Changes In Splicing Patterns Research Articles

Pathomechanisms of Monoallelic variants in TTN causing skeletal muscle disease.

Pathogenic variants in the titin gene (TTN) are known to cause a wide range of cardiac and musculoskeletal disorders, with skeletal myopathy mostly attributed to biallelic variants. We identified monoallelic truncating variants (TTNtv), splice site or internal deletions in TTN in probands with mild, progressive axial and proximal weakness, with dilated cardiomyopathy frequently developing with age. These variants segregated in an autosomal dominant pattern in 7 out of 8 studied families. We investigated the impact of these variants on mRNA, protein levels, and skeletal muscle structure and function. Results reveal that nonsense-mediated decay likely prevents accumulation of harmful truncated protein in skeletal muscle in patients with TTNtvs. Splice variants and an out-of-frame deletion induce aberrant exon skipping, while an in-frame deletion produces shortened titin with intact N- and C-termini, resulting in disrupted sarcomeric structure. All variant types were associated with genome-wide changes in splicing patterns, which represent a hallmark of disease progression. Lastly, RNA-seq studies revealed that GDF11, a member of the TGF-β superfamily, is upregulated in diseased tissue, indicating that it might be a useful therapeutic target in skeletal muscle titinopathies.

Predicting splicing patterns from the transcription factor binding sites in the promoter with deep learning

BackgroundAlternative splicing is a pivotal mechanism of post-transcriptional modification that contributes to the transcriptome plasticity and proteome diversity in metazoan cells. Although many splicing regulations around the exon/intron regions are known, the relationship between promoter-bound transcription factors and the downstream alternative splicing largely remains unexplored.ResultsIn this study, we present computational approaches to unravel the regulatory relationship between promoter-bound transcription factor binding sites (TFBSs) and the splicing patterns. We curated a fine dataset that includes DNase I hypersensitive site sequencing and transcriptomes across fifteen human tissues from ENCODE. Specifically, we proposed different representations of TF binding context and splicing patterns to examine the associations between the promoter and downstream splicing events. While machine learning models demonstrated potential in predicting splicing patterns based on TFBS occupancies, the limitations in the generalization of predicting the splicing forms of singleton genes across diverse tissues was observed with carefully examination using different cross-validation methods. We further investigated the association between alterations in individual TFBS at promoters and shifts in exon splicing efficiency. Our results demonstrate that the convolutional neural network (CNN) models, trained on TF binding changes in the promoters, can predict the changes in splicing patterns. Furthermore, a systemic in silico substitutions analysis on the CNN models highlighted several potential splicing regulators. Notably, using empirical validation using K562 CTCFL shRNA knock-down data, we showed the significant role of CTCFL in splicing regulation.ConclusionIn conclusion, our finding highlights the potential role of promoter-bound TFBSs in influencing the regulation of downstream splicing patterns and provides insights for discovering alternative splicing regulations.

Issue Information

Front cover: Son Goku (Sun Wukong), a god in monkey form, was brandishing a Nyoi‐bo representing the TBXT gene. However, an AluY‐marked dagger hit the Nyoi‐bo and broke it off. Xia et al. showed that the loss of tails in hominoids, including humans and apes, was due in part to changes in splicing patterns caused by the insertion of an AluY element into the ancestral TBXT gene (Xia et al. (2024) Nature 626:1042‐1048, DOI: 10.1038/s41586‐024‐07095‐8). Son Goku cannot hide the look of impatience on his face as his tail begins to disappear, having impaired the function of the Nyoi‐bo, which is necessary to maintain his tail, the source of Son Goku's magical power. Designed by TRAIS Co., Ltd. (Kobe, Japan).image

Design of a Phase 1, Placebo-Controlled Study to Assess the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Single-Ascending Doses of PGN-EDODM1 in Adult Participants with Myotonic Dystrophy Type 1 (DM1) (P6-8.004)

