Inclusion Of Exon Research Articles

Abstract 4435: Functional role of four-and-a-half LIM protein 2 in the alternative splicing of cancer hallmark genes of hepatocellular carcinoma

Abstract Four-and-a-half LIM protein 2 (FHL2) participates in various cellular processes, including regulation of cell survival, transcription and signal transduction. In this study, FHL2 specific antibody was used to immunoprecipitate endogenous FHL2 together with its binding partners from WRL68 and Hep3B liver cell lysates, followed by protein identification using mass spectrometry. The top significant functional clusters of identified proteins were related to the splicing process. To validating the pull down results, the interaction of FHL2 with ASF and U1A in WRL68 cell line was confirmed using co-immunoprecipitation. To study the possible effects of FHL2 on mRNA splicing, we knocked down FHL2 in WRL68, L02 and Hep3B cells and the transcriptomes of knocked-down cells were examined by RNA sequencing. We identified a global impact on regulating differential exon usage by knocking down FHL2. Subsequently, several alternatively spliced cancer hallmark genes were selected for validation using real-time PCR. We found that knocking down FHL2 could significantly promote the extra domain B (EDB) exon inclusion in fibronectin 1 (FN1) and the E3b exon inclusion in Rac1. In addition, knocking down FHL2 significantly decreased the percentage of the epithelial specific transcript CD44E while only very mildly decreased the standard CD44S transcript in total CD44 mRNA. Using an ab initio motif identification approach, the possible splicing factor binding motifs in the alternative spliced exons regulated by over-expressing and knocking down FHL2 were obtained. ASF was found to be the most common splicing regulatory protein. To further explore the functional relationship between FHL2 and ASF, we examined the effects of FHL2 and ASF on exon inclusion/skipping of endogenous EDB exon of FN1 in WRL68 cells. It was found that knocking down FHL2 using siRNA promotes inclusion of EDB while knocking down ASF using siRNA promotes skipping of EDB of FN1. When FHL2 and ASF were knocked down simultaneously, the effect of FHL2 on EDB exon skipping was abolished, implying that ASF mediates the effect of FHL2 on the EDB exon skipping. To explore the clinical relevance of this study, the expression of FHL2 and the EDB exon were determined in 40 paired HCC and normal tissues using real-time PCR. Results showed that the fold changes of EDB inclusion level and the FHL2 expression were significantly correlated. In summary, we have identified many alternative splicing-related protein interacting partners of FHL2 and the downregulated of FHL2 could affect the splicing pattern of many cancer hallmark genes, including CD44, Rac1 and FN1. Using FN1 as a model, we elucidated ASF as one of the mediators of the effect of FHL2 on alternative splicing. Taken together, FHL2 is probably involved in the pre-mRNA splicing in liver cells and modulate genes involved in cancer development through the perturbation of RNA splicing machinery. Citation Format: Stephen Kwok-Wing Tsui, Cyanne Ye Cao. Functional role of four-and-a-half LIM protein 2 in the alternative splicing of cancer hallmark genes of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4435.

Abstract 3294: Functional annotation for alternative splicing to investigate disrupted splicing in cancer

Abstract Alternative splicing (AS) plays a key role to confer human genome complexity. The advent of mRNA high-throughput sequencing facilitates to identify precise differential alternative splicing analysis. Massive result estimated from RNA-Seq should be sorted out whether AS has functional impact or not. Previous studies investigated protein domain or nonsense-mediated decay of AS event region. However other functional sites on exon inclusion region also exist. For example, miRNA binding site, and post-translational modification of AS site already are reported to assign functional impact. Therefore we developed ASpedia (http://combio.snu.ac.kr/aspedia/) to annotation functional impact of AS event. AS events of our database were generated from human genome hg19 ENSEMBL and Refseq. ASpedia provides a database, explore system, and AS browser to annotate functional impact evidence encompassing DNA, RNA, and protein. The user could query multiple AS events from differential AS analysis result. Currently our system supports a jar execution file to convert input query file from MISO or rMATS result. The variant of splicing factor (SF) disrupts normal splicing. It could be assumed that target genes of mutated SF could be transcribed to aberrant isoforms by changed splicing machinery. Therefore we investigated the comprehensive SF variant status from TCGA somatic mutation and copy number. SF3B1 (2% in BRCA) and U2AF1 (4% in AML, and 2% in LUAD) were shown to recurrent mutation including hotspot. ELAVL2 known dispensable splicing factor to brain tissue were highly recurrent in deletion (18% in GBM). In order to examine aberrant AS events caused SF variants, we estimated exon-inclusion proportion Percent-Spliced-In (PSI) values for identifiable AS events using TCGA RNA-Seq dataset. Using differentially AS event analysis, we identified AS events that were significantly differentially spliced in the presence of a U2AF S34F/Y mutation (119 in AML). And we purposed to finding In order to investigate functional site, the differentially spliced AS events were additionally annotated using ASpedia. Finally we identified the landscape of that AS events were altered by SF variants, and we proved that a part of the events was involved in cancer-associated functions. Citation Format: Daejin Hyung, Jihyun Kim, Soo Young Cho, Charny Park. Functional annotation for alternative splicing to investigate disrupted splicing in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3294.

Abstract 4458: Identification of splicing regulators using retroviral vectors carrying intron-containing expression cassettes

Abstract Exon inclusion is controlled by both positive (e.g., SR proteins) and negative (e.g., hnRNPs) regulators; yet high-throughput methods to comprehensively identify all relevant RNA-binding proteins are currently lacking. Thus, our long-term goal is to develop a genome-wide CRISPR sgRNA library-based screen, where cells with desired phenotypes are identified by flow cytometry and analyzed as pools using next generation sequencing. To achieve this goal, we took advantage of previous work in our laboratory demonstrating expression of the Δex2 CD19 isoform in B-cell leukemias and lymphomas resistant to CD19-directed immunotherapy (Sotillo et al., 2015; Yu et al., 2016). Skipping of exon 2 results in the truncated protein which fails to localize to the plasma membrane, yielding cells that are CD19-negative by flow cytometry (Bagashev et al., 2015). To study splicing regulation of exon 2, we designed a series of retroviral vectors containing the entire CD19 open reading frame retaining intronic sequences flanking exon 2 (i.e., intron 1 and intron 2). It is known that the nuclear splicing of provirus containing introns completely removes intron from the packaged viral RNA genome (Li et al., 1998). However, when viruses were stably transducedto B-lymphoid cell lines rendered null for endogenous CD19, the retroviral long terminal repeat (LTR) promoter-driven CD19 transcription resulted in skipping of exon2. Further addition of cognate 5'UTR sequence downstream of the retroviral LTR did not promote inclusion of this alternative exon. Similarly, the retroviral based expression of CD19 gene containing intronic sequences flanking constitutive exon10 did not allow proper splicing of exon10 and instead resulted into transcript retaining both introns (9 and 10). Interestingly, the flanking intronic sequences of exon10 allowed precise splicing of upstream exon2 when the expression cassette contained both exon2 and exon10 flanked by their respective introns. Thus, downstream placement of constitutive exon10 and its surrounding introns affected the splicing of upstream alternative exon2. Subsequent introduction of sgRNA libraries into cells transduced with such intron-containing expression cassettes will allow us to identify both positive and negative regulators of CD19 exon2 inclusion and potentially - novel general regulators of splicing. Bagashev, A., Sotillo, E., Wu, G., and Thomas-Tikhonenko, A. (2015). The importance of CD19 exon 2 for surface localization: Closing the Ig-like loop. Blood 126, 3433. Sotillo, E., Barrett, D.M., Black, K.L., Bagashev, A., Oldridge, D., Lanauze, C., Gazzara, M.R., et al. (2015). Convergence of acquired mutations and alternative splicing of CD19 enables resistance to CART-19 immunotherapy. Cancer Discov 5, 1282-1295. Li, K., Garoff, H. (1998). Packaging of intron-containing genes into retrovirus vectors by alphavirus vectors. Proc Natl Acad Sci USA. Citation Format: Mukta Asnani. Identification of splicing regulators using retroviral vectors carrying intron-containing expression cassettes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4458.

PO-211 Profiling the alternative splicing landscape of senescent cells

IntroductionCellular senescence, defined by an irreversible cell cycle arrest in response to potentially oncogenic stimuli, has been described as a protective mechanism in tumourigenesis and a therapeutic target in cancer. The senescence-associated secretory phenotype (SASP) is a pro-inflammatory response by senescent cells involving the release of cytokines, chemokines, growth factors and proteases that, in a cancer progression context, may be beneficial by the elimination of senescent cells or deleterious when triggering angiogenesis, cell proliferation and epithelial-to-mesenchymal transition. Despite senescence’s importance in cancer and the suggested role of alternative splicing in its regulation, the transcriptional heterogeneity of senescent cells has, to our knowledge, been extensively characterised only at the gene expression level.Material and methodsNext-generation sequencing of RNA (RNA-seq) allows alternative splicing quantification with unprecedented precision. The inclusion level of an exon is commonly quantified by its percent-spliced-in (PSI) value, i.e. the proportion of RNA-seq reads providing evidence supporting its inclusion. However, a PSI ratio does not incorporate information about the number of reads used in the quantification of the cognate alternative splicing event, directly proportional to the precision of its estimate. Beta distributions can be exploited in modelling inclusion levels, using reads supporting exon inclusion and exclusion as surrogates of the distribution’s shape parameters. We employed a computational pipeline, based on fitted beta distributions, to accurately quantify and compare alternative splicing across different types of senescent cells, relying both on public and in-house RNA-seq datasets.Results and discussionsOur analyses reproducibly identified, at a transcriptome-wide level, the alternative splicing changes specifically related with replicative and different types of induced senescence in multiple types of cells. For instance, Ras-induced senescence appears to associate with alterations in the splicing of genes involved in the secretory pathway and intracellular trafficking.ConclusionDifferential splicing analyses based on beta distribution modelling contribute to elucidate the specific alternative splicing signatures of different types of senescent cells, providing insights for targeting senescence in cancer therapies.

Abstract 2268: Differential splicing analysis of doxorubicin treatment response in cancer cells

Abstract Background:Splicing of transcripts is an important epigenetic control mechanism that significantly influences expression of protein isoforms in a cell. Alternative splicing is known to contribute to development and progression of cancer, however the impact of cellular splicing profiles on cancer drug response has been largely unexplored. In this study, we integrated two large public data sets and report findings on the basal splicing profiles between doxorubicin treated sensitive and resistant cancer cell lines. We introduced a new method to analyze of splicing data using the quasi-binomial distribution that is incredibly simple, powerful and well suited for comparison of large groups of samples. We also identified exons whose inclusion or exclusion level is likely to be damaging to intended gene products. Finally, we performed RNA-binding protein motif enrichment at these identified exons to characterize proteins that may play a role in their regulation. Methods:Drug response data from the Cancer Therapeutic Response Portal (CTRP) was integrated with RNAseq data from the Cancer Cell Line Encyclopedia (CCLE). AUC values from drug-response time curves in CTRP were used to classify cells as either doxorubicin sensitive, intermediate or resistant. The upper and lower tertiles provided a total of 253 sensitive and 258 resistant cell lines respectively. Splice junction read counts for skipped-exon events were calculated using the matched cell line data from CCLE. Response groups were then used to train a generalized linear model with quasi-binomial family. Results were filtered by group weight significance at an FDR of <=0.01. The remaining events were again filtered for >=10% difference in mean exon inclusion ratio. Results:In total we identified 277 differentially spliced events between doxorubicin sensitive and resistant groups. When performing GO term over-representation our results demonstrated significantly enriched biological processes supporting the role of alternative splicing in EMT and cellular differentiation. This further confirms previous studies linking alternative splicing to these cancer hallmarks, suggesting that splicing may facilitate acquired drug resistance through survival mechanisms similar to those seen in cancer stem cells. Citation Format: Edward Simpson, Yunlong Liu. Differential splicing analysis of doxorubicin treatment response in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2268.

Abstract 4132: ASO screen uncovers splicing as a therapeutic vulnerability in theinsulin-like growth factor (IGF) signaling pathway

Abstract Alternative splicing is a process contributing to structural transcript variation and proteome diversity but is often disrupted in cancer. Like most other transcripts, the insulin receptor (IN-R) undergoes alternative splicing to produce two isoforms: the full-length IN-RB and exon 11 skipped IN-RA isoform. The expression of these isoforms is tightly regulated during development, however there is an aberrant increase in IN-RA expression in childhood cancers like Rhabdomyo- & Osteo-sarcoma. This increased IN-RA expression is deleterious to the cells because it encodes for a receptor which has high affinity for both insulin & IGF2 growth hormones and it exploits the IGF pathway to accelerate the onset of tumor-cell hallmarks like proliferation & angiogenesis. Rhabdosarcoma is the 3rd most frequent solid tumor and the most common soft-tissue sarcoma in children. Despite improvement in the treatment for this disease, there is substantial inadequacy of less toxic alternative therapies. We found that there is a significantly increased expression of IN-RA levels in a cohort of 40 Rhabdosarcoma (RMS) patients (embryonal & alveolar) as compared to the control samples. We confirmed this increased expression in multiple RMS cell lines. Furthermore, we went on to show that cellular stress such as hypoxia increases alternative splicing to produce more IN-RA isoform. In order to get a mechanistic insight into this phenomenon, we utilized this hypoxia-inducible splicing system and performed an antisense-oligonucleotide (ASO) screen to characterize sequence elements and splicing factors involved in the regulation of IN-R splicing. We found that sequence elements flanking exon 11 are critical to the increased alternative splicing we see under hypoxic conditions. We then performed a refined ASO walk to target the regions important for exon inclusion or exclusion and identified a region known to be a binding site for the splicing-factor CUGBP1. RMS derived cell lines almost exclusively express IN-RA but when treated with our lead ASO compound that targets this region, they show a dramatic decrease in the IN-RA expression. More importantly, the ASO treated cells exhibit a significant reduction in cell proliferation and migration. Application of ASO compound attenuates the IGF signaling pathway measured by decreased pAKT staining. Additionally, when tested in RMS xenograft models, we see decreased extravasation of blood vessels in the grafts cells transfected with the ASO that blocks IN-RA expression compared to controls. Our current data shows promising insight into how we can impede the IGF2 pathway by reducing IN-RA expression and consequently mitigate tumor hallmarks like cell-proliferation, migration and angiogenesis. The goal is to use these ASO compounds as therapeutic interventions in conjunction with already established anti-IGF1 receptor therapies to treat pediatric Rhabdomyosarcoma. Citation Format: Safiya Khurshid, Matias Montes, Brianne Sanford, Chelsea Brown, Daniel Comiskey, Frank Rigo, Peter Houghton, Dawn Chandler. ASO screen uncovers splicing as a therapeutic vulnerability in theinsulin-like growth factor (IGF) signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4132.

Abstract LB-204: SON controls the oncogenic alternative splicing program in glioblastoma by regulating PTBP1/2 switch and RBFOX2 activity

Abstract Alternative RNA splicing is a critical regulatory step of gene expression that generates various spliced isoforms and controls RNA stability. While dysregulation of alternative splicing has been implicated in pathogenesis of several cancers, molecular mechanisms of aberrant RNA splicing in brain tumors remain largely uninvestigated. Here, we identified SON, a nuclear speckle protein containing both RNA and DNA binding domains, as a key regulator of the oncogenic alternative splicing program in glioblastoma. Analyses of brain tumor patient samples as well as publicly available databases demonstrated that SON is significantly upregulated in high grade gliomas and correlated with poor prognosis of the patients. SON depletion in glioblastoma cells resulted in inhibition of cell growth and colony formation, suggesting that SON play a pro-survival and oncogenic role in glioblastoma. Interestingly, we found that the SON level in glioblastoma patients has a strong positive correlation with the expression level of PTBP1, a well-known oncogenic RNA splicing factor, and furthermore exhibits an inverse correlation with PTBP2, a neuronal-specific PTBP1 paralog. RNA splicing analysis together with RNA-IP reveals that SON directly interacts with PTBP1 pre-mRNA, and SON knockdown leads to downregulation of PTBP1 through intron retention in PTBP1 pre-mRNA, resulting in inhibition of oncogenic RNA splicing of PTBP1 target genes. On the other hand, SON knockdown strongly induces PTBP2 cassette exon (exon 10) inclusion, resulting in generation of PTBP2 mRNA that does not bear a pre-mature stop codon and subsequent upregulation of PTBP2. To further identify the molecular mechanism of PTBP2 exon 10 inclusion upon SON knockdown, we searched for binding motifs of RNA splicing factors in the PTBP2 pre-mRNA and identified RBFOX2 binding motifs in intron sequences. Interestingly, RNA-IP experiments revealed that both SON and RBFOX2 are enriched in introns flanking the cassette exon and SON knockdown significantly increased RBFOX2-binding to PTBP2 RNA and subsequent cassette exon inclusion. Conversely, RBFOX2 knockdown facilitated SON-binding to PTBP2 RNA and induced skipping of the cassette exon. These results demonstrate that SON competes with RBFOX2 for RNA binding at PTBP2 introns to regulate exon exclusion, affecting PTBP2 expression. Finally, we verified that SON knockdown in human patient-derived glioblastoma stem cells (GSCs) indeed induces downregulation of PTBP1 and its downstream oncogenic splicing program while enhancing RBFOX2-mediated cassette exon inclusion that are found in healthy brains. Taken together, our study defines SON as a novel master splicing regulator that initiates “the oncogenic alternative splicing program” in glioblastoma by promoting PTBP1-mediated oncogenic splicing while suppressing non-oncogenic splicing mediated by RBFOX2 through its competitive binding to target RNAs. Therefore, our findings strongly implicate SON as a potential therapeutic target in malignant brain tumors. Citation Format: Jung-Hyun Kim, Joshua K. Stone, Jianfeng Li, Alexander Richard, Lana Vukadin, G. Yancey Gillespie, Robert W. Sobol, Steve Lim, Eun-Young Erin Ahn. SON controls the oncogenic alternative splicing program in glioblastoma by regulating PTBP1/2 switch and RBFOX2 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-204.

MBNL splicing activity depends on RNA binding site structural context.

Muscleblind-like (MBNL) proteins are conserved RNA-binding factors involved in alternative splicing (AS) regulation during development. While AS is controlled by distribution of MBNL paralogs and isoforms, the affinity of these proteins for specific RNA-binding regions and their location within transcripts, it is currently unclear how RNA structure impacts MBNL-mediated AS regulation. Here, we defined the RNA structural determinants affecting MBNL-dependent AS activity using both cellular and biochemical assays. While enhanced inclusion of MBNL-regulated alternative exons is controlled by the arrangement and number of MBNL binding sites within unstructured RNA, when these sites are embedded in a RNA hairpin MBNL binds preferentially to one side of stem region. Surprisingly, binding of MBNL proteins to RNA targets did not entirely correlate with AS efficiency. Moreover, comparison of MBNL proteins revealed structure-dependent competitive behavior between the paralogs. Our results showed that the structure of targeted RNAs is a prevalent component of the mechanism of alternative splicing regulation by MBNLs.

Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling.

Diastolic dysfunction is increasingly prevalent in our ageing society and an important contributor to heart failure. The giant protein titin could serve as a therapeutic target, as its elastic properties are a main determinant of cardiac filling in diastole. This study aimed to develop a high throughput pharmacological screen to identify small molecules that affect titin isoform expression through differential inclusion of exons encoding the elastic PEVK domains. We used a dual luciferase splice reporter assay that builds on the titin splice factor RBM20 to screen ~34,000 small molecules and identified several compounds that inhibit the exclusion of PEVK exons. These compounds belong to the class of cardenolides and affect RBM20 dependent titin exon exclusion but did not affect RBFOX1 mediated splicing of FMNL3. We provide evidence that cardenolides do not bind to the RNA interacting domain of RBM20, but reduce RBM20 protein levels and alter transcription of select splicing factors that interact with RBM20.Cardenolides affect titin isoform expression. Understanding their mode of action and harnessing the splice effects through chemical modifications that suppress the effects on ion homeostasis and more selectively affect cardiac splicing has the potential to improve cardiac filling and thus help patients with diastolic heart failure, for which currently no targeted therapy exists.

A Multiplexed Assay for Exon Recognition Reveals That an Unappreciated Fraction of Rare Genetic Variants Cause Large-Effect Disruptions to Splicing

Any individual’s genome contains ~4-5 million genetic variants that differ from reference, and understanding how these variants give rise to trait diversity and disease susceptibility is a central goal of human genetics (Auton et al., 2015). A vast majority (96-99%) of an individual’s variants are common, though at the population level the overwhelming majority of variants are rare (Montgomery et al., 2011; Nelson et al., 2012; Tennessen et al., 2012; UK10K Consortium et al., 2015). Because of their scarcity in an individual’s genome, rare variants that play important roles in complex traits are likely to have large functional effects (Bomba et al., 2017; Gibson, 2012). Mutations that cause an exon to be skipped can have severe functional consequences on gene function, and many known disease-causing mutations reduce or eliminate exon recognition (Baralle and Buratti, 2017). Here we explore the extent to which rare genetic variation in humans results in near complete loss of exon recognition. We developed a Multiplexed Functional Assay of Splicing using Sort-seq (MFASS) that allows us to measure exon inclusion in thousands of human exons and surrounding intronic sequence simultaneously. We assayed 27,733 extant variants in the Exome Aggregation Consortium (ExAC) within or adjacent to 2,339 human exons, and found that 3.8% (1,050) of the variants, almost all of which were extremely rare, led to large-effect defects in exon recognition. Importantly, we find that 83% of these splicedisrupting variants (SDVs) are located outside of canonical splice sites, are distributed evenly across distinct exonic and intronic regions, and are difficult to predict a priori. Our results indicate that loss of exon recognition is an important and underappreciated means by which rare variants exert large functional effects, and that MFASS enables their empirical assessment for large-effect splicing defects at scale.

Alternative splicing analysis in human monocytes and macrophages reveals MBNL1 as major regulator

We report the detailed transcriptomic profiles of human innate myeloid cells using RNA sequencing. Monocytes migrate from blood into infected or wounded tissue to differentiate into macrophages, and control inflammation via phagocytosis or cytokine secretion. We differentiated culture primary monocytes with either GM- or M-CSF to obtain pro- or anti-inflammatory macrophages, and respectively activated them with either LPS/IFNγ or anti-inflammatory cytokines. We also treated the THP-1 monocytic cell line with PMA and similar cytokines to mimic differentiation and activation. We detected thousands of expression and alternative-splicing changes during monocyte-to-macrophage differentiation and activation, and a net increase in exon inclusion. MBNL1 knockdown phenocopies several alternative-splicing changes and strongly impairs PMA differentiation, suggesting functional defects in monocytes from Myotonic Dystrophy patients. This study provides general insights into alternative splicing in the monocyte–macrophage lineage, whose future characterization will elucidate their contribution to immune functions, which are altered in immunodeficiencies, autoimmunity, atherosclerosis and cancer.

Modulation of transcriptional activity in brain lower grade glioma by alternative splicing

Proteins that modify the activity of transcription factors (TFs) are often called modulators and play a vital role in gene transcriptional regulation. Alternative splicing is a critical step of gene processing, and differentially spliced isoforms may have different functions. Alternative splicing can modulate gene function by adding or removing certain protein domains and thereby influence the activity of a protein. The objective of this study is to investigate the role of alternative splicing in modulating the transcriptional regulation in brain lower grade glioma (LGG), especially transcription factor ELK1, which is closely related to various disorders, including Alzheimer’s disease and Down syndrome. The results showed that changes in the exon inclusion ratio of proteins APP and STK16 are associated with changes in the expression correlation between ELK1 and its targets. In addition, the structural features of the two modulators are strongly associated with the pathological impact of exon inclusion. The results of our analysis suggest that alternatively spliced proteins have different functions in modifying transcription factors and can thereby induce the dysregulation of multiple genes.

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts.

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide (AON)-mediated therapy. While SMN1 mutations are responsible for the disease, AONs targeting intronic splice silencer (ISS) sites in SMN2, including FDA-approved nusinersen, have been shown to restore SMN expression and ameliorate the symptoms. Currently, many studies involving AON therapy for SMA focus on investigating novel AON chemistries targeting SMN2 that may be more effective and less toxic than nusinersen. Here, we describe a protocol for in vitro evaluation of exon inclusion using lipotransfection of AONs followed by reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), and Western blotting. This method can be employed for various types of AON chemistries. Using this method, we demonstrate that AONs composed of alternating locked nucleic acids (LNAs) and DNA nucleotides (LNA/DNA mixmers) lead to efficient SMN2 exon inclusion and restoration of SMN protein at a very low concentration, and therefore, LNA/DNA mixmer-based antisense oligonucleotides may be an attractive therapeutic strategy to treat splicing defects caused by genetic diseases. The in vitro evaluation method described here is fast, easy, and sensitive enough for the testing of various novel AONs.

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Histone demethylase JMJD1A promotes alternative splicing of AR variant 7 (AR-V7) in prostate cancer cells

Formation of the androgen receptor splicing variant 7 (AR-V7) is one of the major mechanisms by which resistance of prostate cancer to androgen deprivation therapy occurs. The histone demethylase JMJD1A (Jumonji domain containing 1A) functions as a key coactivator for AR by epigenetic regulation of H3K9 methylation marks. Here, we describe a role for JMJD1A in AR-V7 expression. While JMJD1A knockdown had no effect on full-length AR (AR-FL), it reduced AR-V7 levels in prostate cancer cells. Reexpression of AR-V7 in the JMJD1A-knockdown cells elevated expression of select AR targets and partially rescued prostate cancer cell growth in vitro and in vivo. The AR-V7 protein level correlated positively with JMJD1A in a subset of human prostate cancer specimens. Mechanistically, we found that JMJD1A promoted alternative splicing of AR-V7 through heterogeneous nuclear ribonucleoprotein F (HNRNPF), a splicing factor known to regulate exon inclusion. Knockdown of JMJD1A or HNRNPF inhibited splicing of AR-V7, but not AR-FL, in a minigene reporter assay. JMJD1A was found to interact with and promote the recruitment of HNRNPF to a cryptic exon 3b on AR pre-mRNA for the generation of AR-V7. Taken together, the role of JMJD1A in AR-FL coactivation and AR-V7 alternative splicing highlights JMJD1A as a potentially promising target for prostate cancer therapy.

Intronic antisense Alu elements have a negative splicing effect on the inclusion of adjacent downstream exons

Alu elements occupy 10% of the human genome. However, although they contribute to genomic and transcriptomic diversity, their function is still not fully understood. We hypothesized that intronic Alu elements may contribute to alternative splicing. We therefore examined their effect on splicing using minigene constructs including exon 9–exon 11 inclusive of ACAT1 with truncated introns 9 and 10. These constructs contained a suboptimal splice acceptor site for intron 9. Insertion of AluY–partial AluSz6–AluSx, originally located in ACAT1 intron 5, in an antisense direction within intron 9 had a negative effect on exon 10 inclusion. This effect was additive with that of an exonic splicing enhancer mutation in exon 10, and was canceled by the substitution of G for C at the first nucleotide of exon 10 which optimized the splice acceptor site of intron 9. A sense AluY–partial AluSz6–AluSx insertion had no effect on exon 10 inclusion, and one antisense AluSx insertion had a similar effect to antisense AluY–partial AluSz6–AluSx insertion. The shorter the distance between the antisense Alu element and exon 10, the greater the negative effect on exon 10 inclusion. This distance effect was more evident for suboptimal than optimal splice sites. Based on our data, we propose that intronic antisense Alu elements contribute to alternative splicing and transcriptomic diversity in some genes, especially when splice acceptor sites are suboptimal.

Tracing the evolution of the heterotrimeric G protein \u03b1 subunit in Metazoa

BackgroundHeterotrimeric G proteins are fundamental signaling proteins composed of three subunits, Gα and a Gβγ dimer. The role of Gα as a molecular switch is critical for transmitting and amplifying intracellular signaling cascades initiated by an activated G protein Coupled Receptor (GPCR). Despite their biochemical and therapeutic importance, the study of G protein evolution has been limited to the scope of a few model organisms. Furthermore, of the five primary Gα subfamilies, the underlying gene structure of only two families has been thoroughly investigated outside of Mammalia evolution. Therefore our understanding of Gα emergence and evolution across phylogeny remains incomplete.ResultsWe have computationally identified the presence and absence of every Gα gene (GNA-) across all major branches of Deuterostomia and evaluated the conservation of the underlying exon-intron structures across these phylogenetic groups. We provide evidence of mutually exclusive exon inclusion through alternative splicing in specific lineages. Variations of splice site conservation and isoforms were found for several paralogs which coincide with conserved, putative motifs of DNA-/RNA-binding proteins. In addition to our curated gene annotations, within Primates, we identified 15 retrotranspositions, many of which have undergone pseudogenization. Most importantly, we find numerous deviations from previous findings regarding the presence and absence of individual GNA- genes, nuanced differences in phyla-specific gene copy numbers, novel paralog duplications and subsequent intron gain and loss events.ConclusionsOur curated annotations allow us to draw more accurate inferences regarding the emergence of all Gα family members across Metazoa and to present a new, updated theory of Gα evolution. Leveraging this, our results are critical for gaining new insights into the co-evolution of the Gα subunit and its many protein binding partners, especially therapeutically relevant G protein – GPCR signaling pathways which radiated in Vertebrata evolution.

Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells

BackgroundMounting evidence suggests that one of the ways that cells adapt to hypoxia is through alternative splicing. The aim of this study was firstly to examine the effect of hypoxia on the alternative splicing of cancer associated genes using the prostate cancer cell line PC3 as a model. Secondly, the effect of hypoxia on the expression of several regulators of splicing was examined.MethodsPC3 cells were grown in 1% oxygen in a hypoxic chamber for 48 h, RNA extracted and sent for high throughput PCR analysis at the RNomics platform at the University of Sherbrooke, Canada. Genes whose exon inclusion rate PSI (ψ) changed significantly were identified, and their altered exon inclusion rates verified by RT-PCR in three cell lines. The expression of splice factors and splice factor kinases in response to hypoxia was examined by qPCR and western blotting. The splice factor kinase CLK1 was inhibited with the benzothiazole TG003.ResultsIn PC3 cells the exon inclusion rate PSI (ψ) was seen to change by > 25% in 12 cancer-associated genes; MBP, APAF1, PUF60, SYNE2, CDC42BPA, FGFR10P, BTN2A2, UTRN, RAP1GDS1, PTPN13, TTC23 and CASP9 (caspase 9). The expression of the splice factors SRSF1, SRSF2, SRSF3, SAM68, HuR, hnRNPA1, and of the splice factor kinases SRPK1 and CLK1 increased significantly in hypoxia. We also observed that the splice factor kinase CLK3, but not CLK2 and CLK4, was also induced in hypoxic DU145 prostate, HT29 colon and MCF7 breast cancer cell lines. Lastly, we show that the inhibition of CLK1 in PC3 cells with the benzothiazole TG003 increased expression of the anti-apoptotic isoform caspase 9b.ConclusionsSignificant changes in alternative splicing of cancer associated genes occur in prostate cancer cells in hypoxic conditions. The expression of several splice factors and splice factor kinases increases during hypoxia, in particular the Cdc-like splice factor kinases CLK1 and CLK3. We suggest that in hypoxia the elevated expression of these regulators of splicing helps cells adapt through alternative splicing of key cancer-associated genes. We suggest that the CLK splice factor kinases could be targeted in cancers in which hypoxia contributes to resistance to therapy.

Molecular Cloning of a Novel Brain‐specific RGS6 Isoform Preferentially Expressed in the Substantia Nigra of Humans with Parkinson's Disease

RGS proteins modulate G protein‐coupled receptor (GPCR) signaling by facilitating heterotrimeric G protein inactivation through their GTPase‐activating (GAP) activity toward Gα subunits, a function bestowed by their RGS domain. RGS6 (Gαi/o‐ GAP), a member of the R7 subfamily, is critically involved in several CNS disorders for which it may be a novel therapeutic target. Remarkably, RGS6−/− mice have reduced anxiety/depression, exhibit diminished alcohol seeking/reward behaviors, and develop late‐onset Parkinson's disease (PD). The role of RGS6 in these disorders depends on its ability to inhibit various GPCRs, including: cortical and hippocampal 5‐HT1ARs (anxiety/depression), mesolimbic GABABRs and D2Rs (alcoholism), and D2Rs in the substantia nigra (SNc, PD). Potentially key to RGS6's ability to regulate numerous GPCRs are unidentified domains arising via alternative mRNA splicing. Our initial cloning effort identified 36 RGS6 mRNAs in human brain encoding proteins ≤56kDa. Recently, we identified, in mouse and human, additional brain‐specific RGS6 protein isoforms that are larger (~61, 69kDa) than the ubiquitously expressed ~56kDa RGS6L isoforms. The function of these RGS6 isoforms is unknown, but they may be critical for normal CNS function and pathology as both are expressed in brain regions affected by the disorders described above. Here we report the cloning of a novel RGS6 transcript arising via novel exon (Alternative 3, A3) inclusion. This transcript was named RGS6LA3α1 to indicate it resembles the RGS6Lα1 transcript identified in our initial cloning effort. RGS6LA3α1 exhibits near exclusive CNS expression, encodes a protein that co‐migrates with the 69kDa brain‐specific RGS6, and has a C‐terminal extension near the RGS domain that may be a novel protein interaction site or regulatory domain. Of particular interest, is the role of the 69kDa RGS6 in PD pathogenesis as its expression, as well as its transcript, is upregulated in the SNc of PD patients relative to other RGS6L proteins which are down‐regulated. This latter finding is consistent with our evidence that RGS6 is required for SNc dopamine (DA) neuron survival. However, these data also raise the intriguing possibility that while RGS6L isoforms promote SNc DA neuron survival, RGS6LA3α1/the 69kDa brain‐specific RGS6 protein isoform may fail to do so or even contribute to SNc DA neuron death. We hypothesize that dysregulated splicing of primary RGS6 transcripts, and A3 exon inclusion, could lead to both a physical loss of RGS6L protein isoforms as well as a functional loss of RGS6‐mediated GAP activity due to RGS domain interference caused by C‐terminal extension, a prediction supported by molecular modeling studies. We predict that decreased expression of RGS6L isoforms and increased expression of the 69kDa protein disinhibits SNc D2R signaling culminating in accumulation of cytotoxic DA byproducts as well as decreased mitochondrial number and size ultimately inducing SNc DA neuron death. Together, this research begins to elucidate the functional significance of RGS6 alternative mRNA splicing in brain function and pathology.Support or Funding InformationNIH CA161882 and MJ Fox 11551This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Selective Neddylation facilitates proteasome‐mediated degradation of Serine Rich Splicing Factor 3 (SRSF3) in non‐alcoholic fatty liver disease

BackgroundHepatocellular Carcinoma (HCC) is the most common primary liver cancer. Over 80% of patients with HCC develop liver cirrhosis. Metabolic disturbances such as non‐alcoholic fatty liver disease (NAFLD) and its more severe form non‐alcoholic steatosis (NASH) are becoming more common in the obese population and both are risk factors for cirrhosis. Serine‐rich proteins are RNA‐binding proteins that contain an amino‐terminal RNA recognition motif (RRM) and have a C‐terminal domain enriched in arginine/serine dipeptides (RS domain). Previously, our group has demonstrated that liver‐specific SRSF3 deletion causes a multi‐step dysregulation of liver morphology and hepatocyte function, ultimately leading to hepatocellular carcinoma. Although studies have assessed the mechanism of SRSF3 degradation and its effect on cell metabolism. No investigations have been carried out to study the effect of SRSF3 degradation in liver diseases such as NAFLD, NASH or cirrhosis that predispose to HCC. The work presented here addresses the mechanism of SRSF3 degradation and its impact on liver disease such as NASH.MethodsTo assess the degradation of SRSF3 in NAFLD, mice were maintained on a 60% high‐fat diet (HFD) or western diet (NASH diet) and SRSF3 protein expression in liver was studied. In addition, we also tested the effect of oxidative stress on SRSF3. To understand the molecular mechanism of SRSF3 degradation, we performed experiments with cycloheximide and the proteasome inhibitor MG132. Further, we assessed different SRSF3 lysine residues to determine the SRSF3 lysine site modification. We also performed immunoblot and RT‐PCR of SRSF3 and its target genes to define functions of Lys11.ResultsImmunoblot data showed loss of SRSF3 in HFD and NASH hepatocyte cells. Immunohistochemistry showed a stronger staining for SRSF3 in the nucleus of liver samples from normal chow‐fed mice compared to HFD or NASH fed mice, indicating loss of SRSF3. Fibronectin 1 (Fn1) and insulin receptor (INSR) are direct target for SRSF3. Immunohistochemistry and RT‐PCR results showed higher expression of Fn1 and greater inclusion of exon 33 of the profibrogenic EDA exon in the Fn1 transcript in hepatocytes and liver of HFD and NASH mice. Increased expression of the INSR‐A isoform was evident in HFD and NASH hepatocytes. Further, we found that palmitic acid and H2O2 cause loss of SRSF3 in HepG2 and primary hepatocyte cells. Co‐IP data suggested that SRSF3 is selectively neddylated at Lys11. More importantly, SRSF3 degradation is mediated following Neddylation of the protein and subsequent degradation via a proteasome complex degradation pathway. We observed that, Lys11 impaired the palmitic acid induced FN1‐EDA inclusion and other EMT genes.ConclusionLipid accumulation causes loss of SRSF3 in HFD and NASH liver. Further, neddylation may be a primary mechanism of SRSF3 degradation following modification at Lys11. However, the degradation profile of SRSF3 in a large number of well‐characterized pathological samples needs to be determined. A detailed understanding of the mechanisms involved in SRSF3 degradation might allow the identification of novel therapeutic targets and may aid in the treatment of hepatocellular carcinoma.Support or Funding InformationVMRFThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.