Splicing Patterns Research Articles

S161: ZRSR2 AND TET2 MUTATIONS PROMOTE MDS BY DYSREGULATING GENE EXPRESSION AND ABERRANT ALTERNATIVE SPLICING IN MICE

Background: Mutations in splicing factors and epigenetic regulators are the most frequent genetic alterations in patients with myelodysplastic syndromes (MDS). The minor spliceosome factor ZRSR2 and the epigenetic regulator TET2 appear significantly associated in MDS patients. However, the functional impact of such mutations in the hematopoietic system and MDS have been scarcely studied. To address this question, we established a murine model (Zrsr2m/mTet2−/−) carrying mutations in both genes, which exhibited signs compatible with MDS disease in mice. However, the molecular disease mechanism has not yet been elucidated. Aims: To interrogate the impact of ZRSR2 and TET2 mutations in gene expression and alternative splicing in the context of hematopoiesis and MDS. Methods: Whole transcriptome sequencing was performed to investigate changes in gene expression and alternative splicing patterns. Lin-Sca-1+c-kit+ (LSK) cells (50,000 per sample) were sorted from pools of 3 mice, and mRNA was sequenced in an Illumina NovaSeq 6000 platform. Differential gene expression was determined using DESeq2 with adjusted P-value ≤ 0.05, log2FoldChange ≥ 0. Functional enrichment from differentially spliced genes was identified with KEGG, and the enrichment cut-off p-value was set as adjusted P-value ≤ 0.05. Relevant alternative splicing events were validated by RT-PCR. Results: RNA-seq analysis of Zrsr2m/mTet2−/−cells revealed 2952 differentially expressed genes (DEG), compared to 571 in Zrsr2m/m and 1203 in Tet2−/−. From 2952, 1327 were up-regulated and 1625 were downregulated. Interestingly, relevant genes for hematopoietic lineage specification were significantly dysregulated. In particular, genes related to lymphoid lineage (Flt3, Notch1, and Tlr7) were downregulated, while those related to myeloid lineage (Ccl9, Mpo, Ms4a6b, and Prtn3) and megakaryocytic-erythroid lineage (Pdfgrb, Itgb3, Optn, and Tgfbr3) were upregulated. This suggests an early myeloid and megakaryocyte-erythroid priming of LSK towards the production of cells from these lineages in Zrsr2m/mTet2−/− mice. Enrichment analysis of DEG using Go enrichment analysis identified ribosome function, inflammation, and migration/motility processes as the most significantly altered. Further, KEGG pathway enrichment pointed ribosome, the MAPK family, and pro-inflammatory pathways as significantly enriched in Zrsr2m/mTet2−/− cells. Finally, KEGG pathway enrichment of alternative splicing targets identified the MAPK family and the Fanconi anemia pathway as the most altered targets in Zrsr2m/mTet2−/− cells. Importantly, a total of 9 genes related to the MAPK pathway were identified as mis-spliced, from which Dusp1, Tgfbr2, and Fgf11 were validated in this study. Summary/Conclusion: In this study, we broaden our previous report and show that concurrent mutations in Zrsr2 and Tet2 dysregulate normal gene expression and cause aberrant mRNA splicing. Gene expression analysis identified ribosome function, inflammation, and migration/motility as the most altered processes in Zrsr2m/mTet2−/− cells. Alternative splicing analysis identified the MAPK and the Fanconi Anemia pathway as key targets of aberrant splicing. All in all, gene expression dysregulation and aberrant mRNA splicing disturb important biological pathways and drive the molecular pathomechanism in Zrsr2m/mTet2−/− mice.

Splice site proximity influences alternative exon definition

ABSTRACT Alternative splicing enables higher eukaryotes to expand mRNA diversity from a finite number of genes through highly combinatorial splice site selection mechanisms that are influenced by the sequence of competing splice sites, cis-regulatory elements binding trans-acting factors, the length of exons and introns harbouring alternative splice sites and RNA secondary structures at putative splice junctions. To test the hypothesis that the intron definition or exon definition modes of splice site recognition direct the selection of alternative splice patterns, we created a database of alternative splice site usage (ALTssDB). When alternative splice sites are embedded within short introns (intron definition), the 5′ and 3′ splice sites closest to each other across the intron preferentially pair, consistent with previous observations. However, when alternative splice sites are embedded within large flanking introns (exon definition), the 5′ and 3′ splice sites closest to each other across the exon are preferentially selected. Thus, alternative splicing decisions are influenced by the intron and exon definition modes of splice site recognition. The results demonstrate that the spliceosome pairs splice sites that are closest in proximity within the unit of initial splice site selection.

