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Aberrant splicing is a major hallmark of cancer, affecting tumor progression, metastasis, and therapy resistance. The oncogenic activity of specific cis splicing errors and trans-acting splicing factor misregulation in patient tumors have been demonstrated in multiple studies. As such, cancer-associated splicing dysregulation is a novel source of clinically actionable biomarkers and therapeutic targets, particularly for the treatment of insensitive cancers such as Triple Negative Breast Cancer (TNBC). Envisagenics’ SpliceCore technology is an innovative cloud-based software platform that integrates machine learning (ML) algorithms with high performance computing to analyze large RNA-seq datasets to predict biologically relevant, novel, and highly prevalent tumor specific alternative splicing (AS) changes. Using SpliceCore, we have analyzed >2500 RNAseq samples from different breast cancer subtypes as well as normal breast tissue and identified several AS targets with the potential to translate into therapeutic candidates for TNBC. Interestingly, one of our leading AS targets is an exon skipping isoform that is present in 60.5% of TNBC patients and correlates with poor overall survival, without showing differences in gene expression between all the breast cancer subtypes and healthy patients studied. In addition, SpliceCore was used to predict and design a set of splice switching oligos (SSO) that can efficiently switch the skipping isoform to an inclusion isoform in TNBC cells. The skipping isoform plays a critical role in tumor progression via a TGFβ-dependent mechanism as demonstrated by detailed isoform switching studies using SSO-0205. Pretreatment of the TNBC cells with SSO-0205 24 hours before TGFβ pathway activation modulated TGFβ pathway related protein levels and cellular localization and reversed the cell proliferative response associated with it. This resulted in a strong inhibition of p21 gene expression, accompanied by a 50% decrease on the number of cells in G2, the mitotic phase of the cell cycle, and 40% decrease on cell viability. Additionally, migratory response induced by TGFβ in TNBC cells was also significantly inhibited by SSO-0205 pretreatment, which downregulated ANGPTL4 gene expression followed by a 55% decrease in cell migration. In summary, we were able to uncover a novel therapeutic target for TNBC, whose aberrant splicing contributes to TNBC pathogenesis by promoting an overactivation of the TGFβ pathway. Our results provide experimental proof of concept that demonstrate SpliceCore’s ability to discover novel disease specific AS targets and design splice correcting oligonucleotides for subsequent therapeutic development. Reversal of this aberrant TNBC specific splicing using SSOs represent a new and promising therapeutic approach that will have a significant impact on TNBC treatment and clinical care. Moreover, SpliceCore can be applied to multiple other indications opening a new avenue for therapeutic development in cancer. Citation Format: Miguel A Manzanares, Priyanka Dhingra, Kendall Anderson, Vanessa Frederick, Adam Geier, Alyssa Casill, Martin Akerman, Gayatri Arun. Novel therapeutic target for triple negative breast cancer uncovered by SpliceCore® an innovative platform that identifies disease-specific alternative splicing [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-11.

This study demonstrates the value that artificial intelligence/machine learning (AI/ML) provides for the identification of novel and verifiable splice-switching oligonucleotide (SSO) targetsin-silico. SSOs are antisense compounds that act directly on pre-mRNA to modulate alternative splicing (AS). To leverage the potential of AS research for therapeutic development, we created SpliceLearn™, an AI/ML algorithm for the identification of modulatory SSO binding sites on pre-mRNA. SpliceLearn also predicts the identity of specific splicing factors whose binding to pre-mRNA is blocked by SSOs, adding considerable transparency to AI/ML-driven drug discovery and informing biological insights useful in further validation steps. Here we predictedNEDD4Lexon 13 (NEDD4Le13) as a novel target in triple negative breast cancer (TNBC) and computationally designed an SSO to modulateNEDD4Le13. TargetingNEDD4Le13with this SSO decreased the proliferative and migratory behavior of TNBC cells via downregulation of the TGFβ pathway. Overall, this study illustrates the ability of AI/ML to extract actionable insights from RNA-seq data. SpliceLearn is part of the SpliceCore® platform, an AI/ML predictive ensemble for AS-based drug target discovery.

RNA splicing is a central step in the regulation of gene expression, leading to the accurate assembly of viable mRNA isoforms. Errors in the splicing process are common in cancer and lead to the production of tumor-specific mRNAs that often encode for neoantigen peptides. Such neoantigens cannot be discovered using conventional DNA-based tools; they require specialized software for alternative splicing analysis using RNA-seq data. Envisagenics has developed SpliceIOTM, a proprietary software platform for splicing-derived neoantigen discovery. SpliceIO uses RNA-seq data to uncover extracellular neoantigens encoded by transmembrane proteins or presented to immune cells through the MHC complex. Using SpliceIO, we have analyzed roughly 8,000 RNA-seq samples from 4 different cancers and 14 normal tissue types. Each dataset first undergoes an unsupervised clustering analysis using SpliceSliceTM to stratify patients into subpopulations based on similar aberrations in splicing machinery. We then leverage SpliceIO to identify splicing-derived, tumor-specific neoantigens enriched in these cohorts. SpliceIO incorporates features, such as protein localization, MHC binding affinity, epitope accessibility and cancer relevance of the gene to help select tumor specific targets with the highest therapeutic potential. Here, we summarize the discovery and validation of novel splicing-derived neoantigens identified using SpliceIO. To validate neoantigen candidates, we performed isoform expression quantification at the RNA and protein levels using PCR, RNA-FISH, western blot and mass spectrometry. Our experiments confirmed several neoantigen candidates encoded by tumor-specific RNA isoforms, that are translated and localized to the cellular membrane. Interestingly, one of the candidates is an endoplasmic reticulum protein that exhibits cassette exon skipping in approximately 24% of TNBC tumors. Fluorescence microscopy experiments demonstrate that the TNBC-specific isoform of the protein is localized to plasma membrane but not the ER, suggesting that alternative splicing can alter protein localization. Together, our results demonstrate the potential of RNA splicing analysis for the discovery of novel immunotherapeutic targets. Citation Format: Hasan Ekrem Zumrut, Kendall Anderson, Miguel Manzanares, Shaleigh Smith, Sakshi Gera, Nayab Habib, Sanjana Shah, Devon Trahan, Martin Akerman, Gayatri Arun. Computational discovery and experimental validation of splicing-derived neoantigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 448.