<h3>Objective:</h3> The primary objective of this single-ascending dose (SAD) study is to evaluate the safety and tolerability of PGN-EDODM1 in adults living with DM1. Secondary and exploratory objectives include pharmacokinetics (PK), muscle distribution, pharmacodynamics (changes in splicing pattern of transcripts), and select functional outcome measures. <h3>Background:</h3> PepGen’s Enhanced Delivery Oligonucleotide (EDO) cell-penetrating peptide technology is engineered to optimize tissue delivery and cellular uptake of therapeutic oligonucleotides. PGN-EDODM1 is being evaluated for the treatment of DM1. PGN-EDODM1 binds to the pathogenic CUG repeat expansion in <i>DMPK</i> mRNA and reduces sequestration of Muscleblind (MBNL) proteins through a steric blocking mechanism. Release of MBNL proteins is hypothesized to correct DM1 spliceopathy; the root cause of DM1. <h3>Design/Methods:</h3> Males and females 18–50 years of age, inclusive, with genetically confirmed diagnosis of DM1 will be eligible to participate, and will be randomized 3:1 (6 active, 2 placebo) to receive PGN-EDODM1 or placebo in each dose cohort. A muscle needle biopsy (tibialis anterior) will be performed at baseline and at Weeks 4 and 16 post-dosing for measurement of splicing of selected transcripts. An independent data safety and monitoring committee will oversee dose escalation. <h3>Results:</h3> The doses to be assessed will be based on data from the preclinical data and data from the Phase 1 healthy volunteer safety study of our lead EDO, PGN-EDO51. A plan for analysis of safety measures such as vital signs, electrocardiograms, clinical chemistry, urinalysis, and biomarkers will be presented, along with details on proposed functional endpoints and biomarkers to be explored in this study. <h3>Conclusions:</h3> This Phase 1 SAD clinical study will support continued development of PGN-EDODM1 for the treatment of DM1, and will be initiated in 2023. Participants will be invited to enroll in a planned multiple-ascending dose study (MAD) and a subsequent open-label extension study. <b>Disclosure:</b> Dr. Mellion has received personal compensation for serving as an employee of PepGen. Dr. Mellion has stock in PepGen. Dr. Larkindale has received personal compensation for serving as an employee of PepGen. Jennifer Cormier has nothing to disclose. Ms. Hand has received personal compensation for serving as an employee of 4D Molecular Therapeutics. Ms. Hand has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for Fulcrum Therapeutics, Inc.. Ms. Hand has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for PepGen Inc. Ms. Hand has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for Bluebird Bio. Ms. Vacca has received personal compensation for serving as an employee of PepGen Inc. Ms. Vacca has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for PepGen Inc. Ms. Vacca has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for Agios Pharmaceuticals. Ms. Vacca has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Nocion Therapeutics. Ms. Vacca has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for Relmada Therapeutics. Ms. Vacca has stock in PepGen Inc.. Pallavi Lonkar has nothing to disclose. Dr. Holland has received personal compensation for serving as an employee of PepGen Inc.. Dr. Holland has received intellectual property interests from a discovery or technology relating to health care. Mr. Garg has received personal compensation for serving as an employee of PepGen. Mr. Garg has stock in pepgen. Mr. Foy has received personal compensation for serving as an employee of PepGen Inc.. Dr. McArthur has received personal compensation for serving as an employee of PepGen . Dr. McArthur has stock in PepGen .

Genomically Hardwired Regulation of Gene Activity Orchestrates Cellular Iron Homeostasis in Arabidopsis

ABSTRACT Iron (Fe) is an essential micronutrient which plays pivotal roles as electron donor and catalyst across organisms. In plants, variable, often insufficient Fe supply necessitates mechanisms that constantly attune Fe uptake rates and recalibrate cellular Fe homoeostasis. Here, we show that short-term (0.5, 6, and 12 h) exposure of Arabidopsis thaliana plants to Fe deficiency triggered massive changes in gene activity governed by transcription and alternative splicing (AS), regulatory layers that were to a large extent mutually exclusive. Such preclusion was not observed for genes that are directly involved in the acquisition of Fe, which appears to be concordantly regulated by both expression and AS. Generally, genes with lower splice site strengths and higher intron numbers were more likely to be regulated by AS, no dependence on gene architecture was observed for transcriptionally controlled genes. Conspicuously, specific processes were associated with particular genomic features and biased towards either regulatory mode, suggesting that genomic hardwiring is functionally biased. Early changes in splicing patterns were, in many cases, congruent with later changes in transcript or protein abundance, thus contributing to the pronounced transcriptome-proteome discordance observed in plants.

