Splicing Regulatory Genes Research Articles

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.

Abstract 2197: Dysregulated alternative splicing landscape identifies intron retention as a hallmark and spliceosome as a therapeutic vulnerability in aggressive prostate cancer

Abstract Dysregulation of mRNA alternative splicing (AS) has been implicated in development and progression of hematological malignancies. How the global AS dysregulation contributes to the development and progression of solid tumors is under-studied and remains generally unclear. Here we describe the first comprehensive AS landscape in the spectrum of human prostate cancer (PCa) development, progression and therapy resistance. We find that the severity of splicing dysregulation correlates with disease progression and establish intron retention (IR) as a hallmark of PCa stemness and aggressiveness. Systematic interrogation of 274 splicing-regulatory genes (SRGs) uncovers prevalent SRG mutations associated with, mainly, copy number variations leading to mis-expression of ~68% of SRGs during PCa evolution. Consequently, we identify many SRGs as prognostic markers associated with splicing disruption and patient outcome. Interestingly, androgen receptor (AR) controls a splicing program distinct from its transcriptional regulation. The spliceosome modulator, E7107, reverses cancer aggressiveness and inhibits the growth of castration-resistant PCa (CRPC) xenograft models and of the autochthonous Hi-Myc tumors. Altogether, our studies establish aberrant AS landscape caused by dysregulated SRGs as a hallmark of PCa aggressiveness and a novel therapeutic vulnerability for CRPC. Citation Format: Dingxiao Zhang, qiang Hu, Xiaozhuo Liu, Yibing Ji, Hsueh-Ping Chao, Amanda Tracz, Jason Kirk, Silvia Buonamici, Ping Zhu, Jianmin Wang, song liu, Dean Tang. Dysregulated alternative splicing landscape identifies intron retention as a hallmark and spliceosome as a therapeutic vulnerability in aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2197.

PSV-27 Program Chair Poster Pick: Expression QTL mapping for meat quality in beef cattle

Abstract Expression QTL mapping provides information about genetic variant with modulatory effects on gene expression which are useful for understanding the genetic architecture of complex phenotypes. This mapping allows for uncovering of genomic regions associated with transcription regulation of genes which can be related to phenotypic variation when they colocalize with QTLs (cis and trans effects), providing a molecular basis for the phenotype-genotype association. The objectives of the present research were to perform eQTL mapping for meat quality traits in longissimus dorsi muscle and to uncover genes whose expression is influenced by local or distant genetic variation. A total of 120 steers from the University of Florida Beef Unit multibreed Angus-Brahman herd born between 2013 and 2014 were used in this study. The first three principal components from a principal component analysis for all meat quality phenotypes were used to construct a meat quality index. Eighty animals were selected based on extreme meat quality index for mRNA sequencing and 100 bp paired-end reads were mapped against to the Btau_4.6.1 reference genome. eQTL mapping was performed using 112,042 SNPs and 8,588 genes. A cis QTL was defined as an SNP located no more than 1 Mb upstream of the transcription start site or downstream of the transcription end site of an annotated gene. Polymorphisms associated with expression of at least 20 genes in the case of eQTL were considered hot spots. The harboring or adjacent gene was defined as master regulators. Multiple cis eQTLs and sQTLs effects were identified and genes such as LSM2, SOAT1, TTN and TEK are a few examples of potential expression and splicing regulatory genes. A total of 27 expression and 13 splicing master regulator genes were uncovered, mainly cytoskeletal or membrane-associated proteins, transcription factors and DNA methylases.

Intron retention is a hallmark and spliceosome represents a therapeutic vulnerability in aggressive prostate cancer

The role of dysregulation of mRNA alternative splicing (AS) in the development and progression of solid tumors remains to be defined. Here we describe the first comprehensive AS landscape in the spectrum of human prostate cancer (PCa) evolution. We find that the severity of splicing dysregulation correlates with disease progression and establish intron retention as a hallmark of PCa stemness and aggressiveness. Systematic interrogation of 274 splicing-regulatory genes (SRGs) uncovers prevalent genomic copy number variations (CNVs), leading to mis-expression of ~68% of SRGs during PCa development and progression. Consequently, many SRGs are prognostic. Surprisingly, androgen receptor controls a splicing program distinct from its transcriptional regulation. The spliceosome modulator, E7107, reverses cancer aggressiveness and inhibits castration-resistant PCa (CRPC) in xenograft and autochthonous PCa models. Altogether, our studies establish aberrant AS landscape caused by dysregulated SRGs as a hallmark of PCa aggressiveness and the spliceosome as a therapeutic vulnerability for CRPC.

