogen Mycobacterium tuberculosis (Mtb). We became interested in one of these mRNA processing proteins, a splicing regulatory factor called SRSF7 (formerly known as 9G8), that was differentially phosphorylated at three serine residues during early Mtb infection. Transcriptomics analysis of SRSF7 knockdown RAW 264.7 macrophage-like cells revealed that SRSF7 is required for proper expression of a group of innate immune transcripts including the antiviral genes Mx1 and Ifit3 and the cytosolic DNA sensor Zbp1. Consistent with this phenotype, SRSF7 KD macrophages are permissive to Vesicular Stomatitis Virus (VSV) hyper replication. On the other hand, overexpression of SRSF7 in RAW 264.7 cells leads to elevated ISG abundance in resting cells and enhanced ability to restrict VSV replication. Curiously, it appears that certain ISGs are more reliant on SRSF7 than others, suggesting that SRSF7 acts at the level of the RNA itself. We have started to implicate phosphorylation of SRSF7 in these phenotypes. Together, these studies provide some of the best evidence to date that pathogen sensing cascades can functionalize RNA binding proteins during macrophage activation and highlight a previously unappreciated role for SRSF7 in controlling macrophage antiviral responses. Supported by grants from 1F31GM143893-01 NIH Predoctoral Fellowship and R35GM133720 from NIH/NIGMS.

Clinical Implications of Circulating Circular RNAs in Lung Cancer.

Circular RNAs (circRNAs) are single-stranded RNAs with a covalently closed-loop structure that increases their stability; thus, they are more advantageous to use as liquid biopsy markers than linear RNAs. circRNAs are thought to be generated by back-splicing of pre-mRNA transcripts, which can be facilitated by reverse complementary sequences in the flanking introns and trans-acting factors, such as splicing regulatory factors and RNA-binding factors. circRNAs function as miRNA sponges, interact with target proteins, regulate the stability and translatability of other mRNAs, regulate gene expression, and produce microproteins. circRNAs are also found in the body fluids of cancer patients, including plasma, saliva, urine, and cerebrospinal fluid, and these “circulating circRNAs” can be used as cancer biomarkers. In lung cancer, some circulating circRNAs have been reported to regulate cancer progression and drug resistance. Circulating circRNAs have significant diagnostic value and are associated with the prognosis of lung cancer patients. Owing to their functional versatility, heightened stability, and practical applicability, circulating circRNAs represent promising biomarkers for lung cancer diagnosis, prognosis, and treatment monitoring.

Rapid Regulation of Alternative Splicing in Response to Environmental Stresses.

Plants overcome the changing environmental conditions through diverse strategies and complex regulations. In addition to direct regulation of gene transcription, alternative splicing (AS) also acts as a crucial regulatory mechanism to cope with various stresses. Generating from the same pre-mRNA, AS events allow rapid adjustment of the abundance and function of key stress-response components. Mounting evidence has indicated the close link between AS and plant stress response. However, the mechanisms on how environmental stresses trigger AS are far from understood. The advancing high-throughput sequencing technologies have been providing useful information, whereas genetic approaches have also yielded remarkable phenotypic evidence for AS control of stress responses. It is important to study how stresses trigger AS events for both fundamental science and applications. We review current understanding of stress-responsive AS in plants and discuss research challenges for the near future, including regulation of splicing factors, epigenetic modifications, the shared targets of splice isoforms, and the stress-adjusting ratios between splicing variants.

Functional analyses of human LUC7-like proteins involved in splicing regulation and myeloid neoplasms.

SUMMARYVertebrates have evolved three paralogs, termed LUC7L, LUC7L2, and LUC7L3, of the essential yeast U1 small nuclear RNA (snRNA)-associated splicing factor Luc7p. We investigated the mechanistic and regulatory functions of these putative splicing factors, of which one (LUC7L2) is mutated or deleted in myeloid neoplasms. Protein interaction data show that all three proteins bind similar core but distinct regulatory splicing factors, probably mediated through their divergent arginine-serine-rich domains, which are not present in Luc7p. Knockdown of each factor reveals mostly unique sets of significantly dysregulated alternative splicing events dependent on their binding locations, which are largely non-overlapping. Notably, knockdown of LUC7L2 alone significantly upregulates the expression of multiple spliceosomal factors and downregulates glycolysis genes, possibly contributing to disease pathogenesis. RNA binding studies reveal that LUC7L2 and LUC7L3 crosslink to weak 5′ splice sites and to the 5′ end of U1 snRNA, establishing an evolutionarily conserved role in 5′ splice site selection.

Splicing alterations in healthy aging and disease

Alternative RNA splicing is a key step in gene expression that allows generation of numerous messenger RNA transcripts encoding proteins of varied functions from the same gene. It is thus a rich source of proteomic and functional diversity. Alterations in alternative RNA splicing are observed both during healthy aging and in a number of human diseases, several of which display premature aging phenotypes or increased incidence with age. Age-associated splicing alterations include differential splicing of genes associated with hallmarks of aging, as well as changes in the levels of core spliceosomal genes and regulatory splicing factors. Here, we review the current known links between alternative RNA splicing, its regulators, healthy biological aging, and diseases associated with aging or aging-like phenotypes. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Splicing Regulation/Alternative Splicing.