Abstract PS8-32: Effects of DNA-damaging and demethylating agents on alternate mRNA splicing in BRCA2

Abstract Introduction DNA sequence variants of unknown clinical significance (VUSs) are routinely identified during genetic testing of BRCA2 and other tumor suppressor genes associated with hereditary breast/ovarian cancer syndrome (HBOC). To determine which VUSs could be classified as pathogenic mutations, sequence variants near intron-exon boundaries are tested for effects on normal splicing patterns. However, these investigations can be complicated by potential genome-wide disruptions of normal splicing patterns caused by therapeutic agents. To test the potential contribution of systemic therapies to alternative splicing events in BRCA2, we have tested the effects of two DNA damaging agents and two demethylating agents on the relative levels of the naturally occurring alternative splicing variant BRCA2Δ3. While the protein product of this alternative splicing event has not been characterized, the BRCA2Δ3 mRNA maintains the full-length translational reading frame, and lacks sequence encoding EMSY and PALB binding domains as well as transactivation function. Previous work has shown that, while some VUSs associated with increased levels of BRCA2Δ3 in lymphoblastoid cell lines are not pathogenic, germline deletions that eliminate BRCA2 exon 3 are associated with increased breast cancer risk. Thus, alternative splicing events that alter relative levels of the BRCA2Δ3 mRNA variant may serve both as an indicator of the effects of some systemic therapies on genome-wide splicing defects and also BRCA2 gene function per se. Methods 1) To determine whether therapeutic DNA damaging agents can alter the levels of Δ3 alternate splicing isoforms, the breast cancer cell line MCF7 was treated with either doxorubicin or bleomycin, and isoform-specific RT-PCR was used to compare relative levels of splice junctions containing or skipping exon 3. 2) To determine whether DNA demethylating agents known to promote expression of some tumor suppressors can alter the levels of Δ3, MCF7 and/or the non-cancer breast cell line MCF 10A was treated with 5-aza 2’-deoxycytidine (5-AzadC) or 5-Azacytidine (5-AzaC), and again isoform-specific RT-PCR was used to compare relative levels of splice junctions containing or skipping exon 3. 3) To determine whether the BRCA2 Δ3 isoform was equally accessible to translational machinery in all cell types, RNA was prepared from separated nuclear and cytoplasmic fractions of MCF7 and MCF 10A and isoform-specific RT-PCR was used to estimate levels of cytoplasmic Δ3 available to translational machinery. Results and conclusions 1) While bleomycin decreases the relative levels of Δ3 compared with untreated controls, doxorubicin increases relative levels of Δ3 in MCF7. This suggests that some DNA damaging agents may cause changes in splicing patterns that may affect therapeutic effectiveness. 2) While both 5-AzaC and 5-AzadC reduce relative levels of Δ3 in MCF7, 5-azadC does not reduce relative levels of Δ3 in MCF 10A, suggesting there may be a cell type-specific splicing response to genomic demethylation therapies. 3) MCF 10A limits the level of Δ3 that can accumulate in the cytoplasm, but this regulatory mechanism is absent from MCF7, indicating there is a cell type-specific mechanism that limits the amount of alternately spliced mRNA variants that are available to translational machinery. Citation Format: James D. Fackenthal, Talia Ishfaq, Zaain Ahmad, Nourhan Mohamed, Milica Janosevic, Ziyad Abdelrahim, Jessica Georgopulos. Effects of DNA-damaging and demethylating agents on alternate mRNA splicing in BRCA2 [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS8-32.

A Splicing Variant in OCRL Gene Might Explain the Second Case of Lowe Syndrome in Iran

: Lowe syndrome is a condition that primarily affects eyes, brain, and kidneys. This disorder follows X-linked recessive mode of inheritance and it occurs in males mainly. Mutations in OCRL (located at Xq25) gene can cause accumulation of phosphatidylinositol bisphosphate and disturbed actin cytoskeleton remodeling. There are 268 mutations in OCRL gene causing Lowe syndrome or Dent disease 2 in HGMD database, however 10 - 20% of Lowe syndrome suspects remain undiagnosed at molecular level. Here we present a male case of Lowe syndrome with characteristic features. Comprehensive clinical examination and genetic counseling were performed. Sanger sequencing was employed to investigate the possible OCRL mutations and we identified a donor splice site variant (NM-000276: c.2469 + 1G &gt; A) in hemizygous state. This is a pathogenic variant according to the ACMG standards and guidelines and might explain the clinical features of the patient. This result is in accordance with the clinical diagnosis of Lowe syndrome and it is absent from ExAC, 1000 G, Iranome, GME, gnomAD Genome databases of healthy controls. In-silico analysis of this splicing variant revealed that the position is highly conserved between species. Splicing prediction tools predicted some changes in splicing pattern of the OCRL transcript, elimination of some protein features, and malfunctioning the OCRL protein as a consequence of this variant. Accordingly, we proposed the c.2469 + 1G &gt; A variant might explain the clinical features in studied patient and be employed for prenatal diagnosis of Lowe syndrome in the family.

Aberrant RNA Splicing Events Driven by Mutations of RNA-Binding Proteins as Indicators for Skin Cutaneous Melanoma Prognosis.