Abstract 2724: A combination of short-read and long-read RNA sequencing reveals NOVA1’s role in telomere biology

Abstract Dysregulated alternative splicing underlies many aspects of cancer cell biology including tumor cell telomere maintenance. We recently described a role for the neuronal splicing factor, NOVA1, in non-small cell lung cancer in the regulation of full-length hTERT splicing, telomerase activity, maintenance of telomere length, and general tumor growth characteristics. We wanted to further define the role of NOVA1 in telomere biology. Cells that maintain telomeres with different mechanisms were studied to be able to decipher telomerase specific versus alternative lengthening of telomeres (ALT) specific perturbations by NOVA1. Both short-read (Illumina) and long-read (Pacific Biosciences) sequencing of control and NOVA1 siRNA knockdown cells in H920 non-small cell lung cancer cells (telomerase positive) and U2OS cells (ALT positive cells) were performed to detail the transcriptomes of these cells. A combination of rMATS analysis of the short-read sequencing, SQANTI analysis of the long-read sequencing, and pathway analysis was performed to identify genes related to telomere biology and alternative splicing related to telomerase. Skipped exon events were the primary focus of this study as this is NOVA1’s main role in alternative splicing. rMATS analysis identified 4,530 significant events mapping to 2,950 genes in H920 cells and 2,534 significant events in 1,863 genes in U2OS cells. SQANTI analyzed 15,443 full-length transcripts (7,530 genes) in control and 14,463 transcripts (7,240 genes) in NOVA1 siRNA treated samples of the H920 cells and 12,133 transcripts (6,618 genes) in control and 10,126 transcripts (5,959 genes) in NOVA1 siRNA treated U2OS cells. There were 2,762 and 2,470 novel transcripts in control and NOVA1 siRNA treated H920 cells, respectively. In U2OS cells there were 2,178 novel transcripts in control and 1,688 novel transcripts in NOVA1 siRNA treated cells. The pathway analysis of rMATS cross-referenced with SQANTI revealed five top candidates that were specifically altered in NOVA1 knockdown cells and related telomere length maintenance by telomerase (POLD1, SUN2, ERCC1, SRSF2, and PCBP2). Interestingly, SRSF2 and PCBP2 were identified in our recent hTERT minigene loss of function screen for splicing regulatory genes of hTERT and have binding sites in hTERT near NOVA1s motif in intron 8. Long-read sequencing determined the full-length nature of these splicing events pointed out by the rMATS analysis. These data indicate that telomerase positive cancer cells with dysregulated expression of NOVA1 utilize different isoforms compared to ALT cells with NOVA1 to maintain telomeres. These new observations reveal that alternative splicing regulated in part by NOVA1 plays a larger and unexpected role in telomere biology. Manipulation of alternative splicing to shorten/deprotect telomeres should be pursued as a means to inhibit survival and growth of cancer cells. Citation Format: Andrew T. Ludlow, Mohammed E. Sayed, Aaron L. Slusher, Mark Ribick, Anisha Pancholi, Brian Sereni, Yu Qui, Elizabeth Tseng, Meredith Ashby, David C. Corney. A combination of short-read and long-read RNA sequencing reveals NOVA1’s role in telomere biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2724.

Rapid and Dynamic Alternative Splicing Impacts the Arabidopsis Cold Response Transcriptome.

Plants have adapted to tolerate and survive constantly changing environmental conditions by reprogramming gene expression The dynamics of the contribution of alternative splicing (AS) to stress responses are unknown. RNA-sequencing of a time-series of Arabidopsis thaliana plants exposed to cold determines the timing of significant AS changes. This shows a massive and rapid AS response with coincident waves of transcriptional and AS activity occurring in the first few hours of temperature reduction and further AS throughout the cold. In particular, hundreds of genes showed changes in expression due to rapidly occurring AS in response to cold ("early AS" genes); these included numerous novel cold-responsive transcription factors and splicing factors/RNA binding proteins regulated only by AS. The speed and sensitivity to small temperature changes of AS of some of these genes suggest that fine-tuning expression via AS pathways contributes to the thermo-plasticity of expression. Four early AS splicing regulatory genes have been shown previously to be required for freezing tolerance and acclimation; we provide evidence of a fifth gene, U2B"-LIKE Such factors likely drive cascades of AS of downstream genes that, alongside transcription, modulate transcriptome reprogramming that together govern the physiological and survival responses of plants to low temperature.

The Characterization of GSDMB Splicing and Backsplicing Profiles Identifies Novel Isoforms and a Circular RNA That Are Dysregulated in Multiple Sclerosis.

Abnormalities in alternative splicing (AS) are emerging as recurrent features in autoimmune diseases (AIDs). In particular, a growing body of evidence suggests the existence of a pathogenic association between a generalized defect in splicing regulatory genes and multiple sclerosis (MS). Moreover, several studies have documented an unbalance in alternatively-spliced isoforms in MS patients possibly contributing to the disease etiology. In this work, using a combination of PCR-based techniques (reverse-transcription (RT)-PCR, fluorescent-competitive, real-time, and digital RT-PCR assays), we investigated the alternatively-spliced gene encoding Gasdermin B, GSDMB, which was repeatedly associated with susceptibility to asthma and AIDs. The in-depth characterization of GSDMB AS and backsplicing profiles led us to the identification of an exonic circular RNA (ecircRNA) as well as of novel GSDMB in-frame and out-of-frame isoforms. The non-productive splicing variants were shown to be downregulated by the nonsense-mediated mRNA decay (NMD) in human cell lines, suggesting that GSDMB levels are significantly modulated by NMD. Importantly, both AS isoforms and the identified ecircRNA were significantly dysregulated in peripheral blood mononuclear cells of relapsing-remitting MS patients compared to controls, further supporting the notion that aberrant RNA metabolism is a characteristic feature of the disease.