Splicing Patterns Research Articles

Alternatively Spliced Isoforms of MUC4 and ADAM12 as Biomarkers for Colorectal Cancer Metastasis.

There is a pertinent need to develop prognostic biomarkers for practicing predictive, preventive and personalized medicine (PPPM) in colorectal cancer metastasis. The analysis of isoform expression data governed by alternative splicing provides a high-resolution picture of mRNAs in a defined condition. This information would not be available by studying gene expression changes alone. Hence, we utilized our prior data from an exon microarray and found ADAM12 and MUC4 to be strong biomarker candidates based on their alternative splicing scores and pattern. In this study, we characterized their isoform expression in a cell line model of metastatic colorectal cancer (SW480 & SW620). These two genes were found to be good prognostic indicators in two cohorts from The Cancer Genome Atlas database. We studied their exon structure using sequence information in the NCBI and ENSEMBL genome databases to amplify and validate six isoforms each for the ADAM12 and MUC4 genes. The differential expression of these isoforms was observed between normal, primary and metastatic colorectal cancer cell lines. RNA-Seq analysis further proved the differential expression of the gene isoforms. The isoforms of MUC4 and ADAM12 were found to change expression levels in response to 5-Fluorouracil (5-FU) treatment in a dose-, time- and cell line-dependent manner. Furthermore, we successfully detected the protein isoforms of ADAM12 and MUC4 in cell lysates, reflecting the differential expression at the protein level. The change in the mRNA and protein expression of MUC4 and ADAM12 in primary and metastatic cells and in response to 5-FU qualifies them to be studied as potential biomarkers. This comprehensive study underscores the importance of studying alternatively spliced isoforms and their potential use as prognostic and/or predictive biomarkers in the PPPM approach towards cancer.

Identification of a Novel Non-Canonical Splice-Site Variant in ABCD1.

Cerebral adrenoleukodystrophy (CALD) is a fatal genetic disease characterized by rapid, devastating neurological decline, with a narrow curative treatment window in the early stage. Non-canonical splice-site (NCSS) variants can easily be missed during genomic DNA analyses, and only a few of them in ABCD1 have been explored. Here, we studied a Chinese patient with clinical features similar to those of early-stage CALD but with a negative molecular diagnosis and a sibling who had presumably died of CALD. Trio-based whole-exome sequencing (trio-WES) and RNA sequencing (RNA-Seq) revealed a novel hemizygote NCSS variant c.901-25_901-9 del in ABCD1 intron 1, resulting in a complex splicing pattern. The in vitro minigene assay revealed that the c.901-25_901-9 del construct contained two aberrant transcripts that caused skipping of exon 2 and a small 48-bp deletion on left of the same exon. We identified a novel NCSS variant, that extends the spectrum of the known ABCD1 variants, and demonstrated the pathogenicity of this gene variant. Our findings highlight the importance of combining RNA-Seq and WES techniques for prompt diagnosis of leukodystrophy with NCSS variants.

Identification and transfer of a new Pm21 haplotype with high genetic diversity and a special molecular resistance mechanism

Key messageA new functional Pm21 haplotype, Pm21(8#), was cloned from the new wheat-H. villosa translocation line T6VS(8#)·6DL, which confers the same strong resistance to powdery mildew through a different resistance mechanism.Broad-spectrum disease resistance genes are desirable in crop breeding for conferring stable, durable resistance in field production. Pm21(4#) is a gene introduced from wild Haynaldia villosa into wheat that confers broad-spectrum resistance to wheat powdery mildew and has been widely used in wheat production for approximately 30 years. The discovery and transfer of new functional haplotypes of Pm21 into wheat will expand its genetic diversity in production and avoid the breakdown of resistance conferred by a single gene on a large scale. Pm21(4#) previously found from T6VS(4#)·6AL has been cloned. In this study, a new wheat-H. villosa translocation, T6VS(8#)·6DL, was identified. A new functional Pm21 haplotype, designated Pm21(8#), was cloned and characterized. The genomic structures and the splicing patterns of Pm21(4#) and Pm21(8#) were different, and widespread sequence diversity was observed in the gene coding region and the promoter region. In the field, Pm21(8#) conferred resistance to Blumeria graminis f. sp. tritici (Bgt), similar to Pm21(4#), indicating that Pm21(8#) was also a resistance gene. However, Bgt development during the infection stage was obviously different between Pm21(4#)- and Pm21(8#)-containing materials under the microscopic observation. Pm21(4#) inhibited the formation of haustoria and the development of hyphae in the initial infection stage, while Pm21(8#) limited the growth of hyphae and inhibited the formation of conidiophores in the late infection stage. Therefore, Pm21(8#) is a new functional Pm21 haplotype that provides a new gene resource for wheat breeding.