The worldwide incidence of skin cutaneous melanoma (SKCM) is increasing at a more rapid rate than other tumors. Aberrant alternative splicing (AS) is found to be common in cancer; however, how this process contributes to cancer prognosis still remains largely unknown. Mutations in RNA-binding proteins (RBPs) may trigger great changes in the splicing process. In this study, we comprehensively analyzed DNA and RNA sequencing data and clinical information of SKCM patients, together with widespread changes in splicing patterns induced by RBP mutations. We screened mRNA expression-related and prognosis-related mutations in RBPs and investigated the potential affections of RBP mutations on splicing patterns. Mutations in 853 RBPs were demonstrated to be correlated with splicing aberrations (p < 0.01). Functional enrichment analysis revealed that these alternative splicing events (ASEs) may participate in tumor progress by regulating the modification process, cell-cycle checkpoint, metabolic pathways, MAPK signaling, PI3K-Akt signaling, and other important pathways in cancer. We also constructed a prediction model based on overall survival-related AS events (OS-ASEs) affected by RBP mutations, which exhibited a good predict efficiency with the area under the curve of 0.989. Our work highlights the importance of RBP mutations in splicing alterations and provides effective biomarkers for prediction of prognosis of SKCM.

Alternative Splicing During theChlamydomonasreinhardtiiCell Cycle

Genome-wide analysis of transcriptome data in Chlamydomonas reinhardtii shows periodic patterns in gene expression levels when cultures are grown under alternating light and dark cycles so that G1 of the cell cycle occurs in the light phase and S/M/G0 occurs during the dark phase. However, alternative splicing, a process that enables a greater protein diversity from a limited set of genes, remains largely unexplored by previous transcriptome based studies in C. reinhardtii. In this study, we used existing longitudinal RNA-seq data obtained during the light-dark cycle to investigate the changes in the alternative splicing pattern and found that 3277 genes (19.75% of 17,746 genes) undergo alternative splicing. These splicing events include Alternative 5′ (Alt 5′), Alternative 3′ (Alt 3′) and Exon skipping (ES) events that are referred as alternative site selection (ASS) events and Intron retention (IR) events. By clustering analysis, we identified a subset of events (26 ASS events and 10 IR events) that show periodic changes in the splicing pattern during the cell cycle. About two-thirds of these 36 genes either introduce a pre-termination codon (PTC) or introduce insertions or deletions into functional domains of the proteins, which implicate splicing in altering gene function. These findings suggest that alternative splicing is also regulated during the Chlamydomonas cell cycle, although not as extensively as changes in gene expression. The longitudinal changes in the alternative splicing pattern during the cell cycle captured by this study provides an important resource to investigate alternative splicing in genes of interest during the cell cycle in Chlamydomonas reinhardtii and other eukaryotes.

Abstract 4724: DNA demethylation and double-strand breaks affect levels of the BRCA2 Δ3 alternate splicing isoform

Abstract Introduction: BRCA2 is one of two principal tumor suppressors responsible for hereditary breast/ovarian cancer syndrome (HBOC). Patients with strong family histories of breast/ovarian cancer may carry BRCA2 DNA sequence variants of unknown clinical significance (VUSs). To evaluate which of these VUSs may contribute to cancer risk by altering splicing patterns, naturally occurring BRCA2 mRNA alternate splicing events have been extensively characterized, but it is not yet known which if any of these alternate splicing events plays a role in BRCA2 function. One variant lacking exon 3 (Δ3), which maintains the full length translational reading frame, lacks sequence encoding EMSY and PALB binding domains as well as transactivation function. Previous work has shown that, while some VUSs associated with increased levels of Δ3 in lymphoblastoid cell lines are not pathogenic, germline deletions that eliminate BRCA2 exon 3 are associated with increased breast cancer risk. It is therefore of interest to characterize the factors that influence the frequency of naturally occurring BRCA2 exon 3-skipping, including cell types and therapies. Methods: 1) To determine whether therapeutic DNA damaging agents can alter the levels of Δ3 alternate splicing isoforms, the breast cancer cell line MCF7 was treated with either doxorubicin or bleomycin, and isoform-specific RT-PCR was used to compare relative levels of splice junctions containing or skipping exon 3. 2) To determine whether DNA demethylating agents known to promote expression of some tumor suppressors can alter the levels of Δ3, MCF7 and/or the non-cancer breast cell line MCF 10A was treated with 5-aza 2'-deoxycytidine (5-AzadC) or 5-Azacytidine (5-AzaC), and again isoform-specific RT-PCR was used to compare relative levels of splice junctions containing or skipping exon 3. 3) To determine whether the BRCA2 Δ3 isoform was equally accessible to translational machinery in all cell types, RNA was prepared from separated nuclear and cytoplasmic fractions of MCF7 and MCF 10A and isoform-specific RT-PCR was used to estimate levels of cytoplasmic Δ3 available to translational machinery. Results and conclusions: 1) While bleomycin decreases the relative levels of Δ3 compared with untreated controls, doxorubicin increases relative levels of Δ3 in MCF7. This suggests that some DNA damaging agents may cause changes in splicing patterns that may affect therapeutic effectiveness. 2) While both 5-AzaC and 5-AzadC reduce relative levels of Δ3 in MCF7, 5-azadC does not reduce relative levels of Δ3 in MCF 10A, suggesting there may be a cell type-specific splicing response to genomic demethylation therapies. 3) MCF 10A limits the level of Δ3 that can accumulate in the cytoplasm, but this regulatory mechanism is absent from MCF7, indicating there is a cell type- specific mechanism that limits the amount of alternately spliced mRNA variants that are available to translational machinery. Citation Format: Talia Ishfaq, Zaain Ahmad, Nourhan Mohamed, Milica Janosevic, Ziyad Abdelrahim, Jessica Georgopulos, James Fackenthal. DNA demethylation and double-strand breaks affect levels of the BRCA2 Δ3 alternate splicing isoform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4724.