Genome-wide alternative splicing profile in the posterior kidney of brown trout (Salmo trutta) during proliferative kidney disease

BackgroundThe cnidarian myxozoan parasite Tetracapsuloides bryosalmonae causes chronic proliferative kidney disease (PKD) in salmonids. This parasite is a serious threat to wild and cultured salmonids. T. bryosalmonae undergoes intra-luminal sporogonic development in the kidney of brown trout (Salmo trutta) and the viable spores are released via urine. We investigated the alternative splicing pattern in the posterior kidney of brown trout during PKD.ResultsRNA-seq data were generated from the posterior kidney of brown trout collected at 12 weeks post-exposure to T. bryosalmonae. Subsequently, this data was mapped to the brown trout genome. About 153 significant differently expressed alternatively spliced (DEAS) genes, (delta PSI = 5%, FDR P-value < 0.05) were identified from 19,722 alternatively spliced events. Among the DEAS genes, the least and most abundant alternative splicing types were alternative 5′ splice site (5.23%) and exon skipping (70.59%), respectively. The DEAS genes were significantly enriched for sodium-potassium transporter activity and ion homeostasis (ahcyl1, atp1a3a, atp1a1a.1, and atp1a1a.5). The protein-protein interaction network analysis enriched two local network clusters namely cation transporting ATPase C-terminus and Sodium/potassium ATPase beta chain cluster, and mixed inclusion of Ion homeostasis and EF-hand domain cluster. Furthermore, the human disease-related salmonella infection pathway was significantly enriched in the protein-protein interaction network.ConclusionThis study provides the first baseline information about alternative splicing in brown trout during PKD. The generated data lay a foundation for further functional molecular studies in PKD - brown trout infection model. The information generated from the present study can help to develop therapeutic strategies for PKD in the future.

Abstract 5852: Doxorubicin increases the level of a BRCA2 exon 3-skipping mRNA isoform in MCF7

Abstract Introduction: BRCA2 is a tumor suppressor that participates in DNA repair. Inherited pathogenic mutations in BRCA2 are associated with a 38%-84% risk for breast cancer and a 16.5%-27% risk for ovarian cancer. It is therefore critical to distinguish pathogenic mutations from sequence variants of unknown clinical significance (VUSs). Among the VUSs that require clinical classification are those that could potentially disrupt normal splicing patterns. The challenge in determining the spliceogenic nature of a VUS is the ubiquity of naturally occurring alternative splicing variants. BRCA2 exon 3, which encodes a binding site for the interacting proteins EMSY and PALB2, is frequently skipped during alternative splicing events to different levels in different cell types under different conditions. It is not yet known whether BRCA2 exon 3-skipping is a regulated event contributing to the spectrum of BRCA2 functions or is a stochastic occurrence. A genomic deletion that results in complete allelic loss of exon 3 has been shown to be pathogenic, yet VUSs that result in increased frequency of exon 3-skipping have been shown to be non-pathogenic. Thus, the allele-specific ability of BRCA2 to contribute to DNA repair is affected by the level of exon 3-skipping. Alternative splicing patterns have also been observed in response to DNA damaging agents, potentially linking some therapies to risks from genome-wide aberrant splicing. In this study we explore the effects of the topoisomerase inhibitor doxorubicin on the frequency of BRCA2 exon 3-skipping. Isoform-specific end-point RT-PCR shows that doxorubicin increases the frequency of BRCA2 exon 3-skipping in the breast cancer cell line MCF7. Methods: MCF7 cells were treated with doxorubicin according to standard protocols. RNA was prepared using Qiagen reagents and amplified with Invitrogen One-Step RT-PCR reagents. Isoform-specific RT-PCR primers were designed to amplify the full-length region surrounding BRCA2 exon 3 independently from the region left by an exon-skipping event (Δ3). RT-PCR products were visualized using agarose gel electrophoresis. Results and Conclusion: Preliminary results suggest that doxorubicin increases the level of exon 3-skipping in MCF7. These results suggest doxorubicin, used to treat breast and other cancers, may compromise the repair functions of BRCA2 in addition to other transcriptome-wide effects from aberrant splicing patterns. However, it is not known whether this difference in isoform accumulation between treated and untreated cells is the result of increased frequency of alternative splicing events or changes in relative stabilities of the full-length and Δ3 isoforms, so it cannot yet be determined whether any potential physiological defects associated with alternative splicing of BRCA2 exon 3 result from insufficiency of the full-length isoform. Citation Format: Zoha Hameedi, Talia Ishfaq, Zaain Ahmad, James D. Fackenthal. Doxorubicin increases the level of a BRCA2 exon 3-skipping mRNA isoform in MCF7 [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 5852.