A novel short transcript isoform of chicken IRF7 negatively regulates interferon-β production

Type I interferon (IFN-I) provides an important first line to protect avian species against pathogens invasion. IFN regulatory factor 7 (IRF7) has been identified as the most important regulator for both DNA and RNA virus-induced IFN-I production in chickens. Although four splicing variants of IRF7 have been identified in mammals, it is still unclear whether alternative splicing patterns and the function of IRF7 isoform(s) exist in chickens. In this study, we reported a novel short transcript isoform of chicken IRF7 (chIRF7), termed chIRF7-iso, which contained an intact N-terminal DNA-binding domain (DBD) and 14 amino acids different from chIRF7 in the C-terminal. Overexpression of chIRF7 in chicken leghorn male hepatocellular (LMH) cells activated the IFN-β promoter and significantly inhibited Newcastle disease virus (NDV) and fowl adenovirus serotype 4 (FAdV-4) replication. Conversely, overexpression of chIRF7-iso blocked the IFN-β promoter activity and was favorable for NDV and FAdV-4 replication in vitro. Collectively, our results confirm that a novel chIRF7 isoform-mediated negative regulates IFN-β production, which will contribute to understanding the role of chIRF7 in innate antiviral response in chicken.

In-depth analysis of alternative splicing landscape in multiple myeloma and potential role of dysregulated splicing factors

Splicing changes are common in cancer and are associated with dysregulated splicing factors. Here, we analyzed RNA-seq data from 323 newly diagnosed multiple myeloma (MM) patients and described the alternative splicing (AS) landscape. We observed a large number of splicing pattern changes in MM cells compared to normal plasma cells (NPC). The most common events were alterations of mutually exclusive exons and exon skipping. Most of these events were observed in the absence of overall changes in gene expression and often impacted the coding potential of the alternatively spliced genes. To understand the molecular mechanisms driving frequent aberrant AS, we investigated 115 splicing factors (SFs) and associated them with the AS events in MM. We observed that ~40% of SFs were dysregulated in MM cells compared to NPC and found a significant enrichment of SRSF1, SRSF9, and PCB1 binding motifs around AS events. Importantly, SRSF1 overexpression was linked with shorter survival in two independent MM datasets and was correlated with the number of AS events, impacting tumor cell proliferation. Together with the observation that MM cells are vulnerable to splicing inhibition, our results may lay the foundation for developing new therapeutic strategies for MM. We have developed a web portal that allows custom alternative splicing event queries by using gene symbols and visualizes AS events in MM and subgroups. Our portals can be accessed at http://rconnect.dfci.harvard.edu/mmsplicing/ and https://rconnect.dfci.harvard.edu/mmleafcutter/.

Clustered variants in the 5′ coding region of TRA2B cause a distinctive neurodevelopmental syndrome

PurposeTransformer2 proteins (Tra2α and Tra2β) control splicing patterns in human cells, and no human phenotypes have been associated with germline variants in these genes. The aim of this work was to associate germline variants in the TRA2B gene to a novel neurodevelopmental disorder. MethodsA total of 12 individuals from 11 unrelated families who harbored predicted loss-of-function monoallelic variants, mostly de novo, were recruited. RNA sequencing and western blot analyses of Tra2β-1 and Tra2β-3 isoforms from patient-derived cells were performed. Tra2β1-GFP, Tra2β3-GFP and CHEK1 exon 3 plasmids were transfected into HEK-293 cells. ResultsAll variants clustered in the 5′ part of TRA2B, upstream of an alternative translation start site responsible for the expression of the noncanonical Tra2β-3 isoform. All affected individuals presented intellectual disability and/or developmental delay, frequently associated with infantile spasms, microcephaly, brain anomalies, autism spectrum disorder, feeding difficulties, and short stature. Experimental studies showed that these variants decreased the expression of the canonical Tra2β-1 isoform, whereas they increased the expression of the Tra2β-3 isoform, which is shorter and lacks the N-terminal RS1 domain. Increased expression of Tra2β-3-GFP were shown to interfere with the incorporation of CHEK1 exon 3 into its mature transcript, normally incorporated by Tra2β-1. ConclusionPredicted loss-of-function variants clustered in the 5′ portion of TRA2B cause a new neurodevelopmental syndrome through an apparently dominant negative disease mechanism involving the use of an alternative translation start site and the overexpression of a shorter, repressive Tra2β protein.