Hypoxia-induced changes in intragenic DNA methylation correlate with alternative splicing in breast cancer

The tumor microenvironment is marked by gradients in the level of oxygen and nutrients, with oxygen levels reaching a minimum at the core of the tumor, a condition known as tumor hypoxia. Mediated by members of the HIF family of transcription factors, hypoxia leads to a more aggressive tumor phenotype by transactivation of several genes as well as reprogramming of pre-mRNA splicing. Intragenic DNA methylation, which is known to affect alternative splicing in cancer, could be one of several reasons behind the changes in splicing patterns under hypoxia. Here, we have tried to establish a correlation between intragenicDNA methylation and alternative usage of exons in tumor hypoxia. First, we have generated a customhypoxia signature consisting of 34 genes that are upregulated under hypoxia and are direct targets of HIF-1α. Using this gene expression signature, we have successfully stratified publicly available breast cancer patient samples into hypoxia positive and hypoxia negative groups followed by mining of differentially spliced isoforms between these groups. The Hypoxia Hallmark signature from MSigDB was also used independently to stratify the same tumor samples into hypoxic and normoxic.We found that 821 genes were showing differential splicing between samples stratified using a custom signature, whereas, 911 genes were showing differential splicing between samples stratified using the MSigDB signature. Finally, we performed multiple correlation tests between the methylation levels (β) of microarray probes located within 1 kilo base pairs of isoform-specific exons using those exons' expression levels in the same patient samples in which the methylation level was recorded. We found that the expression level of one of the exons ofDHX32 and BICD2 significantly correlated with the methylation levels, and we were also able to predict patient survival (p-value: 0.02 for DHX32 and 0.0024 for BICD2). Our findings provide new insights into the potential functional role of intragenic DNA methylation in modulating alternative splicing during hypoxia.

Hypothermia induces changes in the alternative splicing pattern of cold-inducible RNA-binding protein transcripts in a non-hibernator, the mouse.

Cold-inducible RNA-binding protein (CIRBP) plays important roles in protection against harmful effects of cold temperature. We previously found that several splicing variants of CIRBP mRNA are constitutively expressed in the heart of non-hibernating euthermic hamsters and that one of the variants is predominantly expressed with remarkable reduction in the expression of other variants in hibernating hypothermic hamsters. The aim of this study was to determine whether the regulation of alternative splicing is a common function in a non-hibernator, the mouse. The expression of CIRBP mRNA was assessed by RT-PCR. In euthermic control mice, several splicing variants of CIRBP mRNA were detected in various organs. When hypothermia was induced in mice by using isoflurane anesthesia, the short form variant, which encodes full-length functional CIRBP, was predominantly detected. Keeping body temperature of anesthetized mice at 37°C prevented changes in the splicing pattern. Exposure of mice to a low temperature did not change the splicing pattern, suggesting that endogenous neuronal and/or humoral pathways activated in response to cold stimuli applied to the body surface play minor roles. In agreement with this, the shift in alternative splicing was reproduced in isolated leukocytes in vitro when they were incubated at 28°C. Since application of a TRPM8 or TRPA1 agonist at 37°C failed to promote the shift in the splicing pattern, it seems likely that cold-sensitive channels are not involved in the splicing regulation. Therefore, it is probable that a substantial reduction of temperature is a major cause of the regulation of alternative splicing of CIRBP transcripts. The regulatory system of CIRBP expression at the level of alternative splicing, which was originally discovered in the hibernating hamster, commonly exists in non-hibernators such as mice.