Abstract SY11-02: Long-read sequencing reveals complex patterns of wraparound transcription in polyomaviruses

Abstract Polyomaviruses (PyV) are ubiquitous pathogens that can cause devastating human diseases. Due to the small size of their genomes, PyV utilize complex patterns of RNA splicing to maximize their coding capacity. Despite the importance of PyV to human disease, their transcriptome architecture is poorly characterized. Here, we compare short- and long-read RNA sequencing data from eight human and non-human PyV. We provide a detailed transcriptome atlas for BK polyomavirus (BKPyV), an important human pathogen, and the prototype PyV, simian virus 40 (SV40). We identify pervasive wraparound transcription in PyV, wherein transcription runs through the polyA site and circles the genome multiple times. Comparative analyses identify novel, conserved transcripts that increase PyV coding capacity. One of these conserved transcripts encodes superT, a T antigen containing two RB-binding LxCxE motifs. We find that superT-encoding transcripts are abundant in PyV-associated human cancers. Together, we show that comparative transcriptomic approaches can greatly expand known transcript and coding capacity in one of the simplest and most well-studied viral families. Citation Format: James A. DeCaprio, Jason Nomburg, Matthew Meyerson. Long-read sequencing reveals complex patterns of wraparound transcription in polyomaviruses [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 SY11-02.

Abstract 1215: pVACsplice: Predicting neoantigens from tumor-specific alternative splicing events derived from cis-acting regulatory mutations using whole exome and RNA sequencing data

Abstract Neoantigens are tumor-specific peptides on the cell surface that can be recognized by the adaptive immune system. Personalized immunotherapies, such as cancer vaccines, rely on neoantigen prediction to identify sequences that can activate T cells to recognize and destroy the tumor. The majority of cancer vaccine trials have utilized neoantigens derived from missense mutations and small insertions and deletions. However, other mutation types could contribute to the overall neoantigen landscape, such as aberrantly spliced transcripts arising from cis-acting regulatory mutations. In this study, we explore the potential immunogenicity of alternative splicing events by creating pVACsplice, a tool to expand the capability of pVACtools, a suite of tools for neoantigen prediction (http://www.pvactools.org). pVACsplice assembles alternative transcripts from tumor-specific splicing patterns, identifies sequence changes by comparison to a reference, and predicts neoantigens from the novel regions. To verify the accuracy of alternative transcript assembly, we ran pVACsplice with HCC1395 cell line samples and performed long-read sequencing to detect the transcripts in vitro. Matched whole exome sequencing and RNA sequencing datasets from glioblastoma, melanoma, and colorectal cancer cohorts will also be analyzed with pVACsplice to obtain binding affinity estimates. We will compare these results to neoantigen predictions from other mutation sources and across cancer types to discover the prevalence of immunogenic splicing events. Finally, we will perform immunogenicity testing with a set of high quality candidates to validate our predictions. We hope to increase the number of candidates for patients’ vaccines by adding this functionality to our standard neoantigen prediction workflow. This tool could help generate a more accurate portrait of the neoantigen landscape in tumors, and in turn, enhance responses to personalized immunotherapies. Citation Format: Megan M. Richters, Kelsy C. Cotto, Susanna Kiwala, Huiming Xia, Beatriz M. Carreno, Gavin P. Dunn, Antoni Ribas, Obi L. Griffith, Malachi Griffith. pVACsplice: Predicting neoantigens from tumor-specific alternative splicing events derived from cis-acting regulatory mutations using whole exome and RNA sequencing data [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 1215.

Abstract 1873: SpliceCore® a novel ML tool for identifying disease-specific alternative splicing uncovered a promising therapeutic target for triple negative breast cancer

Abstract 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 &amp;gt;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 analyses for variants in MMR genes: best practice recommendations from the European Mismatch Repair Working Group

Over 20% of the DNA mismatch repair (MMR) germline variants in suspected Lynch syndrome patients are classified as variants of uncertain significance (VUS). Well-established functional assays are pivotal for assessing the biological impact of these variants and provide relevant evidence for clinical classification. In our collaborative European Mismatch Repair Working Group (EMMR-WG) we compared three different experimental approaches for evaluating the effect of seven variants on mRNA splicing in MMR genes: (i) RT-PCR of full-length transcripts (FLT), (ii) RT-PCR of targeted transcript sections (TTS), both from patient biological samples and (iii) minigene splicing assays. An overall good concordance was observed between splicing patterns in TTS, FLT and minigene analyses for all variants. The FLT analysis depicted a higher number of different isoforms and mitigated PCR-bias towards shorter isoforms. TTS analyses may miss aberrant isoforms and minigene assays may under/overestimate the severity of certain splicing defects. The interpretation of the experimental findings must be cautious to adequately discriminate abnormal events from physiological complex alternative splicing patterns. A consensus strategy for investigating the impact of MMR variants on splicing was defined. First, RNA should be obtained from patient’s cell cultures (such as fresh lymphocyte cultures) incubated with/without a nonsense-mediated decay inhibitor. Second, FLT RT-PCR analysis is recommended to oversee all generated isoforms. Third, TTS analysis and minigene assays are useful independent approaches for verifying and clarifying FLT results. The use of several methodologies is likely to increase the strength of the experimental evidence which contributes to improve variant interpretation.