Expression dynamics of periodic transcripts during cancer cell cycle progression and their correlation with anticancer drug sensitivity

BackgroundThe cell cycle is at the center of cellular activities and is orchestrated by complex regulatory mechanisms, among which transcriptional regulation is one of the most important components. Alternative splicing dramatically expands the regulatory network by producing transcript isoforms of genes to exquisitely control the cell cycle. However, the patterns of transcript isoform expression in the cell cycle are unclear. Therapies targeting cell cycle checkpoints are commonly used as anticancer therapies, but none of them have been designed or evaluated at the alternative splicing transcript level. The utility of these transcripts as markers of cell cycle-related drug sensitivity is still unknown, and studies on the expression patterns of cell cycle-targeting drug-related transcripts are also rare.MethodsTo explore alternative splicing patterns during cell cycle progression, we performed sequential transcriptomic assays following cell cycle synchronization in colon cancer HCT116 and breast cancer MDA-MB-231 cell lines, using flow cytometry and reference cell cycle transcripts to confirm the cell cycle phases of samples, and we developed a new algorithm to describe the periodic patterns of transcripts fluctuating during the cell cycle. Genomics of Drug Sensitivity in Cancer (GDSC) drug sensitivity datasets and Cancer Cell Line Encyclopedia (CCLE) transcript datasets were used to assess the correlation of genes and their transcript isoforms with drug sensitivity. We identified transcripts associated with typical drugs targeting cell cycle by determining correlation coefficients. Cytotoxicity assays were used to confirm the effect of ENST00000257904 against cyclin dependent kinase 4/6 (CDK4/6) inhibitors. Finally, alternative splicing transcripts associated with mitotic (M) phase arrest were analyzed using an RNA synthesis inhibition assay and transcriptome analysis.ResultsWe established high-resolution transcriptome datasets of synchronized cell cycle samples from colon cancer HCT116 and breast cancer MDA-MB-231 cells. The results of the cell cycle assessment showed that 43,326, 41,578 and 29,244 transcripts were found to be periodically expressed in HeLa, HCT116 and MDA-MB-231 cells, respectively, among which 1280 transcripts showed this expression pattern in all three cancer cell lines. Drug sensitivity assessments showed that a large number of these transcripts displayed a higher correlation with drug sensitivity than their corresponding genes. Cell cycle-related drug screening showed that the level of the CDK4 transcript ENST00000547281 was more significantly associated with the resistance of cells to CDK4/6 inhibitors than the level of the CDK4 reference transcript ENST00000257904. The transcriptional inhibition assay following M phase arrest further confirmed the M-phase-specific expression of the splicing transcripts. Combined with the cell cycle-related drug screening, the results also showed that a set of periodic transcripts, for example, ENST00000314392 (a dolichyl-phosphate mannosyltransferase polypeptide 2 isoform transcript), was more associated with drug sensitivity than the levels of their corresponding gene transcripts.ConclusionsIn summary, we identified a panel of cell cycle-related periodic transcripts and found that the levels of transcripts of drug target genes showed different values for predicting drug sensitivity, providing novel insights into alternative splicing-related drug development and evaluation.

A Patient with Corticobasal Syndrome and Progressive Non-Fluent Aphasia (CBS-PNFA), with Variants in ATP7B, SETX, SORL1, and FOXP1 Genes.

Our aim was to analyze the phenotypic-genetic correlations in a patient diagnosed with early onset corticobasal syndrome with progressive non-fluent aphasia (CBS-PNFA), characterized by predominant apraxia of speech, accompanied by prominent right-sided upper-limb limb-kinetic apraxia, alien limb phenomenon, synkinesis, myoclonus, mild cortical sensory loss, and right-sided hemispatial neglect. Whole-exome sequencing (WES) identified rare single heterozygous variants in ATP7B (c.3207C>A), SORL1 (c.352G>A), SETX (c.2385_2387delAAA), and FOXP1 (c.1762G>A) genes. The functional analysis revealed that the deletion in the SETX gene changed the splicing pattern, which was accompanied by lower SETX mRNA levels in the patient's fibroblasts, suggesting loss-of-function as the underlying mechanism. In addition, the patient's fibroblasts demonstrated altered mitochondrial architecture with decreased connectivity, compared to the control individuals. This is the first association of the CBS-PNFA phenotype with the most common ATP7B pathogenic variant p.H1069Q, previously linked to Wilson's disease, and early onset Parkinson's disease. This study expands the complex clinical spectrum related to variants in well-known disease genes, such as ATP7B, SORL1, SETX, and FOXP1, corroborating the hypothesis of oligogenic inheritance. To date, the FOXP1 gene has been linked exclusively to neurodevelopmental speech disorders, while our study highlights its possible relevance for adult-onset progressive apraxia of speech, which guarantees further study.