IsoformSwitchAnalyzeR: analysis of changes in genome-wide patterns of alternative splicing and its functional consequences.

Alternative splicing is an important mechanism involved in health and disease. Recent work highlights the importance of investigating genome-wide changes in splicing patterns and the subsequent functional consequences. Current computational methods only support such analysis on a gene-by-gene basis. Therefore, we extended IsoformSwitchAnalyzeR R library to enable analysis of genome-wide changes in specific types of alternative splicing and predicted functional consequences of the resulting isoform switches. As a case study, we analyzed RNA-seq data from The Cancer Genome Atlas and found systematic changes in alternative splicing and the consequences of the associated isoform switches. Windows, Linux and Mac OS: http://bioconductor.org/packages/IsoformSwitchAnalyzeR. Supplementary data are available at Bioinformatics online.

Proper splicing contributes to visual function in the aging Drosophila eye.

Changes in splicing patterns are a characteristic of the aging transcriptome; however, it is unclear whether these age‐related changes in splicing facilitate the progressive functional decline that defines aging. In Drosophila, visual behavior declines with age and correlates with altered gene expression in photoreceptors, including downregulation of genes encoding splicing factors. Here, we characterized the significance of these age‐regulated splicing‐associated genes in both splicing and visual function. To do this, we identified differential splicing events in either the entire eye or photoreceptors of young and old flies. Intriguingly, aging photoreceptors show differential splicing of a large number of visual function genes. In addition, as shown previously for aging photoreceptors, aging eyes showed increased accumulation of circular RNAs, which result from noncanonical splicing events. To test whether proper splicing was necessary for visual behavior, we knocked down age‐regulated splicing factors in photoreceptors in young flies and examined phototaxis. Notably, many of the age‐regulated splicing factors tested were necessary for proper visual behavior. In addition, knockdown of individual splicing factors resulted in changes in both alternative splicing at age‐spliced genes and increased accumulation of circular RNAs. Together, these data suggest that cumulative decreases in splicing factor expression could contribute to the differential splicing, circular RNA accumulation, and defective visual behavior observed in aging photoreceptors.

Abstract MIP-057: TARGETING THE LONG–NONCODING RNA MALAT1 IN OVARIAN CANCER

Abstract PURPOSE: Ovarian cancer metastasizes via direct seeding, whereby cells must resist anoikis (detachment-induced cell death) in the peritoneal cavity before invading through the mesothelium to metastasize into peritoneal organs. The molecular mechanisms that facilitate these processes are not fully understood. Using an unbiased screen, we identified the long non-coding RNA MALAT1 (metastasis associated lung adenocarcinoma transcript 1) as being a potential regulator of anoikis resistance. We hypothesized that MALAT1 promotes progression through the early steps of metastasis, including anoikis resistance, peritoneal invasion, and metastatic outgrowth, and that therapeutic inhibition of MALAT1 will reduce ovarian tumor burden and metastatic spread. EXPERIMENTAL PROCEDURES: Modified anti-sense oligonucleotides (ASO) targeting MALAT1 were used to inhibit MALAT1 expression in ovarian cancer cell lines (OVCA420, HEY, OV1847). Transfections were performed using lipofectamine as well as novel lipid-based nanoparticles that are minimally toxic and have potential in vivo applications. Transcriptome profiling was performed on Affymetrix arrays. Migration and invasion assays were performed on an XCelligence Real Time Cell Analyzer, and proliferation was measured by crystal violet. Anoikis was measured using Caspase 3/7 assay. Knockdown of RBFOX2 and KIF1B-beta was performed using shRNAs. Gene expression was measured by RT-qPCR. Publicly available datasets including the Cancer Genome Atlas were analyzed for association of MALAT1 gene expression and DNA copy number with clinicopathologic variables. RESULTS: MALAT1 gene expression significantly increased after 24 hours in forced suspension culture versus attached culture in multiple anoikis-resistant ovarian cancer cell lines. MALAT1 knockdown via ASO resulted in decreased proliferation, migration, invasion, anchorage-independent growth, and increased anoikis. Lipid nanoparticle-mediated delivery of MALAT1-targeted ASO resulted in effective gene knockdown with minimal off-target toxicity. Transcriptome profiling of MALAT1-knockdown cells revealed significant changes in splicing patterns, including alternative splicing of the pro-apoptotic gene KIF1B (Kinesin Family Member 1B). MALAT1 knockdown resulted in decreased gene expression of the splicing factor RBFOX2 (RNA Binding Protein Fox-1, Homolog 2), which is associated with epithelial-to-mesenchymal transition (EMT). KIF1B contains RBFOX2 binding sites within spliced regions, suggesting that MALAT1 may control alternative splicing through regulation of RBFOX2. Indeed, RBFOX2 knockdown resulted in increased expression of the pro-apoptotic isoform of KIF1B (KIF1B-beta), as well as increased anoikis. Furthermore, knockdown of KIF1B-beta reversed the apoptotic effects of MALAT1-knockdown. In ovarian cancer patients, high MALAT1 gene expression is associated with higher stage disease, increased risk of recurrence, and reduced overall survival. Additionally, MALAT1 is amplified in 3% of ovarian cancers, and amplification is associated with increased MALAT1 gene expression and reduced overall survival. CONCLUSIONS: The lncRNA MALAT1 facilitates a pro-metastatic phenotype in ovarian cancer cells by promoting alternative splicing and differential expression of anti-apoptotic and EMT-related genes. In vivo studies to determine the efficacy of MALAT1-knockdown via lipid-mediated delivery to inhibit progression of ovarian cancer cell lines and patient derived xenografts are ongoing. Citation Format: Michael A Gordon, Dawn R Cochrane, Jamie Betker, Tom Anchordoquy, Jennifer K Richer. TARGETING THE LONG–NONCODING RNA MALAT1 IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-057.