Splicing deregulation is a major hallmark of cancer, affecting progression, metastasis and therapy resistance. Multiple studies have demonstrated the oncogenic activity of specific splicing events and splicing factors, including SRSF1, SRSF2, ESRP1, and RBFOX1, in human and animal models. As such, cancer-associated splicing deregulation may be a novel source of clinically-applicable biomarkers and therapeutic targets, particularly for treatment-insensitive cancers such as TNBC. Here, we identified and experimentally characterized novel splicing changes in triple negative breast cancer by analyzing TCGA breast cancer RNA-seq data using our software platform SpliceCore. SpliceCore integrates machine learning (ML) algorithms with high-performance computing to analyze large RNA-Seq datasets to predict biologically relevant splicing events. Our analysis of TCGA BRCA datasets has revealed several previously unknown splicing events specific to TNBC patient cohorts, that is subsequently experimentally validated in TNBC cell lines. We provide biological evidence for such TNBC specific splice-switch both at the RNA and protein level. We provide evidence for SpliceCore predicted splice-switching oligonucleotides (SSO) and verify their biological activity in TNBC cell lines. Further, functional studies using potent and specific SSOs against the top candidates reveal a critical role for some of these novel isoforms in TNBC. Together, we provide a proof of concept study using SpliceCore to identify, design, and target novel splicing changes for subsequent clinical translation and therapeutic development. Citation Format: Gayatri Arun, Priyanka Dhingra, Robin Munch, Vanessa Fredrick, Martin Akerman. Identification and characterization of novel splice variants in triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-21.

Summary: Misregulation of alternative splicing is a hallmark of human tumors, yet to what extent and how it contributes to malignancy are only beginning to be unraveled. Here, we define which members of the splicing factor SR and SR-like families contribute to breast cancer and uncover differences and redundancies in their targets and biological functions. We identify splicing factors frequently altered in human breast tumors and assay their oncogenic functions using breast organoid models. We demonstrate that not all splicing factors affect mammary tumorigenesis in MCF-10A cells. Specifically, the upregulation of SRSF4, SRSF6, or TRA2β disrupts acinar morphogenesis and promotes cell proliferation and invasion in MCF-10A cells. By characterizing the targets of these oncogenic splicing factors, we identify shared spliced isoforms associated with well-established cancer hallmarks. Finally, we demonstrate that TRA2β is regulated by the MYC oncogene, plays a role in metastasis maintenance in vivo, and its levels correlate with breast cancer patient survival. : Park et al. demonstrate that >50% of human breast tumors exhibit an alteration in one of the splicing factors from the SR protein family. Using in vitro and in vivo breast cancer models, they identify three splicing factors that promote cell proliferation and invasion by regulating isoforms associated with cancer hallmarks. Keywords: alternative RNA splicing, breast cancer, splicing factor, SR protein, metastasis, MYC, TRA2-beta, triple negative breast cancer

Accumulating evidence highlights aberrant splicing as a disease-driving event in cancer, affecting tumor progression, metastasis, and therapy resistance. Multiple studies have described the tumor-promoting activity of specific aberrant cis-splicing variants and misregulation of trans-acting splicing factors in patients. Hence, the identification of aberrant splicing patterns has the potential to translate into actionable biomarkers and novel therapeutic targets, particularly for treatment insensitive cancers such as Triple Negative Breast Cancer (TNBC). Envisagenics’ SpliceCore is an innovative cloud-based platform that integrates machine learning (ML) algorithms with high performance computing to analyze large RNA-seq datasets to predict biologically relevant, novel, and highly prevalent tumor-specific alternative splicing (AS) changes. Using SpliceCore, we have analyzed >2500 RNA-seq samples from different breast cancer subtypes as well as normal breast tissue and identified several AS derived therapeutic targets with the potential to translate into therapeutic candidates for TNBC. Here, we report a novel, alternatively spliced isoform that is present in 60.5% of TNBC patients and correlates with poor overall survival. This cytoplasmic protein was previously described as a regulator of the TGFβ pathway; therefore, we have validated the isoform switching action in a TGFβ dependent tumor progression model and demonstrated the activity of Splice Switching Oligos (SSO) to modulate its effect. We observed that this AS was correlated to a differential response on subcellular localization for phSmad2/3, Smad2/3 and TGFβRI. Pretreatment of the TNBC cells with SSO before TGFβ pathway activation modulates its proliferation response by affecting gene expression of p21, c-Myc and Smad7, leading to a decrease of cells in the G2 mitotic phase of the cell cycle and a loss of cell viability after the SSO treatment. More importantly, migratory response induced by TGFβ in TNBC cells was significantly inhibited by SSOs. Pre-treatment with SSO-0205 before TGFβ activation downregulated gene expression of Angiopoietin-like 4, Integrins α5 and β3, TGFβ associated migration markers, which was followed by a 55% decrease in cell migration. Our state-of-the-art ML technology, SpliceCore, has proven its ability to uncover novel disease-specific AS targets and to design splice correcting oligonucleotides for subsequent therapeutic development. Our data provides experimental proof of concept that has uncovered a novel therapeutic target for TNBC, whose aberrant splicing contributes to TNBC pathogenesis by misregulation of the TGFβ pathway. Moreover, SSOs predicted by SpliceCore were able to correct the aberrant splicing downstream effect, thereby uncovering a novel alternative splicing targeted approach for treating TNBC patients. Citation Format: Miguel A. Manzanares, Alyssa Casill, Kendall Anderson, Priyanka Dhingra, Vanessa Frederick, Adam Geier, Martin Akerman, Gayatri Arun. SpliceCore® a novel ML tool for identifying disease-specific alternative splicing uncovered a promising therapeutic target for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1873.