L-RAPiT: A Cloud-Based Computing Pipeline for the Analysis of Long-Read RNA Sequencing Data.

Long-read sequencing (LRS) has been adopted to meet a wide variety of research needs, ranging from the construction of novel transcriptome annotations to the rapid identification of emerging virus variants. Amongst other advantages, LRS preserves more information about RNA at the transcript level than conventional high-throughput sequencing, including far more accurate and quantitative records of splicing patterns. New studies with LRS datasets are being published at an exponential rate, generating a vast reservoir of information that can be leveraged to address a host of different research questions. However, mining such publicly available data in a tailored fashion is currently not easy, as the available software tools typically require familiarity with the command-line interface, which constitutes a significant obstacle to many researchers. Additionally, different research groups utilize different software packages to perform LRS analysis, which often prevents a direct comparison of published results across different studies. To address these challenges, we have developed the Long-Read Analysis Pipeline for Transcriptomics (L-RAPiT), a user-friendly, free pipeline requiring no dedicated computational resources or bioinformatics expertise. L-RAPiT can be implemented directly through Google Colaboratory, a system based on the open-source Jupyter notebook environment, and allows for the direct analysis of transcriptomic reads from Oxford Nanopore and PacBio LRS machines. This new pipeline enables the rapid, convenient, and standardized analysis of publicly available or newly generated LRS datasets.

Cell-based analysis reveals that sex-determining gene signals in Ostrinia are pivotally changed by male-killing Wolbachia.

Wolbachia, a maternally transmitted bacterium, shows male-killing, an adaptive phenotype for cytoplasmic elements, in various arthropod species during the early developmental stages. In lepidopteran insects, lethality of males is accounted for by improper dosage compensation in sex-linked genes owing to Wolbachia-induced feminization. Herein, we established Ostrinia scapulalis cell lines that retained sex specificity per the splicing pattern of the sex-determining gene doublesex (Osdsx). We found that Wolbachia transinfection in male cell lines enhanced the female-specific splice variant of Osdsx (OsdsxF ) while suppressing the male-specific variant (OsdsxM ), indicating that Wolbachia affects sex-determining gene signals even in vitro. Comparative transcriptome analysis isolated only two genes that behave differently upon Wolbachia infection. The two genes were respectively homologous to Masculinizer (BmMasc) and zinc finger-2 (Bmznf-2), male-specifically expressed sex-determining genes of the silkworm Bombyx mori that encode CCCH-type zinc finger motif proteins. By using cultured cells and organismal samples, OsMasc and Osznf-2 were found to be sex-determining genes of O. scapulalis that are subjected to sex-specific alternative splicing depending upon the chromosomal sex, developmental stage, and infection status. Overall, our findings expound the cellular autonomy in insect sex determination and the mechanism through which sex is manipulated by intracellular selfish microbes.

ANRIL overexpression globally induces expression and alternative splicing of genes involved in inflammation in HUVECs.

Long non‑coding (lnc)RNAs serve important cellular functions and certain lncRNAs have roles in different mechanisms of gene regulation. lncRNA‑antisense non‑coding RNA in the INK4 locus (ANRIL) affects cell inflammation; however, the potential genes underlying the inflammatory response regulated by ANRIL remain unclear. In the present study, the potential function of ANRIL in regulating gene expression and alternative splicing was assessed. ANRIL‑regulated human umbilical vein endothelial cell (HUVEC) transcriptome was obtained using high‑throughput RNA sequencing (RNA‑seq) to evaluate the potential role of ANRIL. Following plasmid transfection, gene expression profile and alternative splicing pattern of HUVECs overexpressing ANRIL were analyzed using RNA‑seq. ANRIL overexpression affected the transcription levels of genes associated with the inflammatory response, NF‑κB signaling pathway, typeI interferon‑mediated signal transduction pathway and innate immune response. ANRIL regulated the alternative splicing of hundreds of genes with functions such as gene expression, translation, DNA repair, RNA processing and participation in the NF‑κB signaling pathway. Many of these genes serve a key role in the inflammatory response. ANRIL‑regulated inflammatory response may be achieved by regulating alternate splicing and transcription. The present study broadened the understanding of ANRIL‑mediated gene regulation mechanisms and clarified the role of ANRIL in mediating inflammatory response mechanisms.

Chicken GLUT4 undergoes complex alternative splicing events and its expression in striated muscle changes dramatically during development

Glucose transporter protein 4 (GLUT4) plays an important role in regulating insulin-mediated glucose homeostasis in mammals. Until now, studies on GLUT4 have focused on mammals mostly, while chicken GLUT4 has been rarely investigated. In this study, chicken GLUT4 mRNA sequences were obtained by combining conventional amplification, 5'- and 3'- rapid amplification of cDNA ends technique (RACE), then bioinformatics analysis on its genomic structure, splicing pattern, subcellular localization prediction and homologous comparisons were carried out. In addition, the distribution of GLUT4 was detected by RT-qPCR in bird's liver and striated muscles (cardiac muscle, pectoralis and leg muscle) at different ages, including embryonic day 14 (E14), E19, 7-day-old (D7), D21 and D49 (n=3-4). Results showed that chicken GLUT4 gene produced at least 14 transcripts (GenBank accession No: OP491293-OP491306) through alternative splicing and polyadenylation, which predicted encoding 12 types of amino acid (AA) sequences (with length ranged from 65 AA to 519 AA). These proteins contain typical major facilitator superfamily domain of glucose transporters with length variations, sharing a common sequence of 59 AA, and were predicted to have distinct subcellular localization. The dominant transcript (named as T1) consists of 11 exons with an open reading frame being predicted encoding 519 AA. In addition, analyzing on the spatio-temporal expression of chicken GLUT4 showed it dominantly expressed in pectoralis, leg muscles and cardiac muscle, and the mRNA level of chicken GLUT4 dramatically fluctuated with birds' development in cardiac muscle, pectoralis and leg muscles, with the level at D21 significantly higher than that at E14, E19, and D49 (P < 0.05). These data indicated that chicken GLUT4 undergoes complex alternative splicing events, and GLUT4 expression level in striated muscle was subjected to dynamic regulation with birds' development. Results indicate these isoforms may play overlapping and distinct roles in chicken.

CHD8 suppression impacts on histone H3 lysine 36 trimethylation and alters RNA alternative splicing.

Disruptive mutations in the chromodomain helicase DNA-binding protein 8 gene (CHD8) have been recurrently associated with autism spectrum disorders (ASDs). Here we investigated how chromatin reacts to CHD8 suppression by analyzing a panel of histone modifications in induced pluripotent stem cell-derived neural progenitors. CHD8 suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affects highly expressed, CHD8-bound genes and correlates with altered alternative splicing patterns of 462 genes implicated in 'regulation of RNA splicing' and 'mRNA catabolic process'. Mass spectrometry analysis uncovered a novel interaction between CHD8 and the splicing regulator heterogeneous nuclear ribonucleoprotein L (hnRNPL), providing the first mechanistic insights to explain the CHD8 suppression-derived splicing phenotype, partly implicating SETD2, a H3K36me3 methyltransferase. In summary, our results point toward broad molecular consequences of CHD8 suppression, entailing altered histone deposition/maintenance and RNA processing regulation as important regulatory processes in ASD.

Transcriptome analyses in infertile men reveal germ cell-specific expression and splicing patterns.

The process of spermatogenesis-when germ cells differentiate into sperm-is tightly regulated, and misregulation in gene expression is likely to be involved in the physiopathology of male infertility. The testis is one of the most transcriptionally rich tissues; nevertheless, the specific gene expression changes occurring during spermatogenesis are not fully understood. To better understand gene expression during spermatogenesis, we generated germ cell-specific whole transcriptome profiles by systematically comparing testicular transcriptomes from tissues in which spermatogenesis is arrested at successive steps of germ cell differentiation. In these comparisons, we found thousands of differentially expressed genes between successive germ cell types of infertility patients. We demonstrate our analyses' potential to identify novel highly germ cell-specific markers (TSPY4 and LUZP4 for spermatogonia; HMGB4 for round spermatids) and identified putatively misregulated genes in male infertility (RWDD2A, CCDC183, CNNM1, SERF1B). Apart from these, we found thousands of genes showing germ cell-specific isoforms (including SOX15, SPATA4, SYCP3, MKI67). Our approach and dataset can help elucidate genetic and transcriptional causes for male infertility.

M6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site.

Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5' splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the ACAGA box is modified by N6 methylation, but the role of this m6A modification is poorly understood. Here, we show that m6A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIONA1, is required for Arabidopsis U6 snRNA m6A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5' splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m6A influences 3' splice site usage. We generalise these findings to reveal two major classes of 5' splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA ACAGA box. We conclude that U6 snRNA m6A modification contributes to the selection of degenerate 5' splice sites crucial to alternative splicing.

Minigene Assay as an Effective Molecular Diagnostic Strategy in Determining the Pathogenicity of Noncanonical Splice-Site Variants in FLCN

Primary spontaneous pneumothorax (PSP) or pulmonary cyst is one of the manifestations of Birt-Hogg-Dubé syndrome, which is caused by pathogenic variants in FLCN gene. Genetic testing in patients with PSP identifies a certain number of missense or intronic variants. These variants are usually considered as variants of uncertain significance, whose functional interpretations pose a challenge in clinical genetics. To improve recognition of pathogenic splice-altering variants in FLCN gene, computational tools are used to prioritize potential splice-altering variants and then a hybrid minigene assay is performed to verify the RNA splicing pattern. Herein, variants in FLCN exon 11 and its flanking sequence are focused. Eight variants detected in 11 patients with PSP are evaluated, and six variants are prioritized by in silico tools as potential splice-altering variants of uncertain significance. Four variants (c.1177-5_1177-3delCTC, c.1292_1300+4del, c.1300+4C>T, and c.1300+5G>A) are demonstrated by minigene assay to alter RNA splicing of FLCN, and the last three of them are novel. RT-PCR of patient-derived RNA gives consistent results. Genotype-phenotype correlation analysis in patients with PSP with these variants demonstrates good concordance. Our results underline the importance of RNA analysis, which could provide molecular evidence for pathogenicity of a variant, and provide essential information for the clinical interpretation of variants. Combining the clinical information, a definitive diagnosis could be made.

Exploring Splicing Factor Aberrations As a Therapeutic Target in Mature B-Cell Malignancies

Objectives Cancers originating from mature B cells at various stages of terminal differentiation represent a diverse set of cancers. There are numerous pathological subtypes, each comprising genetically and clinically heterogeneous malignancies with distinct biology and clinical outcomes. We have previously identified the recurrence of mutations in splicing factor HNRNPH1 in mantle cell lymphoma (MCL), which were associated with inferior outcomes. Based on growing evidence for the role of splicing factors in mature B cell neoplasms, we sought to extend our characterization of splicing factor aberrations across additional B cell malignancies. We hypothesize that regulation of alternative splicing is essential for B cell differentiation and maturation, and aberrations of splicing factors in B cell malignancies are related to these normal patterns of alternative splicing. Ultimately, we are pursuing alternative splicing as a potential novel therapeutic vulnerability common among mature B cell neoplasms. Methods We leveraged whole genome, exome, and RNA sequencing data from 2999 tumors assembled from controlled-access repositories and ongoing projects at BC Cancer as part of our Genomic Analysis of Mature B-cell Lymphomas (GAMBL) project. RNA-seq data was aligned with STAR and splicing events were quantified with RMATS. Novel splicing events were quantified following de novo transcriptome assembly with StringTie. Simple somatic mutations were detected using a workflow comprised of four algorithms to identify high-confidence variants. We used additional RNA-seq data from distinct B-cell maturation states (naïve, centroblast, centrocyte, memory, bone marrow plasma, and tonsillar plasma cells) to identify annotated and novel splicing events which are differentially regulated throughout B cell maturation. Annotated and novel events were subsequently used to query alternative splicing in a subset of tumor samples using a combination of Illumina-based and Oxford Nanopore sequencing. Results More than 30% of patient samples harboured somatic mutations in at least one of 67 queried splicing factors. HNRNPU, PCBP1, SF3B1, DAZAP1, and HNRNPH1 were most commonly mutated, 13% of cases (Figure 1A). Although only reported in MCL thus far, we noted an abundance of HNRNPH1 mutations in DLBCL showing the same pattern of non-coding mutations, most commonly in the activated B cell subgroup. In non-malignant samples, we identified 129 known and 267 novel splicing events which display differential regulation during B cell maturation and warrant further investigation within subtypes of B cell malignancies (Figure 1B). Based on this observation, we hypothesize that the RNA isoform repertoire may vary across B cell differentiation states and its perturbation may contribute to oncogenesis in a context-dependent manner. Furthermore, the importance of alternative splicing during B cell maturation implies that perturbation of splicing in B cell malignancies could play a role in pathogenesis. We previously demonstrated that non-coding mutations in HNRNPH1 contribute to overexpression of the hnRNP H protein and are associated with poor patient outcomes in MCL. Based on the predominance of these mutations in DLBCL, we sought to evaluate hnRNP H overexpression as a potential therapeutic target. We identified a putative exonic splicing enhancer (ESE) with minimal secondary structure in the essential exon of HNRNPH1 using ESEfinder and designed locked nucleic acid antisense oligos (ASOs) complementary to the ESE. Using ddPCR, we showed that the ESE ASO can effectively decrease the proportion of the productive HNRNPH1 splice variant at nanomolar concentrations in HEK cells; however, a splice site ASO does not display the same efficacy. Conclusions We have identified a growing number of splicing factors in mature B cell malignancies as recurrently mutated, indicating that deregulation of the RNA isoform repertoire in B cells can contribute to oncogenesis. Characterizing the landscape of alternative splicing in normal and malignant B cells will allow us to identify opportunities to exploit this reliance in the development of novel therapeutic strategies. Our data highlight antisense oligonucleotides as a promising therapeutic avenue for targeting splicing aberrations caused by common mutations in B cell malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multi-Omics Data Integration of Genomics and RNA-Splicing in Myeloid Neoplasia

The high occurrence of splicing factor mutations (SFMT) has suggested that RNA splicing dysfunction plays a key pathogenic role in myeloid neoplasia (MN). Nevertheless, transcriptome-wide analysis suggests that MDS is linked with a broad spectrum of splicing lesions also occurring in cases without SFMT. Due to such a vast complexity, traditional approaches for identification of splicing patterns taking into account features in unimodal fashion may not be adequate to dissect the pathophysiologic features in detail, being instead amenable to more complex machine learning (ML) strategies. Indeed, we hypothesize that splicing dysfunction in MDS may be more wide spread and not restricted to SFMT cases consistent with view that MDS is a global spliceopathy. We explore the geno-transcriptomic pattern of MN by merging multi-omics data to build an ML multimodal tool. An integrative analysis of omics data (RNA and WGS of 1258 whole bone marrows of MN patients) along with ML methods coupling variational autoencoders and canonical correlation analysis was applied to identify covarying features including genomic lesions (mutations, deletions, CNV), exon-inclusion ratio (percent spliced in, PSI) and gene expression (GE). CIBERSORT analysis was used to quantify the composition of clonal cells in bulk samples. Patterns of similarities and differences across patients were compared to 63 age-matched healthy controls and an internal validation of the model was also performed (Fig1). Combination of all features followed by unsupervised clustering identified 15 clusters (C-0 to C-14) resulting in discrete differences in MN subtypes distribution. A distinct definition of the clusters was also seen based on molecular signatures. In our cohort, 54% of patients carried SFMT, further stressing that the mere mutational status of a handful of SFs may not be sufficient to assess the real impact of spliceosome changes. Some clusters were dominated by a high frequency of specific mutations, suggesting a strong functional consequence imprinted by the mutational event. MDS patients were enriched in three clusters with SF3B1MT mainly included in C-1, C-7, C-12. C-1 and C-12 had two distinctive PSI patterns showing decreased splicing efficiency of various genes including DDX20 and KDM3B. SRSF2MT followed similar patterns of distinct PSI in the clusters they populated most (C-8, C-14). C-2 and C-13 contained mainly TET2MT and ASXL1MT. AML patients composed six clusters with two of them (C-9, C-10) consisting of >95% of AML diagnoses. Carriers of DNMT3A, TET2 and FLT3 hits were equally represented in C-0, C-5, C-6 while DNMT3AMT and FLT3MT were equal in C-10. PSI score differences based on ΔPSI (difference in PSI between pairwise clusters) showed high correlation for EZH2 in SRSF2MT, LUC7L2MT and PRPF8MT. PSI similarities were found in subclusters with 1) SF3B1MT, DNMT3AMT, and TET2MT; 2) SF3B1MT with other SFs (PRPF8, DDX41, LUC7L2); 3) TP53MT and DDX41MT possibly suggesting a process of phenocopying and common downstream targets. Using Limma for differential GE analysis we also identified mutational groups clustering together based on GE profiles (group 1: SRSF2, RIT1, JAK2, PTPN11, TET2; group 2: LUC7L2, SF3B1, BCORL1, CSF3R). Using Ward's criterion based hierarchical clustering, we created a map of defining splicing features per cluster. Combination of our multimodal ML with survival outcome in a continuous fashion by means of survival forests and permutation of latent features identified components with highest associations with OS. Focusing on MDS as the most paradigmatic group inherently characterized by a high heterogeneity, we stratified patients based on low and high risk IPSS-R and found that our ML model was effective in discriminating high risk IPSS-R clusters. Finally, we also found a further refinement in low risk IPSS-R cases, with C-12 (SF3B1MT cluster) showing significant separation from other two close clusters (C-7, C-11). This is the first study unifying different levels of multi-omics information to examine the genetic picture of MN as a global spliceopathy. The convergence of molecular and transcriptomic pathways might unveil subgroups of seemingly diverse patients sharing common pathogenetic routes and the identification of these pathways might guide rational therapies. To facilitate the use of such integrated approach intertwining PSI, mutations and GE, we created an interactive MutExPSIplot.1 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of WNT4 alternative splicing patterns and effects on proliferation of granulosa cells in goat

Increasing ovulation numbers is one of the most important ways to promote reproduction in mammals, and follicular granulosa cells (GCs) provide the necessary nutrients and microenvironment for oocytes to ovulate. WNT4 has been shown to be a key factor in regulating the proliferation of GCs in mammalian ovarian tissues. Our previous transcriptome sequencing (RNA-seq) results have identified two alternatively spliced products of WNT4;however, little is known about the splicing mechanism and its effect on GC proliferation. In this study, two alternatively spliced products of WNT4, designated WNT4-α and WNT4-β, were identified by cloning and analyzed for their function by bioinformatics. The RT–qPCR and Western blot results showed that the expression of WNT4-α was significantly higher than that of WNT4-β in the ovary tissues and GCs of Yunshang black goats. We therefore hypothesized that WNT4-α was the main isoform affecting the proliferation of goat GCs. Subsequently, goat GC proliferation assays showed that overexpression of WNT4-α significantly promoted GC proliferation, and the opposite was true after WNT4-α inhibition. The expression of marker genes of the Wnt signaling pathway was also examined and WNT4-α was found to affect the proliferation and hormone secretion of goat GCs by regulating the Wnt signaling pathway. In addition, a series of splicing factors were involved in in the alternative splicing; in this study, SRSF6 was found to be involved as a splicing factor in the generation of WNT4 alternative splicing. In summary, WNT4 alternative splicing was mediated by the splicing factor SRSF6, and WNT4-α alternative splicing played an important role in follicle development and had a significant effect on the proliferation of goat GCs. The results of this study provide a theoretical foundation for further understanding the molecular regulatory mechanisms of the WNT4 in follicle development in goats.

Decreased Embryonic Splicing Repressor Expression Enhances Splicing Modulator Sensitivity in Pediatric Acute Myeloid Leukemia Stem Cells

Adults with myeloid malignancies frequently harbor mutations in splicing regulatory genes, such as SRSF2, U2AF1, and SF3B1. In pediatric AML (pAML), splicing factor mutations are relatively infrequent in bulk populations of cells. However, by performing whole exome sequencing, single cell proteogenomics (Tapestri®) as well as whole transcriptome RNA sequencing (RNA-seq) analyses of FACS-purified hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), we discovered an increased frequency of exon skipping, typical of embryonic splicing patterns, in pAML HSCs and HPCs. By employing single cell proteogenomics (Tapestri®) analysis, we discovered SF3B1 splicing factor mutations at the single cell in pAML CD34+ cells that were not readily detectable by whole exome sequencing in bulk populations thereby suggesting that clonal expansion of minor clones may fuel leukemic stem cell (LSC) generation. Further RNA-seq analysis demonstrated that both pAML HSCs and HPCs harbored decreased expression of a repressor of embryonic splicing, RNA-binding Fox protein 2, RBFOX2(also known as RBM9). By employing, rMATS, an algorithm that detects splicing events, we discovered differential splicing of genes involved in cell cycle regulation, DNA repair and chromatin organization in pAML more than in non-leukemic (NL) samples. In addition, differentially spliced RBFOX2 target genes were found to be involved in mRNA processing. In pAML HSCs, we observed differential splicing of THOC7, which regulates mRNA 3' end processing while in pAML HPCs, we uncovered a cluster of splicing regulators, such as SRSF7, SRSF11 and U2AF2. Thus, pAML is typified by widespread splicing deregulation in the setting of RBFOX2 downregulation. Previous reports suggest that splicing deregulation represents a therapeutic vulnerability in a broad array of malignancies. Thus, we examined the response of pAML cell lines in vitro and in lentiviral splicing reporter humanized AML mouse models to Rebecsinib (17S-FD-895; Crews et al, Cell Stem Cell, 2016), a small molecule splicing modulator that is currently undergoing IND enabling studies. In these studies, Rebecsinib treatment reduced pAML cell survival at doses that altered splicing reporter activity and spared normal hematopoietic stem and progenitor cells (HSPCs). Notably, shRNA knockdown of RBFOX2 in cord blood CD34+ HSPCs phenocopied aspects of pAML in that it significantly increased clonogenic capacity and sensitivity to Rebecsinib in replating assays. In summary, decreased RBFOX2 expression and reversion to an embryonic splicing program typified by differential exon usage in pAML HSPCs was associated with increased sensitivity to selective splicing modulation and may represent a new therapeutic paradigm for pediatric patients with relapsed or refractory AML.