A transcriptomic atlas underlying developmental plasticity of seasonal forms of Bicyclus anynana butterflies.

Organisms residing in regions with alternating seasons often develop different phenotypes, or forms, in each season. These forms are often adaptations to each season and result from an altered developmental response to specific environmental cues such as temperature. Although multiple studies have examined form-specific gene expression profiles in a diversity of species, little is known about how environments and developmental transitions, cued by hormone pulses, alter post-transcriptional patterns. In this study, we examine how gene expression, alternative splicing, and miRNA-mediated gene silencing in Bicyclus anynana butterfly hindwing tissue, varies across two rearing temperatures at four developmental timepoints. These timepoints flank two temperature-sensitive periods that coincide with two pulses of the insect hormone 20E. Our results suggest that developmental transitions, coincident with 20E pulses, elicit a greater impact on all these transcriptomic patterns than rearing temperatures per se. More similar transcriptomic patterns are observed pre-20E pulses than those observed post-20E pulses. We also found functionally distinct sets of differentially expressed genes (DEGs) and differentially spliced genes (DSGs) in the seasonal forms. Furthermore, around 10% of DEGs are predicted to be direct targets of, and regulated by, differentially expressed miRNAs (DEmiRs) between the seasonal forms. Many DEGs, DEmiRs, or DSGs potentially regulate eyespot size plasticity, and we validated the differential splicing pattern of one such gene, daughterless. We present a comprehensive and interactive transcriptomic atlas of the hindwing tissue of both seasonal forms of B. anynana throughout development, a model organism of seasonal plasticity.

Genetic Polymorphism of Avian Leukosis Virus Host Receptors in Korean Native Chickens and Establishment of Resistant Line

Avian leukosis virus (ALV) is a highly contagious retrovirus that causes tumors and has resulted in great economic loss worldwide owing to its high transmission rate. Various ALV viral subgroups exist, with infections occurring via specific host receptors. The susceptibility or resistance of avian species to the ALV-A and K subgroups is determined by the host receptor, the tumor virus locus A (<i>tva</i>) gene, while that to ALV-B depends on another host receptor, the tumor virus locus B (<i>tvb</i>) gene. The resistance alleles of <i>tva</i> and <i>tvb</i> have primarily been identified in China, but none have been detected in Korea. We analyzed the frequencies of <i>tva</i> and <i>tvb</i> genotypes in White Leghorn (WL), Korean Ogye (KO), and Korean native chicken (KNC) breeds and assessed the resistance to ALV subgroups. In WL, both <i>tva</i> and <i>tvb</i> had various genotypes, including susceptibility and resistance alleles, whereas in KO, <i>tva</i> and <i>tvb</i> resistance alleles were dominant. In KNC, <i>tva</i> susceptibility and resistance alleles were mixed, whereas <i>tvb</i> resistance alleles were dominant. In addition, we showed that there were differences in the splicing pattern of <i>tva</i> transcripts and the expression level of <i>tvb</i> transcripts within breeds. Finally, we confirmed that ALV resistance depended on KO and KNC genotypes by <i>in vitro</i> infection of chicken embryonic fibroblasts with ALV. These results highlight that some KO and KNC individuals are naturally resistant to ALV subgroups A, B, and K and will facilitate the preservation of economically superior traits through selective breeding.

LncRNA MIAT Promotes Spinal Cord Injury Recovery in Rats by Regulating RBFOX2-Mediated Alternative Splicing of MCL-1.