Splicing dysregulation is a major hallmark of cancer, affecting tumor progression, metastasis, and therapy resistance. Multiple studies have demonstrated the oncogenic activity of specific cis splicing errors and trans-acting splicing factor misregulation in patient tumors. As such, cancer-associated splicing dysregulation is a novel source of clinically actionable biomarkers and therapeutic targets, particularly for treatment insensitive cancers such as TNBC. Envisagenics has developed SpliceCore, an innovative cloud-based software platform that integrates machine learning (ML) algorithms with high performance computing to analyze large RNA-seq datasets to predict biologically relevant, novel, and highly prevalent tumor specific alternative splicing (AS) changes. Using SpliceCore, we have analyzed >2500 RNAseq samples from different breast cancer subtypes as well as normal breast tissue and identified several AS targets with a potential to translate into therapeutic candidates for TNBC. Here, we will discuss one of our most promising AS targets; it is present in 60.5% of TNBC patients and correlates with poor overall survival. Using SpliceCore, we predicted and designed an optimal set of splice switching oligos (SSO) that can efficiently switch the skipping isoform to an inclusion isoform in TNBC cells. Detailed mechanism of action studies of isoform switching by SSO-0205 have demonstrated the critical role of the isoform in a TGFβ dependent tumor progression mechanism. Pretreatment of the TNBC cells with SSO-0205 24 hours before TGFβ pathway activation reversed the cell proliferative response associated with it. This resulted in a strong inhibition of p21 gene expression, accompanied by a 50% decrease on the number of cells in G2, the mitotic phase of the cell cycle, and 40% decrease on cell viability. Additionally, migratory response induced by TGFβ in TNBC cells was also significantly inhibited by SSO-0205 pretreatment, which downregulated ANGPTL4 gene expression followed by a 55% decrease in cell migration. Together, we provide experimental proof of concept to demonstrate SpliceCore’s ability to discover novel disease specific AS targets and design splice correcting oligonucleotides for subsequent correction and therapeutic development. Using this approach, we were able to uncover a novel therapeutic target for TNBC, whose aberrant splicing contributes to TNBC pathogenesis by promoting an overactivation of the TGFβ pathway. Reversal of this aberrant TNBC specific splicing using SSOs represent a new and promising therapeutic approach that will have a significant impact on TNBC treatment and clinical care. Citation Format: Miguel A. Manzanares, Dhingra Priyanka, Kendall Anderson, Vanessa Frederick, Adam Geier, Alyssa Casill, Martin Akerman, Gayatri Arun. SpliceCore® a platform for identifying aberrant alternative splicing in triple negative breast cancer for novel therapeutic development [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P170.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality, characterized by progressive degeneration of spinal-cord motor neurons, leading to atrophy of skeletal muscles. However, accumulating evidence indicates that it is a multi-system disorder, particularly in its severe forms. Several studies delineated structural and functional cardiac abnormalities in SMA patients and mouse models, yet the abnormalities have been primarily attributed to autonomic dysfunction. Here, we show in a severe mouse model that its cardiomyocytes undergo G0/G1 cell-cycle arrest and enhanced apoptosis during postnatal development. Microarray and real-time RT-PCR analyses revealed that a set of genes associated with cell cycle and apoptosis were dysregulated in newborn pups. Of particular interest, the Birc5 gene, which encodes Survivin, an essential protein for heart development, was down-regulated even on pre-symptomatic postnatal day 0. Interestingly, cultured cardiomyocytes depleted of SMN recapitulated the gene expression changes including downregulation of Survivin and abnormal cell-cycle progression; and overexpression of Survivin rescued the cell-cycle defect. Finally, increasing SMN in SMA mice with a therapeutic antisense oligonucleotide improved heart pathology and recovered expression of deregulated genes. Collectively, our data demonstrate that the cardiac malfunction of the severe SMA mouse model is mainly a cell-autonomous defect, caused by widespread gene deregulation in heart tissue, particularly of Birc5, resulting in developmental abnormalities through cell-cycle arrest and apoptosis.

SUMMARYMisregulation of alternative splicing is a hallmark of human tumors; yet to what extent and how it contributes to malignancy are only beginning to be unraveled. Here, we define which members of the splicing factor SR and SR-like families contribute to breast cancer, and uncover differences and redundancies in their targets and biological functions. We first identify splicing factors frequently altered in human breast tumors, and then assay their oncogenic functions using breast organoid models. Importantly we demonstrate that not all splicing factors affect mammary tumorigenesis. Specifically, upregulation of either SRSF4, SRSF6 or TRA2β promotes cell transformation and invasion. By characterizing the targets of theses oncogenic factors, we identify a shared set of spliced genes associated with well-established cancer hallmarks. Finally, we demonstrate that the splicing factor TRA2β is regulated by the MYC oncogene, plays a role in metastasis maintenancein vivo, and its levels correlate with breast-cancer-patient survival.

Splicing factor SRSF1 is upregulated in human breast tumors, and its overexpression promotes transformation of mammary cells. Using RNA-seq, we identified SRSF1-regulated alternative splicing (AS) targets in organotypic three-dimensional MCF-10A cell cultures that mimic a context relevant to breast cancer. We identified and validated hundreds of endogenous SRSF1-regulated AS events. De-novo discovery of the SRSF1 binding motif reconciled discrepancies in previous motif analyses. Using a Bayesian model, we determined positional effects of SRSF1 binding on cassette exons: binding close to the 5′ splice site generally promoted exon inclusion, whereas binding near the 3′ splice site promoted either exon skipping or inclusion. Finally, we identified SRSF1-regulated AS events deregulated in human tumors; overexpressing one such isoform, exon-9-included CASC4, increased acinar size and proliferation, and decreased apoptosis, partially recapitulating SRSF1's oncogenic effects. Thus, we uncovered SRSF1 positive and negative regulatory mechanisms, and oncogenic AS events that represent potential targets for therapeutics development.

Splicing and translation are highly regulated steps of gene expression. Altered expression of proteins involved in these processes can be deleterious. Therefore, the cell has many safeguards against such misregulation. We report that the oncogenic splicing factor SRSF1, which is overexpressed in many cancers, stabilizes the tumor suppressor protein p53 by abrogating its MDM2-dependent proteasomal degradation. We show that SRSF1 is a necessary component of an MDM2/ribosomal protein complex, separate from the ribosome, that functions in a p53-dependent ribosomal-stress checkpoint pathway. Consistent with the stabilization of p53, increased SRSF1 expression in primary human fibroblasts decreases cellular proliferation and ultimately triggers oncogene-induced senescence (OIS). These findings underscore the deleterious outcome of SRSF1 overexpression and identify a cellular defense mechanism against its aberrant function. Furthermore, they implicate the RPL5-MDM2 complex in OIS and demonstrate a link between spliceosomal and ribosomal components, functioning independently of their canonical roles, to monitor cellular physiology and cell-cycle progression.

SummaryThe SR protein splicing factor SRSF1 is a potent proto-oncogene that is frequently upregulated in cancer. Here, we show that SRSF1 is a direct target of the transcription factor oncoprotein MYC. These two oncogenes are significantly coexpressed in lung carcinomas, and MYC knockdown downregulates SRSF1 expression in lung-cancer cell lines. MYC directly activates transcription of SRSF1 through two noncanonical E-boxes in its promoter. The resulting increase in SRSF1 protein is sufficient to modulate alternative splicing of a subset of transcripts. In particular, MYC induction leads to SRSF1-mediated alternative splicing of the signaling kinase MKNK2 and the transcription factor TEAD1. SRSF1 knockdown reduces MYC's oncogenic activity, decreasing proliferation and anchorage-independent growth. These results suggest a mechanism for SRSF1 upregulation in tumors with elevated MYC and identify SRSF1 as a critical MYC target that contributes to its oncogenic potential by enabling MYC to regulate the expression of specific protein isoforms through alternative splicing.

In this study, we explore the diversity and its distribution along the wheat leaf rust resistance protein LR10 three-dimensional structure. Lr10 is a leaf rust resistance gene encoding a coiled coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) class of protein. Lr10 was cloned and sequenced from 58 accessions representing diverse habitats of wild emmer wheat in Israel. Nucleotide diversity was very high relative to other wild emmer wheat genes (π= 0.029). The CC domain was found to be the most diverse domain and subject to positive selection. Superimposition of the diversity on the CC three-dimensional structure showed that some of the variable and positively selected residues were solvent exposed and may interact with other proteins. The LRR domain was relatively conserved, but showed a hotspot of amino acid variation between two haplotypes in the ninth repeat. This repeat was longer than the other LRRs, and three-dimensional modelling suggested that an extensive α helix structure was formed in this region. The two haplotypes also differed in splicing regulation motifs. In genotypes with one haplotype, an intron was alternatively spliced in this region, whereas, in genotypes with the other haplotype, this intron did not splice at all. The two haplotypes are proposed to be ancient and maintained by balancing selection.

Alternative splicing constitutes a major mechanism creating protein diversity in humans. This diversity can result from the alternative skipping of entire exons or by alternative selection of the 5' or 3' splice sites that define the exon boundaries. In this study, we analyze the sequence and evolutionary characteristics of alternative 3' splice sites conserved between human and mouse genomes for distances ranging from 3 to 100 nucleotides. We show that alternative splicing events can be distinguished from constitutive splicing by a combination of properties which vary depending on the distance between the splice sites. Among the unique features of alternative 3' splice sites, we observed an unexpectedly high occurrence of events in which a polypyrimidine tract was found to overlap the upstream splice site. By applying a machine-learning approach, we show that we can successfully discriminate true alternative 3' splice sites from constitutive 3' splice sites. Finally, we propose that the unique features of the intron flanking alternative splice sites are indicative of a regulatory mechanism that is involved in splice site selection. We postulate that the process of splice site selection is influenced by the distance between the competitive splice sites.

Aberrant DNA methylation has long been implicated in cancers. In this letter, we present a highly discriminative DNA methylation biomarker for non-small cell lung cancers (NSCLCs) and 14 other cancers. Based on 69 NSCLC cell lines and 257 cancer-free lung tissues, we identified a CpG island in SCT gene promoter, which was verified by qMSP experiment in 15 NSCLC cell lines and three immortalized human respiratory epithelium cells. In addition, we found that the SCT promoter was methylated in 23 cancer cell lines involving >10 cancer types profiled by ENCODE. We found that the SCT promoter is hypermethylated in primary tumors from TCGA lung cancer cohort. In addition, we found that SCT promoter is methylated at high frequencies in 15 malignancies and is not methylated in $\sim 1000$ non-cancerous tissues across >30 organ types. This letter indicates that SCT promoter methylation is a highly discriminative biomarker for lung and many other cancers.

Genome-wide analyses have identified thousands of long noncoding RNAs (lncRNAs). Malat1 (metastasis-associated lung adenocarcinoma transcript 1) is among the most abundant lncRNAs whose expression is altered in numerous cancers. Here we report that genetic loss or systemic knockdown of Malat1 using antisense oligonucleotides (ASOs) in the MMTV (mouse mammary tumor virus)-PyMT mouse mammary carcinoma model results in slower tumor growth accompanied by significant differentiation into cystic tumors and a reduction in metastasis. Furthermore, Malat1 loss results in a reduction of branching morphogenesis in MMTV-PyMT- and Her2/neu-amplified tumor organoids, increased cell adhesion, and loss of migration. At the molecular level, Malat1 knockdown results in alterations in gene expression and changes in splicing patterns of genes involved in differentiation and protumorigenic signaling pathways. Together, these data demonstrate for the first time a functional role of Malat1 in regulating critical processes in mammary cancer pathogenesis. Thus, Malat1 represents an exciting therapeutic target, and Malat1 ASOs represent a potential therapy for inhibiting breast cancer progression.

Background During spliceosome assembly, protein-protein interactions (PPI) are sequentially formed and disrupted to accommodate the spatial requirements of pre-mRNA substrate recognition and catalysis. Splicing activators and repressors, such as SR proteins and hnRNPs, modulate spliceosome assembly and regulate alternative splicing. However, it remains unclear how they differentially interact with the core spliceosome to perform their functions. Results Here, we investigate the protein connectivity of SR and hnRNP proteins to the core spliceosome using probabilistic network reconstruction based on the integration of interactome and gene expression data. We validate our model by immunoprecipitation and mass spectrometry of the prototypical splicing factors SRSF1 and hnRNPA1. Network analysis reveals that a factor’s properties as an activator or repressor can be predicted from its overall connectivity to the rest of the spliceosome. In addition, we discover and experimentally validate PPIs between the oncoprotein SRSF1 and members of the anti-tumor drug target SF3 complex. Our findings suggest that activators promote the formation of PPIs between spliceosomal sub-complexes, whereas repressors mostly operate through protein-RNA interactions. Conclusions This study demonstrates that combining in-silico modeling with biochemistry can significantly advance the understanding of structure and function relationships in the human spliceosome. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0682-5) contains supplementary material, which is available to authorized users.

Alternative splicing (AS) constitutes a major mechanism creating protein diversity in humans. Previous bioinformatics studies based on expressed sequence tag and mRNA data have identified many AS events that are conserved between humans and mice. Of these events, approximately 25% are related to alternative choices of 3' and 5' splice sites. Surprisingly, half of all these events involve 3' splice sites that are exactly 3 nt apart. These tandem 3' splice sites result from the presence of the NAGNAG motif at the acceptor splice site, recently reported to be widely spread in the human genome. Although the NAGNAG motif is common in human genes, only a small subset of sites with this motif is confirmed to be involved in AS. We examined the NAGNAG motifs and observed specific features such as high sequence conservation of the motif, high conservation of approximately 30 bp at the intronic regions flanking the 3' splice site and overabundance of cis-regulatory elements, which are characteristic of alternatively spliced tandem acceptor sites and can distinguish them from the constitutive sites in which the proximal NAG splice site is selected. Our findings imply that AS at tandem splice sites and constitutive splicing of the distal NAG are highly regulated.

Eight amino acid permease genes from the protozoan parasite Leishmania donovani (AAPLDs) were cloned, sequenced, and shown to be expressed in promastigotes. Seven of these belong to the amino acid transporter-1 and one to the amino acid polyamino-choline superfamilies. Using these sequences as well as known and characterized amino acid permease genes from all kingdoms, a training set was established and used to search for motifs, using the MEME motif discovery tool. This study revealed two motifs that are specific to the genus Leishmania, four to the family trypanosomatidae, and a single motif that is common between trypanosomatidae and mammalian systems A1 and N. Interestingly, most of these motifs are clustered in two regions of 50-60 amino acids. Blast search analyses indicated a close relationship between the L. donovani and Trypanosoma brucei amino acid permeases. The results of this work describe the cloning of the first amino acid permease genes in parasitic protozoa and contribute to the understanding of amino acid permease evolution in these organisms. Furthermore, the identification of genus-specific motifs in these proteins might be useful to better understand parasite physiology within its hosts.

Genome-wide studies have identified thousands of long non-coding RNAs (lncRNAs) lacking protein-coding capacity. MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) is among the most abundant and highly conserved nuclear restricted lncRNAs whose expression is mis-regulated in many cancers including breast cancer. RNA-FISH experiments on primary tumor and metastatic nodules from patients with luminal type breast cancer revealed that MALAT1 lncRNA is 4-5 times up regulated in metastatic nodules compared to primary tumors. This strongly suggests that MALAT1 plays an important role in the metastatic progression of luminal breast cancers. We have used the MMTV-PyMT mouse model of luminal B breast cancer to characterize the role of Malat1 in primary breast cancer and its subsequent metastasis. Malat1 lncRNA was knocked down via subcutaneous administration of antisense oligonucleotides (ASOs) at a dose of 125mg/kg/week over a period of 7 weeks after which animals were sacrificed and primary tumors and lungs were removed for molecular and histological analyses. Malat1 ASO treatment resulted in ~60% knockdown in the primary tumor concomitant with a significant reduction in tumor progression rate as well as a change in the differentiation status. Detailed histo-pathological analysis of ASO treated tumors showed an increase in well-differentiated ductular tumors, whereas scrambled ASO treated tumors progressed to solid carcinomas. Most interestingly, a marked decrease was observed in the incidence of lung metastases; ~70% fewer metastatic nodules in Malat1 ASO treated animals than scrambled ASO treated animals. Further, Malat1 ASO treated ex-vivo generated mammary gland organoids from MMTV-PyMT mice, resulted in an inhibition of branching morphogenesis, which recapitulates the invasive process that initiate metastases in vivo. RNA-seq analysis of the primary tumors and tumor derived organoids treated with Malat1 ASO showed up-regulation of genes involved in differentiation and down regulation of genes involved in migration and proliferation. Further, Malat1 knock-down also resulted in aberrant splicing of many genes including critical transcription factors. Together, our data indicates that Malat1 lncRNA regulates critical processes in breast cancer pathogenesis and represents a promising therapeutic target for treatment. This abstract is also presented as Poster B02. Citation Format: Gayatri Arun, Sarah Diermeier, Martin Akerman, Kung-Chi Chang, J.Erby Wilkinson, Stephen Hearn, Youngsoo Kim, A.Robert MacLeod, Adrian R. Krainer, Larry Norton, Edi Brogi, Mikala Egeblad, David L. Spector. Differentiation of mammary tumors and reduction in metastasis upon Malat1 LncRNA loss. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR11.

