Identified Splicing Factor Research Articles

Alternative Splicing Is a Major Factor Shaping Transcriptome Diversity in Mild and Severe Chronic Obstructive Pulmonary Disease.

The role of alternative splicing in chronic obstructive pulmonary disease (COPD) is still largely unknown. We aimed to investigate the differences in alternatively splicing events between patients with mild-to-moderate and severe COPD compared with non-COPD control subjects and to identify splicing factors associated with aberrant alternative splicing in COPD. For this purpose, we performed genome-wide RNA-sequencing analysis of bronchial brushings from 23 patients with mild-to-moderate COPD, 121 with severe COPD, and 23 non-COPD control subjects. We found a significant difference in the frequency of alternative splicing events in patients with mild-to-moderate and severe COPD compared with non-COPD control subjects. There were from two to eight times (depending on event type) more differential alternative splicing events in the severe than in the mild-to-moderate stage. The severe COPD samples showed less intron retention and more exon skipping. It is interesting that the transcript levels of the top 10 differentially expressed splicing factors were significantly correlated with the percentage of many alternatively spliced transcripts in severe COPD. The aberrant alternative splicing in severe COPD was predicted to increase the overall protein-coding capacity of gene products. In conclusion, we observed large and significant differences in alternative splicing between bronchial samples of patients with COPD and control subjects, with more events observed in severe than in mild-to-moderate COPD. The changes in the expression of several splicing factors correlated with prevalence of alternative splicing in severe COPD. Alternative splicing can indirectly impact gene expression by changing the relative abundance of protein-coding isoforms potentially influencing pathophysiological changes. The results provide a better understanding of COPD-related alternative splicing changes.

Interplays between cis- and trans-Acting Factors for Alternative Splicing in Response to Environmental Changes during Biological Invasions of Ascidians.

Alternative splicing (AS), a pivotal biological process contributing to phenotypic plasticity, creates a bridge linking genotypes with phenotypes. Despite its importance, the AS mechanisms underlying environmental response and adaptation have not been well studied, and more importantly, the cis- and trans-acting factors influencing AS variation remain unclear. Using the model invasive congeneric ascidians, Ciona robusta, and Ciona savignyi, we compared their AS responses to environmental changes and explored the potential determinants. Our findings unveiled swift and dynamic AS changes in response to environmental challenges, and differentially alternative spliced genes (DASGs) were functionally enriched in transmembrane transport processes. Interestingly, both the prevalence and level of AS in C. robusta were lower than those observed in C. savignyi. Furthermore, these two indices were higher under temperature stresses compared to salinity stresses in C. savignyi. All the observed patterns underscore the species-specific and environmental context-dependent AS responses to environmental challenges. The dissimilarities in genomic structure and exon/intron size distributions between these two species likely contributed to the observed AS variation. Moreover, we identified a total of 11 and 9 serine/arginine-rich splicing factors (SRSFs) with conserved domains and gene structures in the genomes of C. robusta and C. savignyi, respectively. Intriguingly, our analysis revealed that all detected SRSFs did not exhibit prevalent AS regulations. Instead, we observed AS control over a set of genes related to splicing factors and spliceosome components. Altogether, our results elucidate species-specific and environmental challenge-dependent AS response patterns in closely related invasive ascidians. The identified splicing factors and spliceosome components under AS control offer promising candidates for further investigations into AS-mediated rapid responses to environmental challenges complementary to SRSFs.