21P U2AF1 (S34F) mutation leads to altered global splicing patterns and dysregulation of cell cycle and mitosis

The U2AF1, also known as U2AF35, forms a heterodimeric splicing factor with U2AF2. In cancer, the recurrent somatic mutations in U2AF1 have been identified at least two different cancer types, acute myeloid leukemia and lung adenocarcinoma. The S34F-mutation in U2AF1 has been found frequently in lung adenocarcinoma, but the function of U2AF1-S34F mutants is not fully understood in lung cancer development. Specifically, it is not known whether the mutation represents the loss- or gain-of-function with regards to carcinogenesis. We collected transcriptome data from the U2AF1-S34F-overexpressed or the U2AF1-depleted A549 cells. Alternatively spliced transcripts were compared with those from 8 patients with U2AF1-S34F mutation in lung adenocarcinoma (LUAD) from The Cancer Genome Atlas (TCGA). The Gene Ontology (GO) analysis was carried out subsequently for alternatively spliced genes. We also analyzed the cell cycle by flow cytometry and mitotic progression by confocal microscopy in U2AF1-S34F-overexpressed and U2AF1-depleted cells. Both overexpression of mutant U2AF1-S34F and knockdown by RNAi led to alternative splicing in large numbers of genes. Notably, numerous genes involved in regulation of cell cycle and mitosis were found. Consistently, both overexpression of mutant U2AF1-S34F and knockdown of U2AF1 led to G2M arrest as well as problems in mitotic progression. However, there was a very limited overlap in alternatively spliced genes between these two groups. In fact, integrating data from LUAD patients revealed that as far as cell cycle and mitosis are concerned, ectopic expression of mutant led to a splicing pattern similar to those seen from the lung cancer patients. Based on our analysis of changes in splicing pattern, it is the expression of mutant U2AF1-S34F protein rather than the loss of a wild type allele that contributes carcinogenic development of lung adenocarcinoma.

Differentiation of mammary tumors and reduction in metastasis upon Malat1 lncRNA loss.

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.