Alternative splicing is a key control point in gene expression, whose misregulation contributes to cancer malignancy. Although certain splicing factors (SFs) and their targets are altered in human tumors, the functional significance of these alterations remains unclear. We previously demonstrated that the splicing factor SRSF1 is upregulated in human breast tumors and promotes transformation in vivo and in vitro. SRSF1 is a prototypical member of the SR protein family, composed of 12 structurally related proteins. However, little is known about differences and redundancies in their splicing targets and biological functions. Here, we investigated whether additional SFs also promoted breast cancer, using transformation models that mimic the relevant biological context. In parallel, we used RNA sequencing (RNA-seq) to systematically identify their oncogenic splicing targets. By mining a large collection of human tumors from the TCGA project, we defined the molecular portraits of SFs alterations in breast tumors. We identified five SFs amplified and/or overexpressed in at least 10% of breast tumors. We then used SF-overexpressing human mammary epithelial MCF-10A cells grown in organotypic 3-D culture; these cells form polarized growth-arrested acinar structures, similar to the terminal units of mammary ducts. Various breast-cancer oncogenes are known to disrupt acinar growth and/or architecture. Interestingly, only certain SFs were oncogenic in this context, differentially affecting cell proliferation, apoptosis, or acinar organization, suggesting non-redundant functions. We then characterized the splicing targets relevant for SF-mediated transformation. We developed a bioinformatics pipeline to identify and quantify splicing variation in RNA-seq data. We defined the global repertoire of SF-regulated splicing events in 3-D culture and compared the target specificities of various SR proteins. In addition, we identified splicing targets regulated both in 3-D culture as well as in human breast tumors. Strikingly, SFs that promoted similar phenotypic changes shared a significant number of splicing targets, suggesting that they regulate common genes to promote tumor initiation. Furthermore, specific SFs affected targets previously associated with epithelial to mesenchymal transition, and increased cell migration or invasion. Finally, we uncovered that the splicing regulator TRA2β is required for the maintenance of metastatic properties of human breast-cancer cells in 3-D culture and in mouse orthotopic models. Furthermore, TRA2β levels correlate with increased metastatic incidence in breast cancer patients. Thus TRA2β represent a potential target for therapeutics development. In summary, we gained new insights into the biological functions of SR proteins and identified novel oncogenic SF-regulated splicing events involved in tumor initiation and metastasis. Citation Format: Olga Anczuków, Shipra Das, Kuan-Ting Lin, Jie Wu, Martin Akerman, Senthil K. Muthuswamy, Adrian R. Krainer. Nonredundant functions of splicing factors in breast-cancer initiation and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A50.

An established paradigm in pre-mRNA splicing is the recognition of the 5′ splice site (5′ss) by canonical base-pairing to the 5′ end of U1 small nuclear RNA (snRNA). We recently reported that a small subset of 5′ss base-pair to U1 in an alternate register that is shifted by 1 nucleotide. Using genetic suppression experiments in human cells, we now demonstrate that many other 5′ss are recognized via noncanonical base-pairing registers involving bulged nucleotides on either the 5′ss or U1 RNA strand, which we term “bulge registers.” By combining experimental evidence with transcriptome-wide free-energy calculations of 5′ss/U1 base-pairing, we estimate that 10,248 5′ss (∼5% of human 5′ss) in 6577 genes use bulge registers. Several of these 5′ss occur in genes with mutations causing genetic diseases and are often associated with alternative splicing. These results call for a redefinition of an essential element for gene expression that incorporates these registers, with important implications for the molecular classification of splicing mutations and for alternative splicing. Published version

Alternative splicing of the pyruvate kinase M gene (PK-M) can generate the M2 isoform and promote aerobic glycolysis and tumor growth. However, the cancer-specific alternative splicing regulation of PK-M is not completely understood. Here, we demonstrate that PK-M is regulated by reciprocal effects on the mutually exclusive exons 9 and 10, such that exon 9 is repressed and exon 10 is activated in cancer cells. Strikingly, exonic, rather than intronic, cis-elements are key determinants of PK-M splicing isoform ratios. Using a systematic sub-exonic duplication approach, we identify a potent exonic splicing enhancer in exon 10, which differs from its homologous counterpart in exon 9 by only two nucleotides. We identify SRSF3 as one of the cognate factors, and show that this serine/arginine-rich protein activates exon 10 and mediates changes in glucose metabolism. These findings provide mechanistic insights into the complex regulation of alternative splicing of a key regulator of the Warburg effect, and also have implications for other genes with a similar pattern of alternative splicing.

In this study, we explore the diversity and its distribution along the wheat leaf rust resistance protein LR10 three‐dimensional structure. Lr10 is a leaf rust resistance gene encoding a coiled coil–nucleotide‐binding site–leucine‐rich repeat (CC–NBS–LRR) class of protein. Lr10 was cloned and sequenced from 58 accessions representing diverse habitats of wild emmer wheat in Israel. Nucleotide diversity was very high relative to other wild emmer wheat genes (π= 0.029). The CC domain was found to be the most diverse domain and subject to positive selection. Superimposition of the diversity on the CC three‐dimensional structure showed that some of the variable and positively selected residues were solvent exposed and may interact with other proteins. The LRR domain was relatively conserved, but showed a hotspot of amino acid variation between two haplotypes in the ninth repeat. This repeat was longer than the other LRRs, and three‐dimensional modelling suggested that an extensive α helix structure was formed in this region. The two haplotypes also differed in splicing regulation motifs. In genotypes with one haplotype, an intron was alternatively spliced in this region, whereas, in genotypes with the other haplotype, this intron did not splice at all. The two haplotypes are proposed to be ancient and maintained by balancing selection.

Motivation: Alternative splicing (AS) is a pre-mRNA maturation process leading to the expression of multiple mRNA variants from the same primary transcript. More than 90% of human genes are expressed via AS. Therefore, quantifying the inclusion level of every exon is crucial for generating accurate transcriptomic maps and studying the regulation of AS. Results: Here we introduce SpliceTrap, a method to quantify exon inclusion levels using paired-end RNA-seq data. Unlike other tools, which focus on full-length transcript isoforms, SpliceTrap approaches the expression-level estimation of each exon as an independent Bayesian inference problem. In addition, SpliceTrap can identify major classes of alternative splicing events under a single cellular condition, without requiring a background set of reads to estimate relative splicing changes. We tested SpliceTrap both by simulation and real data analysis, and compared it to state-of-the-art tools for transcript quantification. SpliceTrap demonstrated improved accuracy, robustness and reliability in quantifying exon-inclusion ratios. Conclusions: SpliceTrap is a useful tool to study alternative splicing regulation, especially for accurate quantification of local exon-inclusion ratios from RNA-seq data. Availability and Implementation: SpliceTrap can be implemented online through the CSH Galaxy server http://cancan.cshl.edu/splicetrap and is also available for download and installation at http://rulai.cshl.edu/splicetrap/. Contact: michael.zhang@utdallas.edu Supplementary Information: Supplementary data are available at Bioinformatics online.

The splicing-factor oncoprotein SRSF1 (also known as SF2/ASF or ASF/SF2) is upregulated in breast cancers. We investigated the ability of SRSF1 to transform human and mouse mammary epithelial cells in vivo and in vitro. SRSF1-overexpressing COMMA-1D cells formed tumors, following orthotopic transplantation to reconstitute the mammary gland. In three-dimensional (3D) culture, SRSF1-overexpressing MCF-10A cells formed larger acini than control cells, reflecting increased proliferation and delayed apoptosis during acinar morphogenesis. These effects required the first RNA-recognition motif and nuclear functions of SRSF1. SRSF1 overexpression promoted alternative splicing of BIM (also known as BCL2L11) and BIN1 to produce isoforms that lack pro-apoptotic functions and contribute to the phenotype. Finally, SRSF1 cooperated specifically with MYC to transform mammary epithelial cells, in part by potentiating eIF4E activation, and these cooperating oncogenes are significantly coexpressed in human breast tumors. Thus, SRSF1 can promote breast cancer, and SRSF1 itself or its downstream effectors may be valuable targets for the development of therapeutics.

Thymidine phosphorylase (TP) catalyzes the conversion of thymidine to thymine and 2-deoxyribose-1-phosphate. The latter plays an important role in induction of angiogenesis. As such, many human malignancies exhibit TP overexpression that correlates with increased microvessel density, formation of aggressive tumors, and dismal prognosis. Because TP is frequently overexpressed in cancer, pro-drugs were developed that utilize TP activity for their bioactivation to cytotoxic drugs. In this respect, TP is indispensable for the pharmacologic activity of the chemotherapeutic drug capecitabine, as it converts its intermediary metabolite 5′-deoxyfluorouridine to 5-fluorouracil. Thus, loss of TP function confers resistance to the prodrug capecitabine, currently used for the treatment of metastatic colorectal cancer and breast cancer. However, drug resistance phenomena may frequently emerge that compromise the pharmacologic activity of capecitabine. Deciphering the molecular mechanisms underlying resistance to TP-activated prodrugs is an important goal toward the overcoming of such drug resistance phenomena. Here, we discovered that lack of TP protein in drug-resistant tumor cells is due to unsplicing of its pre-mRNA. Advanced bioinformatics identified the family of heterogeneous nuclear ribonucleoproteins (hnRNP) H/F as candidate splicing factors potentially responsible for impaired TP splicing. Indeed, whereas parental cells lacked nuclear localization of hnRNPs H1/H2 and F, drug-resistant cells harbored marked levels of these splicing factors. Nuclear RNA immunoprecipitation experiments established a strong binding of hnRNP H1/H2 to TP pre-mRNA, hence implicating them in TP splicing. Moreover, introduction of hnRNP H2 into drug-sensitive parental cells recapitulated aberrant TP splicing and 5′-deoxyfluorouridine resistance. Thus, this is the first study identifying altered function of hnRNP H1/H2 in tumor cells as a novel determinant of aberrant TP splicing thereby resulting in acquired chemoresistance to TP-activated fluoropyrimidine anticancer drugs.

Alternative splicing (AS) is a post-transcriptional process considered to be responsible for the huge diversity of proteins in higher eukaryotes. AS events are regulated by different splicing factors (SFs) that bind to sequence elements on the RNA. SFmap is a web server for predicting putative SF binding sites in genomic data (http://sfmap.technion.ac.il). SFmap implements the COS(WR) algorithm, which computes similarity scores for a given regulatory motif based on information derived from its sequence environment and its evolutionary conservation. Input for SFmap is a human genomic sequence or a list of sequences in FASTA format that can either be uploaded from a file or pasted into a window. SFmap searches within a given sequence for significant hits of binding motifs that are either stored in our database or defined by the user. SFmap results are provided both as a text file and as a graphical web interface.

Heterogeneous cell populations form an interconnected network that determine their collective output. One example of such a heterogeneous immune population is tumor-infiltrating lymphocytes (TILs), whose output can be measured in terms of its reactivity against tumors. While the degree of reactivity varies considerably between different TILs, ranging from null to a potent response, the underlying network that governs the reactivity is poorly understood. Here, we asked whether one can predict and even control this reactivity. To address this we measured the subpopulation compositions of 91 TILs surgically removed from 27 metastatic melanoma patients. Despite the large number of subpopulations compositions, we were able to computationally extract a simple set of subpopulation-based rules that accurately predict the degree of reactivity. This raised the conjecture of whether one could control reactivity of TILs by manipulating their subpopulation composition. Remarkably, by rationally enriching and depleting selected subsets of subpopulations, we were able to restore anti-tumor reactivity to nonreactive TILs. Altogether, this work describes a general framework for predicting and controlling the output of a cell mixture.

Cancer cells display aberrant alternative splicing profiles, leading to the production of isoforms that can increase cell proliferation and migration, resistance to apoptosis, or alter cell metabolism. Recently, recurrent somatic mutations in components of the splicing machinery have been identified in human tumors, raising a new interest in the field and suggesting that alterations in splicing factors are a new hallmark of cancer. Splicing factors elicit changes in splicing in a concentration-dependent manner. Thus, changes in the expression of these proteins, as reported in different types of cancers, can affect the splicing of multiple genes and are likely involved in splicing deregulation in cancer, even in the absence of mutations. Changes in the expression of splicing factors have been reported in various types of cancers, including breast. We demonstrated previously that the splicing factor SRSF1 is upregulated in human breast cancers and can transform mammary epithelial cells in vivo and in vitro. SRSF1 is a prototypical member of the SR protein family, composed of 12 members sharing structural similarities. However, little is known about differences and redundancies in their splicing targets and thus in their specific biological functions. We are now investigating whether additional splicing factors also promote mammary epithelial cell transformation by using models that mimic the correct biological context in which tumors arise. We first identified splicing factors overexpressed at the transcript and protein levels in a large collection of human breast tumors and cancer cell lines. We then compared the ability of 10 selected splicing factors to transform human mammary epithelial MCF-10A cells grown in organotypic 3-D culture. These cells undergo a 16-day differentiation program, forming growth-arrested, hollow acinar structures with polarized architecture, similar to the terminal units of mammary ducts. Various breast cancer oncogenes are known to disrupt acinar growth and/or architecture. We assessed how splicing-factor overexpression affects differentially cell proliferation and apoptosis, as well as cell migration and invasion. Interestingly, only certain splicing factors are oncogenic in this context, suggesting functional differences. Furthermore, specific splicing factors increase cell migration and invasion, without promoting transformation. We are now characterizing the splicing targets relevant for specific splicing-factor-mediated transformation by next-generation RNA-sequencing. Furthermore, we are investigating which splicing factors are necessary or sufficient to promote metastatic properties of human breast cancer cell lines in vitro and in vivo. Finally, we have identified splicing factors that cooperate specifically with the MYC oncogene and are co-expressed with MYC in human breast tumors. In conclusion, by identifying oncogenic splicing factors, their targets and their regulators, we hope to establish novel targets for targeted cancer therapies. Citation Format: Olga Anczukow, Kuan-Ting Lin, Shipra Das, Jie Wu, Martin Akerman, Senthil K. Muthuswamy, Adrian R. Krainer. Differential functions of splicing factors in breast cancer initiation and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A078.