NF-YAl drives EMT in Claudinlow tumours

NF-Y is a trimeric transcription factor whose binding site -the CCAAT box- is enriched in cancer-promoting genes. The regulatory subunit, the sequence-specificity conferring NF-YA, comes in two major isoforms, NF-YA long (NF-YAl) and short (NF-YAs). Extensive expression analysis in epithelial cancers determined two features: widespread overexpression and changes in NF-YAl/NF-YAs ratios (NF-YAr) in tumours with EMT features. We performed wet and in silico experiments to explore the role of the isoforms in breast -BRCA- and gastric -STAD- cancers. We generated clones of two Claudinlow BRCA lines SUM159PT and BT549 ablated of exon-3, thus shifting expression from NF-YAl to NF-YAs. Edited clones show normal growth but reduced migratory capacities in vitro and ability to metastatize in vivo. Using TCGA, including upon deconvolution of scRNA-seq data, we formalize the clinical importance of high NF-YAr, associated to EMT genes and cell populations. We derive a novel, prognostic 158 genes signature common to BRCA and STAD Claudinlow tumours. Finally, we identify splicing factors associated to high NF-YAr, validating RBFOX2 as promoting expression of NF-YAl. These data bring three relevant results: (i) the definition and clinical implications of NF-YAr and the 158 genes signature in Claudinlow tumours; (ii) genetic evidence of 28 amino acids in NF-YAl with EMT-promoting capacity; (iii) the definition of selected splicing factors associated to NF-YA isoforms.

Alternative Splicing of lncRNAs From SNHG Family Alters snoRNA Expression and Induces Chemoresistance in Hepatoblastoma

Hepatoblastoma (HB) is a common pediatric malignant liver tumor that is characterized by a low level of genetic mutations. Alternative splicing (AS) has been shown to be closely associated with cancer progression, especially in tumors with a low mutational burden. However, the role of AS in HB remains unknown. Transcriptome sequencing was performed on 5 pairs of HB tissues and matched non-tumor tissues to delineate the AS landscape in HB. AS events were validated in 92 samples from 46 patients. RNA pull-down and RNA immunoprecipitation assays were carried out to identify splicing factors that regulate the AS of small nucleolar RNA host genes (SNHG). Patient-derived organoids (PDOs) were established to investigate the role of the splicing factor polyadenylate-binding nuclear protein 1 (PABPN1). This study uncovered aberrant alternative splicing in HB, including lncRNAs from SNHG family that undergo intron retention in HB. Further investigations revealed that PABPN1, a significantly upregulated RNA binding protein, interacts with splicing machinery in HB, inducing the intron retention of these SNHG RNAs and the downregulation of intronic small nucleolar RNAs (snoRNAs). Functionally, PABPN1 acts as an oncofetal splicing regulator in HB by promoting cell proliferation and DNA damage repair via inducing the intron retention of SNHG19. Knock-down of PABPN1 increases the cisplatin sensitivity of HB PDOs. Our findings revealed the role of intron retention in regulating snoRNA expression in hepatoblastoma, explained detailed regulatory mechanism between PABPN1 and the intron retention of SNHG RNAs, and provided insight into the development of new HB treatment options.

Intronic Cis-Element DR8 in hTERT Is Bound by Splicing Factor SF3B4 and Regulates hTERT Splicing in Non-Small Cell Lung Cancer.

Manipulation of a core spliceosome protein reduces telomerase/hTERT splicing in lung cancer cells and results in slowed cancer cell growth and cell death, revealing a potential therapeutic strategy.

Effects of Inhibitors of SLC9A-Type Sodium-Proton Exchangers on Survival Motor Neuron 2 (SMN2) mRNA Splicing and Expression.