MBNL1 As a New Therapeutic Target in MLL-Fusion Gene Leukemia

Translocations of the Mixed Lineage Leukemia (MLL) gene located on chromosome 11 are commonly found in infants with AML or ALL and in secondary leukemia at all ages. A majority of patients with these translocations have a poor prognosis. Gene expression profiling studies demonstrate that one of the most consistently overexpressed genes in these leukemias (compared to all other leukemias) is muscleblind-like 1 (MBNL1). Further, MBNL1 was also identified as a direct transcriptional target of MLL-fusion proteins. An RNA-binding protein, MBNL1 is known to be a key factor in the pathophysiology of Myotonic Dystrophy Type I (DM), where sequestration of MBNL1 leads to splicing defects in muscle and neuronal cells. However, the role of MBNL1 in hematopoiesis and leukemogenesis is unknown. To determine the role of MBNL1 in normal hematopoiesis we studied MBNL1-/- mice. Compared to littermate controls, MBNL1-/- mice showed no differences in peripheral blood counts or bone marrow cellularity. When challenged with 5-FU, both MBNL1-/- and wild type mice displayed similar kinetics of peripheral blood cytopenia and recovery. Next we examined the role of MBNL1 in hematopoietic stem cell function using a competitive transplantation assay. Lethally irradiated mice were transplanted with a 1:1 mix of CD45.1 and CD45.2 bone marrow, with the latter being wild-type or MBNL1-/-. Flow cytometry analysis of peripheral blood at 4 weeks post-transplant showed donor chimerism being 53±4.14% in recipients of wild type marrow and 25±5.41 % in the MBNL1-/- recipients. Successive analyses every 4 weeks showed the chimerism to be stable over the next 16 weeks. To determine the role of MBNL1 in leukemia, we transformed MBNL1-/- or wild type bone marrow cells with various oncogenes delivered via retroviral transduction and compared them in methylcellulose colony replating assays. Absence of MBNL1 significantly reduced colony formation in MLL-AF9 and E2A-HLF transformed cells by 59.5% (± 27.1) and 50.7% (± 23) respectively, compared to controls. To assess the role of MBNL1 in leukemia in vivo, we transplanted MLL-AF9-transformed wild type or MBNL1-/- cells into irradiated mice. All recipients injected with wild-type MLL-AF9-transformed cells succumbed to leukemia with a median time of 106 days. In contrast, the majority of recipients of MBNL1-/- cells survived leukemia-free for at least 140 days post-transplantation (p=0.0017, log rank test). We next assessed the role of MBNL1 in human leukemia cells. Lentiviral-shRNA knockdown of MBNL1 in leukemia cell lines (MV4;11, THP-1) significantly inhibited cell growth, both in liquid culture and methylcellulose colony forming assays. To determine the requirement of MBNL1 for leukemia propagation in vivo, we used cord blood-derived leukemia cells bearing the MLL-AF9 fusion gene and mutant NRAS (MA9NRAS). MA9NRAS cells transduced with MBNL1-specific or control (non-targeting, NT) shRNA were transplanted into immunodeficient mice. Six weeks after transplant, bone marrow aspirates showed persistence of lentiviral-transduced cells in 85% of the NT-group. On the other hand, MBNL1-shRNA transduced cells were not detected in any of the recipient mice. These results suggest that MBNL1 is essential for leukemia cell propagation in vivo. Finally, we tested therapeutic targeting of MBNL1 in MLL-fusion gene leukemia. A lead inhibitor that prevents binding of MBNL1 to its targets was recently identified. Treatment of MA9NRAS cells with the inhibitor for 48 hours led to significant apoptosis whereas normal cord blood CD34+ cells were relatively less sensitive. Blockade of MBNL1 in leukemia cells either by shRNA-knockdown or by the inhibitor showed identical changes in splicing patterns of known MBNL1 target genes. Collectively, our data suggest that MBNL1 is required for the initiation and propagation of MLL-fusion gene leukemia while it appears relatively dispensable for normal hematopoiesis. Further, we have identified a promising lead inhibitor that could be developed for novel treatments for therapy-resistant leukemias. DisclosuresNo relevant conflicts of interest to declare.

The Quest for More Tolerant Rice: How High Concentrations of Iron Affect Alternative Splicing?

Rice (Oryza sativa L.) is a global staple food crop and an important model organism for plant studies. Recent reports have shown that alternative splicing is affected by many stressful conditions, suggesting its importance for the adaptation to adverse environments. Due to the little information on this subject, this study aimed to explore changes in splicing patterns that occur in response to high iron concentration in nutrient solutions. Here we quantified different kind of junctions and splicing events in the transcriptome of a relatively tolerant rice cultivar BRS Querencia, under iron excess with concentration of 300 mg L-1 Fe+2. Plants kept under standard conditions (control) presented 127,781 different splicing junctions, while stressed plants had 123,682 different junctions. Canonical (98.85% and 98.91%), semi-canonical (0.73% and 0.70%) and non-canonical (0.42% and 0.40%) junctions were found in control and stressed plants, respectively. Intron retention was the most frequent event (44.1% and 47.4%), followed by 3’ splice site (22.6% and 21.9%), exon skipping (18.9% and 17.3%) and alternative 5’ splice site (14.4% and 13.4%) in control and stressed plants, respectively. We also found 25 differentially expressed genes (five up and 20 down regulated) that are related to post-translational modifications. These results represent an important step in the understanding of how plant stress responses occur in an iron tolerant genotype, uncovering novel genes involved in iron stress response.

Genome-Wide Survey of Cold Stress Regulated Alternative Splicing in Arabidopsis thaliana with Tiling Microarray

Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression.