Aberrant Alternative Splicing Research Articles

Alternative splicing-related long noncoding RNA ANRIL facilitates hepatocellular carcinoma by targeting the miR-199a-5p/SRSF1 axis and impacting Anillin.

Hepatocellular carcinoma (HCC) is characterized by aberrant alternative splicing (AS), which plays an important part in the pathological process of this disease. However, available reports about genes and mechanisms involved in AS process are limited. Our previous research has identified ANRIL as a long noncoding RNA related to the AS process of HCC. Here, we investigated the exact effect and the mechanism of ANRIL on HCC progress.The ANRIL expression profile was validated using the real-time quantitative polymerase chain reactionassay. The western blot analysis and IHC assay were conducted on candidate targets, including SRSF1 and Anillin. The clinicopathological features of 97 patients were collected and analyzed. Loss-of and gain-of-function experiments were conducted. The dual-luciferase reporter assay was applied to verify the interaction between ANRIL, miR-199a-5p, and SRSF1.Anomalous upregulation of ANRIL in HCC was observed, correlating with worse clinicopathological features of HCC. HCC cell proliferation, mobility, tumorigenesis, and metastasis were impaired by depleting ANRIL. We found that ANRIL acts as a sponger of miRNA-199a-5p, resulting in an elevated level of its target protein SRSF1. The phenotypes induced by ANRIL/miR-199a-5p/SRSF1 alteration are associated with Anillin, a validated HCC promoter.ANRIL is an AS-related lncRNA promoting HCC progress by modulating the miR-199a-5p/SRSF1 axis. The downstream effector of this axis in the development of HCC is Anillin.

Alteration of RNA m6A methylation mediates aberrant RNA binding protein expression and alternative splicing in condyloma acuminatum.

Condyloma acuminatum (CA) is caused by low-risk human papillomavirus, and is characterized by high recurrence after treatment. The RNA modification N6-methyladenosine (m6A) plays an important role during diverse viral infections, including high-risk HPV infection in cervical cancer. However, it is unclear whether low-risk HPV infection changes the RNA m6A methylation in CA. High-throughputm6A-sequencing was performed to profile the transcriptome-wide mRNA modifications of CA tissues infected by LR-HPVs and the paired normal tissues from CA patients. We further investigated the regulation of alternative splicing by RNA binding proteins (RBPs) with altered m6A modification and constructed a regulatory network among these RBPs, regulated alternative splicing events (RASEs) and regulated alternative splicing genes (RASGs) in CA. The results show that the m6A level in CA tissues differed from that in the paired controls. Furthermore, cell cycle- and cell adhesion- associated genes with m6A modification were differentially expressed in CA tissues compared to the paired controls. In particular, seven RNA binding protein genes with specific m6A methylated sites, showed a higher or lower expression at the mRNA level in CA tissues than in the paired normal tissues. In addition, these differentially expressed RNA binding protein genes would regulate the alternative splicing pattern of apoptotic process genes in CA tissue. Our study reveals a sophisticated m6A modification profile in CA tissue that affects the response of host cells to HPV infection, and provides cues for the further exploration of the roles of m6A and the development of a novel treatment strategy for CA.

Screening Splice-Switching Antisense Oligonucleotides in Pancreas-Cancer Organoids.

Aberrant alternative splicing is emerging as a cancer hallmark and a potential therapeutic target. It is the result of dysregulated or mutated splicing factors, or genetic alterations in splicing-regulatory cis-elements. Targeting individual altered splicing events associated with cancer-cell dependencies is a potential therapeutic strategy, but several technical limitations need to be addressed. Patient-derived organoids are a promising platform to recapitulate key aspects of disease states, and to facilitate drug development for precision medicine. Here, we report an efficient antisense-oligonucleotide (ASO) lipofection method to systematically evaluate and screen individual splicing events as therapeutic targets in pancreatic ductal adenocarcinoma organoids. This optimized delivery method allows fast and efficient screening of ASOs, e.g., those that reverse oncogenic alternative splicing. In combination with advances in chemical modifications of oligonucleotides and ASO-delivery strategies, this method has the potential to accelerate the discovery of antitumor ASO drugs that target pathological alternative splicing.

Molecular targets and mechanisms of different aberrant alternative splicing in metastatic liver cancer.

Metastasis remains a major challenge in the successful management of malignant diseases. The liver is a major site of metastatic disease and a leading cause of death from gastrointestinal malignancies such as colon, stomach, and pancreatic cancers, as well as melanoma, breast cancer, and sarcoma. As an important factor that influences the development of metastatic liver cancer, alternative splicing drives the diversity of RNA transcripts and protein subtypes, which may provide potential to broaden the target space. In particular, the dysfunction of splicing factors and abnormal expression of splicing variants are associated with the occurrence, progression, aggressiveness, and drug resistance of cancers caused by the selective splicing of specific genes. This review is the first to provide a detailed summary of the normal splicing process and alterations that occur during metastatic liver cancer. It will cover the role of alternative splicing in the mechanisms of metastatic liver cancer by examining splicing factor changes, abnormal splicing, and the contribution of hypoxia to these changes during metastasis.

RNA splicing regulator EIF3D regulates the tumor microenvironment through immunogene-related alternative splicing in head and neck squamous cell carcinoma.

Study finds that eukaryotic translation initiation factor 3 subunit D (EIF3D) may play an important role in aberrant alternative splicing (AS) events in tumors. AS possesses a pivotal role in both tumour progression and the constitution of the tumour microenvironment (TME). Regrettably, our current understanding of AS remains circumscribed especially in the context of immunogene-related alternative splicing (IGAS) profiles within Head and Neck Squamous Cell Carcinoma (HNSC). In this study, we comprehensively analyzed the function and mechanism of action of EIF3D by bioinformatics analysis combined with in vitro cellular experiments, and found that high expression of EIF3D in HNSC was associated with poor prognosis of overall survival (OS) and progression-free survival (PFS). The EIF3D low expression group had a higher degree of immune infiltration and better efficacy against PD1 and CTLA4 immunotherapy compared to the EIF3D high expression group. TCGA SpliceSeq analysis illustrated that EIF3D influenced differentially spliced alternative splicing (DSAS) events involving 105 differentially expressed immunogenes (DEIGs). We observed an induction of apoptosis and a suppression of cell proliferation, migration, and invasion in EIF3D knock-down FaDu cells. RNA-seq analysis unveiled that 531 genes exhibited differential expression following EIF3D knockdown in FaDu cells. These include 52 DEIGs. Furthermore, EIF3D knockdown influenced the patterns of 1923 alternative splicing events (ASEs), encompassing 129 IGASs. This study identified an RNA splicing regulator and revealed its regulatory role in IGAS and the TME of HNSC, suggesting that EIF3D may be a potential target for predicting HNSC prognosis and immunotherapeutic response.

Abstract 5894: Novel splicing-derived neoantigen in triple negative breast cancer uncovered by SpliceIO™ a robust platform for the discovery and validation of disease-specific immunotherapies

Abstract Splicing dysregulation is a major hallmark of cancer, affecting tumor progression, metastasis, and therapy resistance. Aberrant alternative splicing (AS) plays a critical role in generating tumor-specific neoantigens targetable through an arsenal of immunotherapeutic modalities. Such neoantigens cannot be discovered using conventional DNA-based tools; they require specialized software for AS analysis using RNA-seq data. SpliceIO is Envisagenics’ AI platform for AS-derived neoantigen discovery. SpliceIO uses a “predictive ensemble” approach to uncover neoantigens that are both stably expressed and located on the extracellular membrane of cancer cells. Using SpliceIO, we have analyzed more than 10,000 RNA-seq samples from 5 patient cohorts covering 5 breast cancer subtypes as well as 2,647 normal RNA-seq samples from GTEx. Our analysis has identified tumor-specific neoantigens present in AS transmembrane proteins specific for Triple Negative Breast Cancer. SpliceIO target candidates were validated experimentally using our well-established pipeline in which isoform specific transcripts are tested in cancer cell lines by PCR and primary tumor tissues by qPCR to confirm RNA expression. Tumor heterogeneity and prevalence is confirmed by RNA in-situ hybridization in Tissue Microarrays. Extracellular localization of the novel epitope is confirmed by clone generation with GFP and c-Myc tagging followed by microscopy, Western blotting, and proteomic analysis. Our top validated target presents high TNBC tumor specificity and heterogeneous distribution, and contains a novel extracellular N-terminal domain. These results strongly support the role of aberrant AS as a major source of tumor-specific neoantigens. Our combined computational and experimental approach promotes novel target discovery, enabling the development of novel immunotherapeutic for TNBC patients who are in critical need of better treatment options. Citation Format: Nicole Williams, Miguel A. Manzanares, Kendall Anderson, Hasan Zunrut, Sakshi Gera, Nayab Habib, Shaleigh Stanton, Taylor Floyd, Sanjana Shah, Devon Trahan, Martin Akerman, Gayatri Arun. Novel splicing-derived neoantigen in triple negative breast cancer uncovered by SpliceIO™ a robust platform for the discovery and validation of disease-specific immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5894.

Abstract 4608: Combinations of Cdc-like kinase (CLK) inhibitors with targeted oncology agents or standard chemotherapy in patient-derived multi-cell type tumor spheroids

Abstract The alternative splicing of mRNA precursors allows one gene the capacity to yield multiple protein isoforms, each with distinct functions. Although this intricate process is subject to stringent regulation under normal physiological conditions, aberrant alternative splicing can generate atypical proteins and contribute to various diseases, including cancer. By activating splicing factors, Cdc-like kinases (CLKs) serve as pivotal regulators of alternative splicing and are considered promising therapeutic targets for various tumors. In this study, we investigated the activity of two CLK inhibitors, cirtuvivint (SM08502) and CC-671, in combination with precision oncology agents or conventional chemotherapeutics. Thirty highly characterized patient-derived cancer cell lines were selected from the National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov/models/database.htm). The cell lines were derived from a range of cancer types including head and neck, bladder, pancreas, endometrial, colon, and melanoma, and contained clinically relevant variants of KRAS (G12C, G12D). Multi-cell type tumor spheroids were grown from a ratio of 6:2.5:1.5 malignant cells, endothelial cells, and mesenchymal stem cells, respectively. Following three days of growth, the spheroids were treated with single agents or combinations at concentrations up to their clinical Cmax value, if reported. After seven days of continuous drug exposure, cell viability was assessed using the CellTiter-Glo 3D assay. As a single agent, cirtuvivint showed concentration-dependent activity in all spheroid types with 1 – 3 logs of cytotoxicity, whereas CC-671 did not show activity in all spheroid types and achieved no more than 1 log of cytotoxicity. Among the standard chemotherapeutic drugs, doxorubicin and SN-38 demonstrated additive and greater-than-additive cytotoxicity in various spheroid types when combined with either CLK inhibitor. Several of the targeted oncology drugs exhibited noteworthy additive and greater-than-additive cytotoxicity when combined with a CLK inhibitor. These agents included the XPO1 inhibitor, eltanexor, the MCL-1 inhibitor, tapotoclax, the α-isoform-specific PI3K inhibitor, inavolisib, and the pan-PI3K inhibitor, copanlisib. Notably, combinations of the CLK inhibitors with the KRAS G12D variant-specific inhibitor, MRTX-1133, showed selective activity against all tumor cell lines harboring this genetic variant. Interestingly, a strong antagonistic interaction between paclitaxel and CC-671 was observed in all thirty spheroid types, while no such interaction was observed with cirtuvivint. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Thomas Steven Dexheimer, Thomas Silvers, Nathan P. Coussens, Steven D. Gore, James H. Doroshow, Beverly A. Teicher. Combinations of Cdc-like kinase (CLK) inhibitors with targeted oncology agents or standard chemotherapy in patient-derived multi-cell type tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4608.

Abstract 5105: Aberrant CLK1 splicing is a key dependency factor in pediatric high-grade gliomas

Abstract Pediatric brain cancer is the leading cause of disease-related mortality in children. With a paucity of therapeutically targetable alterations, there is a pressing need to identify effective strategies for many of these tumors. In this study, we systematically examine aberrant alternative splicing in pediatric brain tumors from the Open Pediatric Brain Tumor Atlas, and additional tumors from the Children's Brain Tumor Network and the Pacific Pediatric Neuro-Oncology Consortium (n = 1831). Notably, high-grade gliomas (HGGs), including those with and without H3 histone mutations, were enriched in the upper quartile of all tumors with high splicing burden (SB). HGGs demonstrated the greatest SB heterogeneity of all tumor types and we identified a total of 19,229 aberrant splicing events in 8,577 genes in midline tumors compared to non-tumor brainstem controls (ΔPSI >= |.20|, p < 0.05). We next integrated protein annotations from the UniProt Knowledgebase to assess and prioritize differential alternative splicing events affecting protein function. This approach led to the discovery of 3,754 splice variants in 5,036 genes resulting in the gain or ablation of functional sites. The protein kinase CDC-like kinase 1 (CLK1), a recognized master splicing factor and cell-cycle modulator, exhibited aberrant splicing of exon 4, whereby inclusion promoted the gain of two known phosphorylation sites. Additionally, we observed a strong correlation between CLK1 splicing, its own RNA expression and proteomic activity of the Serine/Arginine-rich (SR) family of splicing factors. The distribution of exon 4 splicing was significantly different (p = 0.019) between tumors with low vs high splicing burden; tumors with high splicing burden had increased exon 4 inclusion. Further, we performed experimental modulation of CLK1 exon 4 aberrant splicing in the brain tumor cell line KNS42 and demonstrated that forced exon 4 skipping leads to reduced CLK1 protein abundance and significantly reduces cell proliferation (p < 0.01 vs. control). Following bulk RNA-Seq analysis of these cell lines, we identified that forced CLK1 exon 4 skipping resulted in 4,518 dysregulated genes enriched for known cancer pathways such as mitotic spindle, E2F targets, and G2M checkpoint. Our data suggest a role for CLK1 in driving transcriptional dysregulation in pediatric high-grade gliomas and uncover a potential therapeutic vulnerability. Citation Format: Ammar S. Naqvi, Priyanka Seghal, Ryan Corbett, Komal Rathi, Brian Ennis, Bo Zhang, Miguel Brown, Daniel Miller, Adam Kraya, Joseph Dybas, Katharina Hayer, Shehbeel Arif, Zhuangzhuang Geng, Antonia Chroni, Aditya Lahiri, Karina Conkrite, Madison Hollawell, Sharon Diskin, Peter Madsen, Jessica B. Foster, Mateusz P. Koptyra, Andrei Thomas-Tikhonenko, Adam Resnick, Phillip J. Storm, Jo Lynne Rokita. Aberrant CLK1 splicing is a key dependency factor in pediatric high-grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5105.

Alternative Splicing Is a Major Factor Shaping Transcriptome Diversity in Mild and Severe Chronic Obstructive Pulmonary Disease.

The role of alternative splicing in chronic obstructive pulmonary disease (COPD) is still largely unknown. We aimed to investigate the differences in alternatively splicing events between patients with mild-to-moderate and severe COPD compared with non-COPD control subjects and to identify splicing factors associated with aberrant alternative splicing in COPD. For this purpose, we performed genome-wide RNA-sequencing analysis of bronchial brushings from 23 patients with mild-to-moderate COPD, 121 with severe COPD, and 23 non-COPD control subjects. We found a significant difference in the frequency of alternative splicing events in patients with mild-to-moderate and severe COPD compared with non-COPD control subjects. There were from two to eight times (depending on event type) more differential alternative splicing events in the severe than in the mild-to-moderate stage. The severe COPD samples showed less intron retention and more exon skipping. It is interesting that the transcript levels of the top 10 differentially expressed splicing factors were significantly correlated with the percentage of many alternatively spliced transcripts in severe COPD. The aberrant alternative splicing in severe COPD was predicted to increase the overall protein-coding capacity of gene products. In conclusion, we observed large and significant differences in alternative splicing between bronchial samples of patients with COPD and control subjects, with more events observed in severe than in mild-to-moderate COPD. The changes in the expression of several splicing factors correlated with prevalence of alternative splicing in severe COPD. Alternative splicing can indirectly impact gene expression by changing the relative abundance of protein-coding isoforms potentially influencing pathophysiological changes. The results provide a better understanding of COPD-related alternative splicing changes.

AEP promotes aberrant RNA splicing through DDX3X cleavage in solid tumors.

Aberrant alternative splicing (AS) events have been identified in a variety of cancers. Although somatic mutations of splicing factors and dysregulation of RNA-binding proteins (RBPs) have been linked to AS and tumor malignancy, it remains unclear how upstream mechanisms contribute to cancer development via alternative gene splicing. In this issue of the JCI, Wenrui Zhang and colleagues identified the role of asparagine endopeptidase (AEP), an intracellular cysteine endopeptidase, in promoting solid tumor-associated RNA splicing. The authors demonstrated that tumor environmental factors such as oxygen and nutrient deprivation induce the activity of AEP in a HIF1A-dependent manner. The activated AEP, in turn, cleaves an RNA helicase DDX3X to promote its nuclear retention. The authors further showed that this DDX3X nuclear fraction engages with splicing machinery to induce AS events in several cancer cells. These findings suggest that targeting an AEP-dependent aberrant RNA splicing cascade may facilitate therapeutics for solid tumors.

Abstract C093: Dysregulated spliceosomal components promote pancreatic cancer progression

Abstract Dysregulated RNA splicing factors have been widely reported in tumorigenesis, but their involvement in pancreatic ductal adenocarcinoma (PDAC) is not well understood. We found that two proteins involved in different stages of spliceosome assembly contribute to PDAC tumorigenesis by regulating alternative splicing, either through direct or indirect interaction with RNA. First, we showed that the splicing factor SRSF1—an RNA-binding protein involved in early spliceosome assembly—can induce pancreatitis and accelerate KRASG12D-mediated PDAC by activating the MAPK pathway. Moreover, the expression of SRSF1 decreased in morphologically normal pancreatic cells expressing KRASG12D to maintain cellular homeostasis. Second, we found that breast cancer amplified sequence 2 (BCAS2, also known as SPF27)—a spliceosome scaffold protein associated with conformational changes—also contributes to PDAC progression. Although BCAS2 does not bind RNA directly, it can regulate alternative splicing events in a sequence-specific manner by promoting the usage of specific 5’ splice sites. BCAS2 promotes the inclusion of exon 21, which has a weak 5' splice site, in the SOS Ras/Rac Guanine Nucleotide Exchange Factor 1 (SOS1) pre-mRNA, resulting in a more oncogenic isoform. These studies not only improve our understanding of the oncogenic role of aberrant alternative splicing, but also provide insights into the mechanisms of alternative-splicing regulation at different stages of spliceosome assembly. Citation Format: Ledong Wan, Kuan-Ting Lin, Mohammad A. Rahman, Yuma Ishigami, Alexander J. Kral, Dillon Voss, Zhikai Wang, Mads A. Jensen, John E. Wilkinson, Youngkyu Park, David A. Tuveson, Adrian R. Krainer. Dysregulated spliceosomal components promote pancreatic cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C093.

Abstract A024: Epigenetic control of topoisomerase 1 activity presents a cancer vulnerability

Abstract Genome integrity is inherently at risk due to the torsional constraints imposed by DNA transactions. Topoisomerase 1 (TOP1) is essential for genomic stability by removing DNA supercoils generated during replication and transcription. Aberrant stabilization of TOP1:DNA cleavage complexes (TOP1ccs), however, result in DNA lesions that drive transcription-associated mutation burden and are a cytotoxic consequence of many clinically used chemotherapeutic agents. What protects genomic regions prone to elevated torsional stress from the cytotoxic potential of TOP1 enzymes remains unknown. Here, we identify macroH2A1.1, the only nucleosome component with an inherent ability to bind the TOP1cc repair-essential poly(ADP-ribose) (PAR) protein modification, as an essential means to establish a TOP1-permissive chromatin environment. MacroH2A1.1 is one of two alternatively spliced isoforms of the macro-histone variant macroH2A1, and comparative proteomics between the two isoforms uncovered a macroH2A1.1-specific and PARP activity-dependent interaction with the TOP1cc repair effector TDP1 as well as downstream base excision repair (BER) factors XRCC1 and LIG3. Consequently, macroH2A1.1 loss or inactivation of its PAR-binding domain resulted in impaired XRCC1 recruitment to damaged DNA and defective TOP1cc turnover, while the macroH2A1.2 splice isoform is unable to bind PAR or protect from TOP1ccs. Genome-wide mapping of macroH2A1.1, TOP1 and TOP1-DNA adducts identified macroH2A1.1 as a regulatory hub for TOP1 activity that ensures transcriptional integrity by preventing excessive TOP1cc accumulation at transcription start sites. Impaired macroH2A1.1 splicing, a frequent cancer feature, correlated with sensitivity to TOP1 poisons in a pharmaco-genomic screen in breast cancer cells, and macroH2A1.1 inactivation mirrored this effect. Finally, we find that aberrant macroH2A1 alternative splicing in cancer correlates with BER-related mutation signatures. We propose that macroH2A1 alternative splicing serves as an epigenetic modulator of TOP1-associated genome maintenance and a potential cancer vulnerability. Citation Format: Tae-Hee Lee, Xuan C. Qiao, Tongyu Wu, Vladislav Kuzin, Xianzhen Zhou, Vijayalalitha Ramanarayanan, Laura Baranello, Philipp Oberdoerffer. Epigenetic control of topoisomerase 1 activity presents a cancer vulnerability [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A024.

Nuclear pore pathology underlying multisystem proteinopathy type 3-related inclusion body myopathy.

Multisystem proteinopathy type 3 (MSP3) is an inherited, pleiotropic degenerative disorder caused by a mutation in heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), which can affect the muscle, bone, and/or nervous system. This study aimed to determine detailed histopathological features and transcriptomic profile of HNRNPA1-mutated skeletal muscles to reveal the core pathomechanism of hereditary inclusion body myopathy (hIBM), a predominant phenotype of MSP3. Histopathological analyses and RNA sequencing of HNRNPA1-mutated skeletal muscles harboring a c.940G > A (p.D314N) mutation (NM_031157) were performed, and the results were compared with those of HNRNPA1-unlinked hIBM and control muscle tissues. RNA sequencing revealed aberrant alternative splicing events that predominantly occurred in myofibril components and mitochondrial respiratory complex. Enrichment analyses identified the nuclear pore complex (NPC) and nucleocytoplasmic transport as suppressed pathways. These two pathways were linked by the hub genes NUP50, NUP98, NUP153, NUP205, and RanBP2. In immunohistochemistry, these nucleoporin proteins (NUPs) were mislocalized to the cytoplasm and aggregated mostly with TAR DNA-binding protein 43 kDa and, to a lesser extent, with hnRNPA1. Based on ultrastructural observation, irregularly shaped myonuclei with deep invaginations were frequently observed in atrophic fibers, consistent with the disorganization of NPCs. Additionally, regarding the expression profiles of overall NUPs, reduced expression of NUP98, NUP153, and RanBP2 was shared with HNRNPA1-unlinked hIBMs. The shared subset of altered NUPs in amyotrophic lateral sclerosis (ALS), as demonstrated in prior research, HNRNPA1-mutated, and HNRNPA1-unlinked hIBM muscle tissues may provide evidence regarding the underlying common nuclear pore pathology of hIBM, ALS, and MSP.

Alternative splicing in breast cancer

Alternative splicing allows a gene to produce multiple proteins and is the main source of human proteome diversity. Aberrant regulation of alternative splicing produces proteins with different structures and can lead to altered protein function, inactivation, or deleterious variants, which can have severe effects on normal cellular function, leading to many diseases, including cancer. Alternative splicing abnormalities are often found in diseases, so alternative splicing is highly correlated with diseases and treatment. According to relevant statistics, breast cancer is an important factor that threatens women’s lives and health. A common sign of tumors in humans is abnormal alternative splicing. An increasing amount of evidence shows that abnormal alternative splicing plays an important role in the occurrence and development of breast cancer. At the same time, in the related treatment of breast cancer, alternative splicing can lead to the failure of existing treatment methods and induce drug resistance. Currently, targeted therapy is still the key strategy for breast cancer treatment. In this review, we discuss the five classifications of alternative splicing and aberrant alternative splicing in breast cancer. The role of alternative splicing in the drug resistance of breast cancer is also discussed. Evidence has shown that aberrant alternative splicing can lead to drug resistance to current therapies. Because splicing factors are involved in many aspects of cancer, splicing factors may provide a new strategy for cancer treatment and a powerful tool for progress in the fight against breast cancer. Antisense oligonucleotides form a double-stranded structure by complementary pairing with mRNA, which prevents the binding of mRNA and ribosomes, thus inhibiting protein synthesis. Therefore, after finding effective targets, antisense oligonucleotides can be applied in gene therapy, drug research and development, biological research, and other fields to improve therapeutic effects.

Abstract 18698: PCBP1 Regulates Alternative Splicing of Aars2 in Congenital Cardiomyopathy

Rationale: Alanyl-transfer RNA synthetase 2 (AARS2) is a nuclear encoded mitochondrial tRNA synthetase that is responsible for charging of tRNA-Ala with alanine during mitochondrial translation. Homozygous or compound heterozygous mutations in the Aars2 gene, including those affecting its alternative splicing, are linked to congenital cardiomyopathy in humans. However, the underlying molecular mechanism of how Aars2 regulates heart development remains unknown. Hypothesis: The alternative splicing of Aars2 and its function is essential for cardiac development. Methods and Results: We found that alternative splicing of the Aars2 transcript is mediated by poly(rC) binding protein 1 (PCBP1), an RNA binding protein, through direct interaction. Cardiomyocyte-specific deletion of Pcbp1 in mice resulted in defects in heart development that are reminiscent of human congenital cardiac defects, including noncompaction cardiomyopathy and a disruption of the cardiomyocyte maturation trajectory. Loss of Pcbp1 led to an aberrant alternative splicing and a premature termination of Aars2 in cardiomyocytes. Therefore, Pcbp1 is critical for the expression and function of Aars2. Additionally, Aars2 mutant mice with exon-16 skipping recapitulated heart developmental defects observed in Pcbp1 mutant mice. Mechanistically, we found dysregulated gene and protein expression of the oxidative phosphorylation pathway in both Pcbp1 and Aars2 mutant hearts; these data provide further evidence that the infantile hypertrophic cardiomyopathy associated with the disorder oxidative phosphorylation defect type 8 (COXPD8) is mediated by Aars2. Conclusion: Our study therefore identifies Pcbp1 and Aars2 as critical regulators of heart development and provides important molecular insights into the role of disruptions in metabolism on congenital heart defects.

Comparative transcriptome profiling reveals RNA splicing alterations and biological function in patients exposed to occupational noise.

Genetic factors play an important role in susceptibility to noise-induced hearing loss (NIHL). Alternative splicing (AS) is an essential mechanism affecting gene expression associated with disease pathogenesis at the post-transcriptional level, but has rarely been studied in NIHL. To explore the role of AS in the development of NIHL, we performed a comprehensive analysis of RNA splicing alterations by comparing the RNA-seq data from blood samples from NIHL patients and subjects with normal hearing who were exposed to the same noise environment. A total of 356 differentially expressed genes, including 23 transcription factors, were identified between the two groups. Of particular note was the identification of 56 aberrant alternative splicing events generated by 41 differentially expressed genes between the two groups, with exon skipping events accounting for 54% of all the differentially alternative splicing (DAS) events. The results of functional enrichment analysis showed that these intersecting DAS genes and differentially expressed genes were significantly enriched in autophagy and mitochondria-related pathways. Together, our findings provide insights into the role of AS events in susceptibility and pathogenesis of NIHL.

Long-read sequencing reveals the landscape of aberrant alternative splicing and novel therapeutic target in colorectal cancer

BackgroundAlternative splicing complexity plays a vital role in carcinogenesis and cancer progression. Improved understanding of novel splicing events and the underlying regulatory mechanisms may contribute new insights into developing new therapeutic strategies for colorectal cancer (CRC).MethodsHere, we combined long-read sequencing technology with short-read RNA-seq methods to investigate the transcriptome complexity in CRC. By using experiment assays, we explored the function of newly identified splicing isoform TIMP1 Δ4-5. Moreover, a CRISPR/dCasRx-based strategy to induce the TIMP1 exon 4–5 exclusion was introduced to inhibit neoplasm growth.ResultsA total of 90,703 transcripts were identified, of which > 62% were novel compared with current transcriptome annotations. These novel transcripts were more likely to be sample specific, expressed at relatively lower levels with more exons, and oncogenes displayed a characteristic to generate more transcripts in CRC. Clinical outcome data analysis showed that 1472 differentially expressed alternative splicing events (DEAS) were tightly associated with CRC patients’ prognosis, and many novel isoforms were likely to be important determinants for patient survival. Among these, newly identified splicing isoform TIMP1 Δ4-5 was significantly downregulated in CRC. Further in vitro and in vivo assays demonstrated that ectopic expression of TIMP1 Δ4-5 significantly suppresses tumor cell growth and metastasis. Serine/arginine-rich splicing factor 1 (SRSF1) acts as a onco-splicing regulator through sustaining the inclusion of TIMP1 exon 4–5. Furthermore, CRISPR/dCasRx-based strategies designed to induce TIMP1 exon 4–5 exclusion have the potential to restrain the CRC growth.ConclusionsThis data provides a rich resource for deeper studies of gastrointestinal malignancies. Newly identified splicing isoform TIMP1 Δ4-5 plays an important role in mediating CRC progression and may be a potential therapy target in CRC.

Dysregulation of alternative splicing underlies synaptic defects in familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by the degeneration of upper and lower motor neurons, progressive wasting and paralysis of voluntary muscles. A hallmark of ALS is the frequent nuclear loss and cytoplasmic accumulation of RNA binding proteins (RBPs) in motor neurons (MN), which leads to aberrant alternative splicing regulation. However, whether altered splicing patterns are also present in familial models of ALS without mutations in RBP-encoding genes has not been investigated yet. Herein, we found that altered splicing of synaptic genes is a common trait of familial ALS MNs. Similar deregulation was also observed in hSOD1G93A MN-like cells. In silico analysis identified the potential regulators of these pre-mRNAs, including the RBP Sam68. Immunofluorescence analysis and biochemical fractionation experiments revealed that Sam68 accumulates in the cytoplasmic insoluble ribonucleoprotein fraction of MN. Remarkably, the synaptic splicing events deregulated in ALS MNs were also affected in Sam68-/- spinal cords. Recombinant expression of Sam68 protein was sufficient to rescue these splicing changes in ALS hSOD1G93A MN-like cells. Hence, our study highlights an aberrant function of Sam68, which leads to splicing changes in synaptic genes and may contribute to the MN phenotype that characterizes ALS.

Tau Protein Induces Aberrant Alternative Splicing Changes in PS19 Transgenic Mice

To explore through big data analysis whether aberrant alternative splicing (AS) events precede tau P301S-induced neurodegenerative phenotype in 6-month-old PS19 mice. The original sequencing files of the GSE182170 dataset was downloaded from the European Nucleotide Archive (ENA) database with axel, aligned to the reference genome of the ENSEMBL database by using STAR software, and common AS event analysis and visualization were performed with rMATS and rmats2sashimiplot R packages. RSEM software was utilized for gene transcript quantification, Deseq2, edgeR, and limma R packages were used for differential expression analysis, and clusterProfiler R package was applied for GO enrichment analysis. String and Cytoscape were used for protein-protein interaction (PPI) analysis. Gene expression correlation analysis was performed with ggcorrplot R package. AS events were validated using PCR followed by agarose electrophoresis. A total of 8 079 AS events were identified with rMATS and 117 significant AS events (ΔPSI>0.1, sequencing coverage >1) were selected eventually. The most frequent type of AS event was skipped exon (SE) (50.43%), followed by alternative 3' splice site (A3SS) and mutually exclusive exons (MXE). GO enrichment analysis revealed that synapse organization genes were aberrantly spliced in SE events and spliceosome genes were spliced in A3SS events. PPI and correlation analyses showed that the splicing factor Snrpn was significantly associated with the largest number of transcripts. Agarose electrophoresis confirmed the aberrant AS event of the Lrp8 gene in PS19 mice. Dysregulated splicing factors may contribute to tau P301S-induced aberrant AS changes. The study also increases the understanding of the cycling of tau protein and splicing factors in tauopathies.

Antisense oligonucleotide rescue of CGG expansion–dependent FMR1 mis-splicing in fragile X syndrome restores FMRP

Aberrant alternative splicing of mRNAs results in dysregulated gene expression in multiple neurological disorders. Here, we show that hundreds of mRNAs are incorrectly expressed and spliced in white blood cells and brain tissues of individuals with fragile X syndrome (FXS). Surprisingly, the FMR1 (Fragile X Messenger Ribonucleoprotein 1) gene is transcribed in >70% of the FXS tissues. In all FMR1-expressing FXS tissues, FMR1 RNA itself is mis-spliced in a CGG expansion-dependent manner to generate the little-known FMR1-217 RNA isoform, which is comprised of FMR1 exon 1 and a pseudo-exon in intron 1. FMR1-217 is also expressed in FXS premutation carrier-derived skin fibroblasts and brain tissues. We show that in cells aberrantly expressing mis-spliced FMR1, antisense oligonucleotide (ASO) treatment reduces FMR1-217, rescues full-length FMR1 RNA, and restores FMRP (Fragile X Messenger RibonucleoProtein) to normal levels. Notably, FMR1 gene reactivation in transcriptionally silent FXS cells using 5-aza-2'-deoxycytidine (5-AzadC), which prevents DNA methylation, increases FMR1-217 RNA levels but not FMRP. ASO treatment of cells prior to 5-AzadC application rescues full-length FMR1 expression and restores FMRP. These findings indicate that misregulated RNA-processing events in blood could serve as potent biomarkers for FXS and that in those individuals expressing FMR1-217, ASO treatment may offer a therapeutic approach to mitigate the disorder.