Regulation of Fetal Genes by Transitions among RNA-Binding Proteins during Liver Development

Transcripts of alpha-fetoprotein (Afp), H19, and insulin-like growth factor 2 (Igf2) genes are highly expressed in mouse fetal liver, but decrease drastically during maturation. While transcriptional regulation of these genes has been well studied, the post-transcriptional regulation of their developmental decrease is poorly understood. Here, we show that shortening of poly(A) tails and subsequent RNA decay are largely responsible for the postnatal decrease of Afp, H19, and Igf2 transcripts in mouse liver. IGF2 mRNA binding protein 1 (IMP1), which regulates stability and translation efficiency of target mRNAs, binds to these fetal liver transcripts. When IMP1 is exogenously expressed in mouse adult liver, fetal liver transcripts show higher expression and possess longer poly(A) tails, suggesting that IMP1 stabilizes them. IMP1 declines concomitantly with fetal liver transcripts as liver matures. Instead, RNA-binding proteins (RBPs) that promote RNA decay, such as cold shock domain containing protein E1 (CSDE1), K-homology domain splicing regulatory protein (KSRP), and CUG-BP1 and ETR3-like factors 1 (CELF1), bind to 3′ regions of fetal liver transcripts. These data suggest that transitions among RBPs associated with fetal liver transcripts shift regulation from stabilization to decay, leading to a postnatal decrease in those fetal transcripts.

The splicing regulatory factor hnRNPU is a novel transcriptional target of c-Myc in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common liver cancer with high mortality. Here, we found that hnRNPU is overexpressed in HCC tissues and is correlated with the poor prognosis of HCC patients. Besides, hnRNPU is of high significance in regulating the proliferation, apoptosis, self-renewal, and tumorigenic potential of HCC cells. Mechanismly, c-Myc regulates hnRNPU expression at the transcriptional level, and meanwhile, hnRNPU stabilizes the mRNA of c-MYC. We found that the hnRNPU and c-Myc regulatory loop exerts a synergistic effect on the proliferation and self-renewal of HCC, and promotes the HCC progression. Taken together, hnRNPU functions as a novel transcriptional target of c-Myc and promotes HCC progression, which may become a promising target for the treatment of c-Myc-driven HCC.

An interactive network of alternative splicing events with prognostic value in geriatric lung adenocarcinoma via the regulation of splicing factors.

Alternative splicing (AS), an essential process for the maturation of mRNAs, is involved in tumorigenesis and tumor progression, including angiogenesis, apoptosis, and metastasis. AS changes can be frequently observed in different tumors, especially in geriatric lung adenocarcinoma (GLAD). Previous studies have reported an association between AS events and tumorigenesis but have lacked a systematic analysis of its underlying mechanisms. In the present study, we obtained splicing event information from SpliceSeq and clinical information regarding GLAD from The Cancer Genome Atlas. Survival-associated AS events were selected to construct eight prognostic index (PI) models. We also constructed a correlation network between splicing factors (SFs) and survival-related AS events to identify a potential molecular mechanism involved in regulating AS-related events in GLAD. Our study findings confirm that AS has a strong prognostic value for GLAD and sheds light on the clinical significance of targeting SFs in the treatment of GLAD.

Posttranslational Regulation of the Exon Skipping Machinery Controls Aberrant Splicing in Leukemia.

Splicing alterations are common in diseases such as cancer, where mutations in splicing factor genes are frequently responsible for aberrant splicing. Here we present an alternative mechanism for splicing regulation in T-cell acute lymphoblastic leukemia (T-ALL) that involves posttranslational stabilization of the splicing machinery via deubiquitination. We demonstrate there are extensive exon skipping changes in disease, affecting proteasomal subunits, cell-cycle regulators, and the RNA machinery. We present that the serine/arginine-rich splicing factors (SRSF), controlling exon skipping, are critical for leukemia cell survival. The ubiquitin-specific peptidase 7 (USP7) regulates SRSF6 protein levels via active deubiquitination, and USP7 inhibition alters the exon skipping pattern and blocks T-ALL growth. The splicing inhibitor H3B-8800 affects splicing of proteasomal transcripts and proteasome activity and acts synergistically with proteasome inhibitors in inhibiting T-ALL growth. Our study provides the proof-of-principle for regulation of splicing factors via deubiquitination and suggests new therapeutic modalities in T-ALL. SIGNIFICANCE: Our study provides a new proof-of-principle for posttranslational regulation of splicing factors independently of mutations in aggressive T-cell leukemia. It further suggests a new drug combination of splicing and proteasomal inhibitors, a concept that might apply to other diseases with or without mutations affecting the splicing machinery.This article is highlighted in the In This Issue feature, p. 1241.