Cancer cells often display aberrant profiles of alternative splicing, leading to the production of isoforms that can stimulate cell proliferation and migration, increase resistance to apoptosis, or alter cell metabolism. Recently, recurrent somatic mutations in components of the splicing machinery have been identified in human tumors, raising a new interest in the field and suggesting that alterations in splicing factors are a new hallmark of cancer. Splicing factors elicit changes in splicing in a concentration-dependent manner. Thus, changes in the expression of these proteins, as reported in different types of cancers, can affect the splicing of multiple genes and are likely involved in splicing deregulation in cancer, even in the absence of mutations. We previously demonstrated that the splicing factor SRSF1'formerly SF2/ASF' is often upregulated in human breast cancers and can transform mammary epithelial cells in vivo and in vitro. SRSF1 is a prototypical member of the SR protein family, composed of 12 members sharing structural similarities. However, little is known about differences and redundancies in their splicing targets and thus in their specific biological functions. We are now investigating wherever additional splicing factors can promote mammary epithelial cell transformation by using models that mimic the correct biological context in which tumors arise. We have mined data from a large collection of human breast tumors and cancer cell lines to identify splicing factors overexpressed at the transcript and protein levels. We then compared the ability of 10 selected splicing factors to transform human mammary epithelial MCF-10A cells grown in organotypic 3-D culture. Under these conditions, the cells undergo a 16-day differentiation program, forming growth-arrested, hollow acinar structures with polarized architecture, similar to the terminal units of mammary ducts. Various oncogenes associated with breast cancer are known to disrupt acinar growth and/or architecture. We assessed how splicing-factor overexpression affects cell proliferation and apoptosis, as well as cell migration and invasion. Interestingly, only certain splicing factors are oncogenic in this context, suggesting functional differences. Furthermore, specific splicing factors increase cell migration and invasion but are unable to promote transformation. We have demonstrated specificity in splicing-factor mediated transformation, and we are now investigating what are the relevant splicing targets for transformation by several of these oncogenic splicing factors. Furthermore, we are investigating which splicing factors are necessary or sufficient to promote cell invasion and metastatic potential in human breast cancer cell lines in vitro and in vivo. Finally, we have identified splicing factors that cooperate specifically with the MYC oncogene and are significantly co-expressed with MYC in human breast tumors. By identifying oncogenic splicing factors that can promote breast cancer, and their downstream effectors, we hope to establish candidate targets for therapeutics development based on the modulation of oncogenic splicing events and their regulators. Citation Format: Olga Anczukow, Kuan-Ting Lin, Martin Akerman, Shipra Das, Senthil K. Muthuswamy, Adrian Krainer. Differential functions of splicing factors in breast-cancer initiation and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B53.

Udgivelsesdato: 2010-Oct-11 Alternative splicing is a crucial step in the generation of protein diversity and its misregulation is observed in many human cancer types. By analyzing 143 colorectal samples using exon arrays, SLC39A14, a divalent cation transporter, was identified as being aberrantly spliced in tumor samples. SLC39A14 contains two mutually exclusive exons 4A and 4B and the exon 4A/4B ratio was significantly altered in adenomas (p=3.6x10-10) and cancers (p=9.4x10-11), independent of microsatellite stability status. The findings were validated in independent exon array data sets and by quantitative real-time reverse-transcription PCR (qRT-PCR). Aberrant Wnt signaling is a hallmark of colorectal tumorigenesis and is characterized by nuclear β-catenin. Experimental inactivation of Wnt signaling in DLD1 and Ls174T cells by knockdown of β-catenin or overexpression of dominant negative TCFs (TCF1 and TCF4) altered the 4A/4B ratio, indicating that SLC39A14 splicing is regulated by the Wnt pathway. An altered 4A/4B ratio was also observed in gastric and lung cancer where Wnt signaling is also known to be aberrantly activated. The splicing factor SRSF1 and its regulator, the kinase SRPK1, were found to be deregulated upon Wnt inactivation in colorectal carcinoma cells. SRPK1 was also found upregulated in both adenoma samples (p=1.5x10-5) and cancer samples (p=5x10-4). In silico splicing factor binding analysis predicted SRSF1 to bind predominantly to the cancer associated exon 4B, hence, it was hypothesized that SRPK1 activates SRSF1 through phosphorylation, followed by SRSF1 binding to exon 4B and regulation of SLC39A14 splicing. Indeed, siRNA-mediated knockdown of SRPK1 and SRSF1 in DLD1 and SW480 colorectal cancer cells led to a change in the 4A/4B isoform ratio, supporting a role of these factors in the regulation of SLC39A14 splicing. In conclusion, alternative splicing of SLC39A14 was identified in colorectal tumors and found to be regulated by the Wnt pathway, most likely through regulation of SRPK1 and SRSF1.

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A computational method is presented for genome-wide mapping of splicing factor binding sites that considers both the genomic environment and evolutionary conservation., Alternative splicing is regulated by splicing factors that serve as positive or negative effectors, interacting with regulatory elements along exons and introns. Here we present a novel computational method for genome-wide mapping of splicing factor binding sites that considers both the genomic environment and the evolutionary conservation of the regulatory elements. The method was applied to study the regulation of different alternative splicing events, uncovering an interesting network of interactions among splicing factors.