LncRNA myocardial infarction-associated transcript (MIAT) alleviates acute spinal cord injury (ASCI)-induced neuronal cell apoptosis, but the specific mechanism of it involved in regulating SCI progression needs further exploration. Here, a SCI rat model was established, followed by administration with adenovirus-mediated MIAT overexpression vector (Ad-MIAT) alone or together with Ad-RBFOX2 (RNA binding fox-1 homolog 2). The data indicated that MIAT overexpression promoted motor function recovery, improved morphology of injured tissues, and restrained neuron loss and cell apoptosis in SCI rats. Then, PC-12 cells were treated with H2O2 to induce cell injury. And highly expressed MIAT suppressed H2O2-caused decrease in cell viability and increase in cell apoptosis. MIAT stabilized RBFOX2 protein expression by binding to RBFOX2, thereby promoting RBFOX2-induced upregulation of anti-apoptotic MCL-1L (myeloid cell leukemia sequence 1) and reduction of pro-apoptotic MCL-1S. And silencing RBFOX2 in vitro blocked the inhibitory effect of MIAT on cell apoptosis. Moreover, MCL-1-specific steric-blocking oligonucleotides (SBOs) were used to transfer the MCL-1 pre-mRNA splicing pattern from MCL-1L to MCL-1S. SBOs reversed the protection effect of RBFOX2 overexpression on H2O2-induced cell injury. Furthermore, overexpression of MCL-1L instead of MCL-1S facilitated autophagy activation in H2O2-stimulated cells. Interestingly, co-overexpression of MIAT and RBFOX2 had a better promoting effect on SCI recovery. In conclusion, MIAT mitigated SCI by promoting RBFOX2-mediated alternative splicing of MCL-1. Our findings might provide a promising therapeutic target for SCI.

Identification of a Novel Canonical Splice Site Variant TSC2 c.2967-1G&gt;T That is Not Associated With Tuberous Sclerosis Pathogenesis

Tuberous sclerosis, also known as tuberous sclerosis complex (TSC), is an autosomal dominant defect characterized by hamartomas in multiple organ systems. Inactivating variants cause this defect in either the TSC1 gene or the TSC2 gene, leading to hamartin or tuberin protein dysfunction, thus resulting in TSC. The diagnostic criteria for TSC suggest that it can be diagnosed by identifying a heterozygous pathogenic variant of TSC1 or TSC2, even in the absence of clinical signs. In a 4-year-old girl, we identified a splicing variant (NM_000548.4: c.2967-1G>T) that she inherited from her father. Neither the girl (patient) nor her father showed typical features of TSC. This variant is located in a NAGNAG acceptor, which can produce mRNA isoforms that differ by a three-nucleotide indel. Reverse transcription polymerase chain reaction analysis of the patient and both parents’ blood RNA samples suggested two different splicing patterns, and these two splicing patterns differed in the presence or absence of the first codon of exon 27, thus providing two splicing products designated as isoforms A and B, respectively. Furthermore, the proportions of these two patterns varied between the patient and either parent. A minigene assay further confirmed that the c.2967-1G>T variant led to the absence of isoform A (including the first codon of exon 27). The finding of our study demonstrates this variant, c.2967-1G>T, disrupts the balance of an alternative splice event which involves the use of two tandem alternatives acceptors and is not associated with typical symptoms of tuberous sclerosis. Our finding is of importance for genetic counseling and suggests that we need to be vigilant to avoid misdiagnosis when we encounter such a site.

Revealing diverse alternative splicing variants of the highly homologous SMN1 and SMN2 genes by targeted long-read sequencing.

The survival of motor neuron (SMN) genes, SMN1 and SMN2, are two highly homologous genes related to spinal muscular atrophy (SMA). Different patterns of alternative splicing have been observed in the SMN genes. In this study, the long-read sequencing technique for distinguishing SMN1 and SMN2 without any assembly were developed and applied to reveal multiple alternative splicing patterns and to comprehensively identify transcript variants of the SMN genes. In total, 36 types of transcript variants were identified, with an equal number of variants generated from both SMN1 and SMN2. Of these, 18 were novel SMN transcripts that have never been reported. The structures of SMN transcripts were revealed to be much more complicated and diverse than previously discovered. These novel transcripts were derived from diverse splicing events, including skipping of one or more exons, intron retention, and exon shortening or addition. SMN1 mainly produces FL-SMN1, SMN1Δ7, SMN1Δ5 and SMN1Δ3. The distribution of SMN2 transcripts was significantly different from those of SMN1, with the majority transcripts to be SMN2Δ7, followed by FL-SMN2, SMN2Δ3,5 and SMN2Δ5,7. Targeted long-read sequencing approach could accurately distinguish sequences of SMN1 from those of SMN2. Our study comprehensively addressed naturally occurring SMN1 and SMN2 transcript variants and splicing patterns in peripheral blood mononuclear cells (PBMCs). The novel transcripts identified in our study expanded knowledge of the diversity of transcript variants generated from the SMN genes and showed a much more comprehensive profile of the SMN splicing spectrum. Results in our study will provide valuable information for the study of low expression level of SMN proteins and SMA pathogenesis based on transcript levels.

Impact of DYRK1A Expression on TNNT2 Splicing and Daunorubicin Toxicity in Human iPSC-Derived Cardiomyocytes

Cardiac troponin T (encoded by TNNT2) is involved in the contraction of cardiomyocytes during beating. The alternative splicing of TNNT2 results in four transcript variants with differential Ca2+ sensitivity. The splicing of TNNT2 involves phosphorylation of the splicing factor SRSF6 by DYRK1A. Altered TNNT2 splicing patterns have been identified in failing human hearts. There is a paucity of studies describing DYRK1A-SRSF6-TNNT2 interplays in human cardiomyocytes. Also, it is not known whether the sensitivity of cardiomyocytes to cardiotoxic anthracyclines is modified in the context of variable DYRK1A-TNNT2 expression. In this study, we investigated the impact of DYRK1A on the endogenous expression of TNNT2 splicing variants in iPSC-derived cardiomyocytes. We also examined whether DYRK1A expression modifies the sensitivity of cardiomyocytes to the cardiotoxic drug daunorubicin (DAU). DYRK1A over-expression increased the abundance of TNNT2 fetal variants by ~ 58% whereas the abundance of the adult cTnT3 variant decreased by ~ 27%. High DYRK1A expression increased the phosphorylation of SRSF6 by ~ 25–65%. DAU cytotoxicity was similar between cardiomyocytes with variable levels of DYRK1A expression. DYRK1A over-expression ameliorated the impact of DAU on beating frequency. This study lays the foundation to further investigate the contribution of variable DYRK1A-TNNT2 expression to Ca2+ handling and beating in human cardiomyocytes.

Reliability analysis of exonic-breakpoint fusions identified by DNA sequencing for predicting the efficacy of targeted therapy in non-small cell lung cancer

BackgroundDiverse genomic breakpoints of fusions that localize to intronic, exonic, or intergenic regions have been identified by DNA next-generation sequencing (NGS), but the role of exonic breakpoints remains elusive. We investigated whether exonic-breakpoint fusions could predict matched targeted therapy efficacy in non-small cell lung cancer (NSCLC).MethodsNSCLC samples were analyzed by DNA NGS, RNA NGS, immunohistochemistry (IHC), and fluorescence in situ hybridization.ResultsUsing DNA NGS, kinase fusions were identified in 685 of 7148 (9.6%) NSCLCs, with 74 harboring exonic-breakpoint fusions, mostly anaplastic lymphoma kinase (ALK) fusions. RNA NGS and IHC revealed that 11 of 55 (20%) exonic-breakpoint fusions generated no aberrant transcript/protein, possibly due to open reading frame disruption or different gene transcriptional orientations. Four cases of genomic-positive but RNA/protein-negative fusions were treated with matched targeted therapy, but progressive disease developed within 2 months. Nevertheless, 44 of 55 (80%) exonic-breakpoint fusions produced chimeric transcripts/proteins, possibly owing to various alternative splicing patterns, including exon skipping, alternative splice site selection, and intron retention. Most of these genomic- and RNA/protein-positive fusion cases showed a clinical response to matched targeted therapy. Particularly, there were no differences in objective response rate (P = 0.714) or median progression-free survival (P = 0.500) between intronic-breakpoint (n = 56) and exonic-breakpoint ALK fusion subtypes (n = 11) among ALK RNA/protein-validated patients who received first-line crizotinib.ConclusionsExonic-breakpoint fusions may generate in-frame fusion transcripts/proteins or not, and thus are unreliable for predicting the efficacy of targeted therapy, which highlights the necessity of implementing RNA or protein assays for functional validation in exonic-breakpoint fusion cases.

The alternative matrisome: Alternative splicing of ECM proteins in development, homeostasis and tumor progression

The extracellular matrix (ECM) is a fundamental component of the tissue of multicellular organisms that is comprised of an intricate network of multidomain proteins and associated factors, collectively known as the matrisome. The ECM creates a biophysical environment that regulates essential cellular processes such as adhesion, proliferation and migration and impacts cell fate decisions. The composition of the ECM varies across organs, developmental stages and diseases. Interestingly, most ECM genes generate transcripts that undergo extensive alternative splicing events, producing multiple protein variants from one gene thus enhancing ECM complexity and impacting matrix architecture. Extensive studies over the past several decades have linked ECM remodeling and expression of alternatively spliced ECM isoforms to cancer, and reprogramming of the alternative splicing patterns in cells has recently been proposed as a new hallmark of tumor progression. Indeed, tumor-associated alternative splicing occurs in both malignant and non-malignant cells of the tumor environment and growing evidence suggests that expression of specific ECM splicing variants could be a key step for stromal activation. In this review, we present a general overview of alternative splicing mechanisms, featuring examples of ECM components. The importance of ECM variant expression during essential physiological processes, such as tissue organization and embryonic development is discussed as well as the dysregulation of alternative splicing in cancer. The overall aim of this review is to address the complexity of the ECM by highlighting the importance of the yet-to-be-fully-characterized “alternative” matrisome in physiological and pathological states such as cancer.

Macula Densa Intracellular Alkalinization Activates NOS1β but Suppresses NOS1α during Tubuloglomerular Feedback

Increased NaCl at the macula densa, the same stimulus of the tubuloglomerular feedback (TGF), also induces intracellular alkalinization (pH: from 7.4 to 7.8) and enhances nitric oxide (NO) generation. Neuronal NO synthase (NOS1) is the primary isoform of NOS expressed in the macula densa and its activity is strongly pH‐dependent. Nevertheless, the impact of pH on the catalytic capacity of NOS1 remains controversial with inconsistent observations. A potential explanation for this discrepancy may be due to the presence of different splice variants of NOS1 (NOS1α and NOS1β). Therefore, we hypothesized that the macula densa intracellular alkalinization during TGF activates NOS1β but suppresses NOS1α, which modulates the NO production and thus the TGF responsiveness. To test this hypothesis, we measured the NOS activity of purified NOS1α and NOS1β proteins in various pH environments using the Griess method. The pH‐dependent NO production curves of NOS1α and NOS1β were different. The optimal pH was ~6.5 for NOS1α, but ~8.5 for NOS1β. The maximum activities of NOS1α and NOS1β were similar at their optimal pH of 6.5 and 8.5, respectively. Then, we measured and compared the renal cortical NOS1 expression, macula densa NO generation, TGF response in vitro and in vivo, renal vascular resistance (RVR), and mean arterial pressure (MAP) in the Sprague Dawley (SD) rats and the spontaneously hypertensive rats (SHRs). While the total NOS1 expression was not significantly different between SD rats and SHRs, the alternative splicing patterns were contradictory. The ratio of NOS1α/NOS1β was greater in SHRs than SD rats (2.94±0.48 vs 0.36±0.07; n=6, p&lt;0.01 vs SD), which was associated with the reduced NO production (21.6±4.5 vs 38.3±5.6 units/min; n=8, p&lt;0.01 vs SD), enhanced TGF in vitro (4.9±0.4 vs 3.5±0.5 µm; n=10, p&lt;0.05 vs SD) and in vivo (12.8±2.1 vs 9.1±1.7 mmHg; n=9, p&lt;0.01 vs SD), increased RVR (23.9±1.3 vs 17.8±1.5 mmHg.ml‐1.min‐1.g kidney wt‐1; n=6, p&lt;0.05 vs SD), and elevated MAP (127.3±6.6 vs 101.7±4.2 mmHg; n=6, p&lt;0.05 vs SD). We also measured and compared the NOS1 expression in the renal biopsy (cortex) tissues of Caucasians (CAs) and African Americans (AAs). There was no significant difference in the total NOS1 expression between CAs and AAs, whereas the ratio of NOS1α/NOS1β was greater in AAs than CAs (3.53±1.64 vs 0.43±0.31; n=10‐11, p&lt;0.01 vs CAs), which was associated with the higher serum creatinine level (1.25±0.08 vs 1.03±0.12 mg/dl; n=10‐11, p&lt;0.05 vs CAs). Lastly, we determined the effects of pH on the macula densa NO generation as well as the TGF responsiveness in SD rats and SHRs. When the pH of the tubular perfusate was switched from 7.4 to 8.0, the macula densa NO production increased by 29.5±6.9% (from 39.7±4.5 to 51.4±5.7 units/min) in SD rats, whereas it decreased by 26.2±4.3% (from 22.5±3.4 to 16.6±4.2 units/min) in SHRs (n=7, p&lt;0.05 vs 7.4); the TGF response in vitro decreased by 24.3±5.4% (from 3.7±0.4 to 2.8±0.6 µm) in SD rats, while it increased by 19.1±3.9% (from 4.7±0.4 to 5.6±0.5 µm) in SHRs (n=7, p&lt;0.05 vs 7.4). In conclusion, the present study demonstrates that the macula densa intracellular alkalinization during TGF activates NOS1β but suppresses NOS1α, which regulates the NO generation, the TGF response and potentially the renal hemodynamics.

Abstract 516: Splice Factor Polypyrimidine Tract-binding Protein 1 (PTBP1) Primes Endothelial Inflammation In Atherogenic Disturbed Flow Conditions

NFκB mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through upregulation of adhesion molecules Icam1 and Vcam. The endothelium is primed for cytokine activation of NFκB by exposure to low and disturbed blood flow (LDF). While priming leads to an exaggerated expression of Icam1 and Vcam following cytokine stimulation, the molecular underpinnings are not understood. In a model of LDF, platelets were required for the increased expression of several RNA-binding splice factors, including Polypyrimidine tract binding protein (Ptbp1). This was coordinated with changes in RNA splicing in the NFκB pathway in primed cells, leading us to examine splice factors as mediators of priming. Using Icam1 and Vcam induction by TNFα stimulation as a readout, we performed a CRISPR-Cas9 screen of these factors and identified a requirement for Ptbp1 in priming. Deletion of Ptbp1 had no obvious effect on cell proliferation or viability, but reversed LDF splicing patterns and inhibited NFκB nuclear translocation and transcriptional activation of nearly all downstream targets, including Icam1 and Vcam. In human coronary arteries, elevated PTBP1 correlates with expression of TNF pathway genes and plaque. In vivo , endothelial specific deletion of Ptbp1 reduced Icam1 expression and myeloid cell infiltration at regions of LDF in atherosclerotic mice and limited atherosclerosis. Together, our data show that Ptbp1, which is induced in a subset of the endothelium by innate immune cell recruitment at regions of LDF, is required for priming of the endothelium for subsequent NFκB activation, myeloid cell recruitment and atherosclerosis.

Expression complementation of gene presence/absence polymorphisms in hybrids contributes importantly to heterosis in sunflower

IntroductionNumerous crops have transitioned to hybrid seed production to increase yields and yield stability through heterosis. However, the molecular mechanisms underlying heterosis and its stability across environments are not yet fully understood. ObjectivesThis study aimed to (1) elucidate the genetic and molecular mechanisms underlying heterosis in sunflower, and (2) determine how heterosis is maintained under different environments. MethodsGenome-wide association (GWA) analyses were employed to assess the effects of presence/absence variants (PAVs) and stop codons on 16 traits phenotyped in the sunflower association mapping population at three locations. To link the GWA results to transcriptomic variation, we sequenced the transcriptomes of two sunflower cultivars and their F1 hybrid (INEDI) under both control and drought conditions and analyzed patterns of gene expression and alternative splicing. ResultsThousands of PAVs were found to affect phenotypic variation using a relaxed significance threshold, and at most such loci the “absence” allele reduced values of heterotic traits, but not those of non-heterotic traits. This pattern was strengthened for PAVs that showed expression complementation in INEDI. Stop codons were much rarer than PAVs and less likely to reduce heterotic trait values. Hybrid expression patterns were enriched for the GO category, sensitivity to stimulus, but all genotypes responded to drought similarily – by up-regulating water stress response pathways and down-regulating metabolic pathways. Changes in alternative splicing were strongly negatively correlated with expression variation, implying that alternative splicing in this system largely acts to reinforce expression responses. ConclusionOur results imply that complementation of expression of PAVs in hybrids is a major contributor to heterosis in sunflower, consistent with the dominance model of heterosis. This mechanism can account for yield stability across different environments. Moreover, given the much larger numbers of PAVs in plant vs. animal genomes, it also offers an explanation for the stronger heterotic responses seen in the former.

Splicing Modulation as a Promising Therapeutic Strategy for Lysosomal Storage Disorders: The Mucopolysaccharidoses Example

Over recent decades, the many functions of RNA have become more evident. This molecule has been recognized not only as a carrier of genetic information, but also as a specific and essential regulator of gene expression. Different RNA species have been identified and novel and exciting roles have been unveiled. Quite remarkably, this explosion of novel RNA classes has increased the possibility for new therapeutic strategies that tap into RNA biology. Most of these drugs use nucleic acid analogues and take advantage of complementary base pairing to either mimic or antagonize the function of RNAs. Among the most successful RNA-based drugs are those that act at the pre-mRNA level to modulate or correct aberrant splicing patterns, which are caused by specific pathogenic variants. This approach is particularly tempting for monogenic disorders with associated splicing defects, especially when they are highly frequent among affected patients worldwide or within a specific population. With more than 600 mutations that cause disease affecting the pre-mRNA splicing process, we consider lysosomal storage diseases (LSDs) to be perfect candidates for this type of approach. Here, we introduce the overall rationale and general mechanisms of splicing modulation approaches and highlight the currently marketed formulations, which have been developed for non-lysosomal genetic disorders. We also extensively reviewed the existing preclinical studies on the potential of this sort of therapeutic strategy to recover aberrant splicing and increase enzyme activity in our diseases of interest: the LSDs. Special attention was paid to a particular subgroup of LSDs: the mucopolysaccharidoses (MPSs). By doing this, we hoped to unveil the unique therapeutic potential of the use of this sort of approach for LSDs as a whole.