Spinal muscular atrophy (SMA) is an autosomal recessive, pediatric-onset disorder caused by the loss of spinal motor neurons, thereby leading to muscle atrophy. SMA is caused by the loss of or mutations in the survival motor neuron 1 (SMN1) gene. SMN1 is duplicated in humans to give rise to the paralogous survival motor neuron 2 (SMN2) gene. This paralog is nearly identical except for a cytosine to thymine transition within an exonic splicing enhancer element within exon 7. As a result, the majority of SMN2 transcripts lack exon 7 (SMNΔ7), which produces a truncated and unstable SMN protein. Since SMN2 copy number is inversely related to disease severity, it is a well established target for SMA therapeutics development. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of sodium/proton exchangers (NHEs), has previously been shown to increase exon 7 inclusion and SMN protein levels in SMA cells. In this study, NHE inhibitors were evaluated for their ability to modulate SMN2 expression. EIPA as well as 5-(N,N-hexamethylene)amiloride (HMA) increase exon 7 inclusion in SMN2 splicing reporter lines as well as in SMA fibroblasts. The EIPA-induced exon 7 inclusion occurs via a unique mechanism that does not involve previously identified splicing factors. Transcriptome analysis identified novel targets, including TIA1 and FABP3, for further characterization. EIPA and HMA are more selective at inhibiting the NHE5 isoform, which is expressed in fibroblasts as well as in neuronal cells. These results show that NHE5 inhibition increases SMN2 expression and may be a novel target for therapeutics development. SIGNIFICANCE STATEMENT: This study demonstrates a molecular mechanism by which inhibitors of the sodium-protein exchanger increase the alternative splicing of SMN2 in spinal muscular atrophy cells. NHE5 selective inhibitors increase the inclusion of full-length SMN2 mRNAs by targeting TIA1 and FABP3 expression, which is distinct from other small molecule regulators of SMN2 alternative splicing. This study provides a novel means to increase full-length SMN2 expression and a novel target for therapeutics development.

The Prognostic Significance of Spliceosomal Proteins for Patients with Glioblastoma

Glioblastoma (GBM) is considered one of the most aggressive human cancers. Earlier, our group have demonstrated that alternative RNA splicing plays an important role in the regulation of the GBM phenotype. To continue this study, we analyzed the type of RNA splicing and the expression levels of the spliceosomal genes in a large number of tumor tissue samples and patient-derived GBM sphere lines. We demonstrated that the expression level of splicing factors allows dividing GBM patients into groups with different survival prognosis and also reflects the phenotype of the tumor. In addition, we identified the alternative splicing events that may regulate the GBM phenotype. Finally, we for the first time compared the expression profiles of the spliceosomal genes in different regions of the same tumor and identified splicing factors whose expression most significantly correlates with GBM patients’ survival. Aforementioned data emphasize the important role of pre-mRNA splicing in GBM progression.

HNRNPM controls circRNA biogenesis and splicing fidelity to sustain cancer cell fitness.

High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and backsplicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM-dependent linear-splicing events using splice-switching-antisense-oligonucleotides was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors.

Splicing factors control triple-negative breast cancer cell mitosis through SUN2 interaction and sororin intron retention

BackgroundTriple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic opportunities. Recently, splicing factors have gained attention as potential targets for cancer treatment. Here we systematically evaluated the role of RNA splicing factors in TNBC cell proliferation.MethodsIn this study, we performed an RNAi screen targeting 244 individual splicing factors to systematically evaluate their role in TNBC cell proliferation. For top candidates, mechanistic insight was gained using amongst others western blot, PCR, FACS, molecular imaging and cloning. Pulldown followed by mass spectrometry were used to determine protein-protein interactions and patient-derived RNA sequencing data was used relate splicing factor expression levels to proliferation markers.ResultsWe identified nine splicing factors, including SNRPD2, SNRPD3 and NHP2L1, of which depletion inhibited proliferation in two TNBC cell lines by deregulation of sister chromatid cohesion (SCC) via increased sororin intron 1 retention and down-regulation of SMC1, MAU2 and ESPL1. Protein-protein interaction analysis of SNRPD2, SNRPD3 and NHP2L1 identified that seven out of the nine identified splicing factors belong to the same spliceosome complex including novel component SUN2 that was also critical for efficient sororin splicing. Finally, sororin transcript levels are highly correlated to various proliferation markers in BC patients.ConclusionWe systematically determined splicing factors that control proliferation of breast cancer cells through a mechanism that involves effective sororin splicing and thereby appropriate sister chromatid cohesion. Moreover, we identified SUN2 as an important new spliceosome complex interacting protein that is critical in this process. We anticipate that deregulating sororin levels through targeting of the relevant splicing factors might be a potential strategy to treat TNBC.

Identification and subcellular localization of splicing factor arginine/serine-rich 10 in the microsporidian Nosema bombycis

Splicing factors are important components of RNA editing in eukaryotic organisms and can produce many functional and coding genes, which is an indispensable step for the correct expression of corresponding proteins. In this study, we identified splicing factor arginine/serine-rich 10 protein in the microsporidian Nosema bombycis and named it NbSRSF10. The NbSRSF10 gene contains a complete ORF of 1449 bp in length that encodes a 482-amino acid polypeptide. The isoelectric point (pI) of the protein encoded by NbSRSF10 gene was 4.94. NbSRSF10 has a molecular weight of 54.6 kD and has no signal peptide. NbSRSF10 is comprised of arginine (11.41%), glutamic acid (11.41%) and serine (9.54%) among the total amino acids, and 7 α-helix, 7 β-sheet and 15 random coils in secondary structure, and contains 71 phosphorylation sites, 22 N-glycosylation sites and 20 O-glycosylation sites. The three-dimensional structure of NbSRSF10 is similar to that of transformer-2 beta of Homo sapiens (hTra2-β). Indirect immunofluorescence showed that the NbSRSF10 is localized in the cytoplasm of the dormant microsporidian spore and is transferred to the nuclei when N. bombycis develops into the proliferative and sporogonic phase. qPCR revealed that the relative expression of NbSRSF10 increased in the meronts stage and was found at a relatively low level in the sporogonic phase of development of N. bombycis, and was up-regulated again during infection in the host cell and early proliferative phase of second life cycle. These results suggested that the NbSRSF10 may participate in the whole life cycle and play an important role in transcription regulation of N. bombycis.

Evolutionarily conserved regulation of immunity by the splicing factor RNP-6/PUF60.

Splicing is a vital cellular process that modulates important aspects of animal physiology, yet roles in regulating innate immunity are relatively unexplored. From genetic screens in C. elegans, we identified splicing factor RNP-6/PUF60 whose activity suppresses immunity, but promotes longevity, suggesting a tradeoff between these processes. Bacterial pathogen exposure affects gene expression and splicing in a rnp-6 dependent manner, and rnp-6 gain and loss-of-function activities reveal an active role in immune regulation. Another longevity promoting splicing factor, SFA-1, similarly exerts an immuno-suppressive effect, working downstream or parallel to RNP-6. RNP-6 acts through TIR-1/PMK-1/MAPK signaling to modulate immunity. The mammalian homolog, PUF60, also displays anti-inflammatory properties, and its levels swiftly decrease after bacterial infection in mammalian cells, implying a role in the host response. Altogether our findings demonstrate an evolutionarily conserved modulation of immunity by specific components of the splicing machinery.

Poison-Exon Inclusion in DHX9 Reduces Its Expression and Sensitizes Ewing Sarcoma Cells to Chemotherapeutic Treatment.

Alternative splicing is a combinatorial mechanism by which exons are joined to produce multiple mRNA variants, thus expanding the coding potential and plasticity of eukaryotic genomes. Defects in alternative splicing regulation are associated with several human diseases, including cancer. Ewing sarcoma is an aggressive tumor of bone and soft tissue, mainly affecting adolescents and young adults. DHX9 is a key player in Ewing sarcoma malignancy, and its expression correlates with worse prognosis in patients. In this study, by screening a library of siRNAs, we have identified splicing factors that regulate the alternative inclusion of a poison exon in DHX9 mRNA, leading to its downregulation. In particular, we found that hnRNPM and SRSF3 bind in vivo to this poison exon and suppress its inclusion. Notably, DHX9 expression correlates with that of SRSF3 and hnRNPM in Ewing sarcoma patients. Furthermore, downregulation of SRSF3 or hnRNPM inhibited DHX9 expression and Ewing sarcoma cell proliferation, while sensitizing cells to chemotherapeutic treatment. Hence, our study suggests that inhibition of hnRNPM and SRSF3 expression or activity could be exploited as a therapeutic tool to enhance the efficacy of chemotherapy in Ewing sarcoma.

Differential Functions of Splicing Factors in Mammary Transformation and Breast Cancer Metastasis

Misregulation of alternative splicing is a hallmark ofhuman tumors, yet to what extent and how it contributes to malignancy are only beginning to be unraveled. Here, we define which members of the splicing factor SR and SR-like families contribute to breast cancer and uncover differences and redundancies in their targets and biological functions. We identify splicing factors frequently altered in human breast tumors and assay their oncogenic functions using breast organoid models. We demonstrate that not all splicing factors affect mammary tumorigenesis in MCF-10A cells. Specifically, the upregulation of SRSF4, SRSF6, or TRA2β disrupts acinar morphogenesis and promotes cell proliferation and invasion in MCF-10A cells. By characterizing the targets of these oncogenic splicing factors, we identify shared spliced isoforms associated with well-established cancer hallmarks. Finally, we demonstrate that TRA2β is regulated by the MYC oncogene, plays a role in metastasis maintenance invivo, and its levels correlate with breast cancer patient survival.

Systematic proteomics of endogenous human cohesin reveals an interaction with diverse splicing factors and RNA-binding proteins required for mitotic progression

The cohesin complex regulates sister chromatid cohesion, chromosome organization, gene expression, and DNA repair. Cohesin is a ring complex composed of four core subunits and seven regulatory subunits. In an effort to comprehensively identify additional cohesin-interacting proteins, we used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in cultured human cells, and we performed MS analyses on dual-affinity purifications. In addition to reciprocally identifying all known components of cohesin, we found that cohesin interacts with a panoply of splicing factors and RNA-binding proteins (RBPs). These included diverse components of the U4/U6.U5 tri-small nuclear ribonucleoprotein complex and several splicing factors that are commonly mutated in cancer. The interaction between cohesin and splicing factors/RBPs was RNA- and DNA-independent, occurred in chromatin, was enhanced during mitosis, and required RAD21. Furthermore, cohesin-interacting splicing factors and RBPs followed the cohesin cycle and prophase pathway of cell cycle-regulated interactions with chromatin. Depletion of cohesin-interacting splicing factors and RBPs resulted in aberrant mitotic progression. These results provide a comprehensive view of the endogenous human cohesin interactome and identify splicing factors and RBPs as functionally significant cohesin-interacting proteins.

Splicing Factor 3B Subunit 1 Interacts with HIV Tat and Plays a Role in Viral Transcription and Reactivation from Latency.

The main obstacle to an HIV cure is the transcriptionally inert proviruses that persist in resting CD4 T cells and other reservoirs. None of the current approaches has significantly reduced the size of the viral reservoir. Hence, alternative approaches, such as permanent blocking of viral transcription, to achieve a sustained remission, need urgent attention. To identify cellular factors that may be important for this approach, we sought for host targets that when altered could block HIV transcription and reactivation. Here, we identified splicing factor 3B subunit 1 (SF3B1) as a critical HIV dependency factor required for viral replication. SF3B1 is a splicing factor involved in directing chromatin and nascent gene transcripts to appropriate splice sites. Inhibitors of SF3B1 are currently in development for cancer and have been found to be nontoxic to normal cells compared to malignant cells. Knockdown of SF3B1 abrogated HIV replication in all cell types tested. SF3B1 interacted with viral protein Tat in vitro and in vivo Genetic or pharmacologic inhibition of SF3B1 prevented Tat-mediated HIV transcription and RNA polymerase II association with the HIV promoter. In addition, an inhibitor of SF3B1 prevented HIV reactivation from latency irrespective of the latency-reversing agent used. The data show that SF3B1 is involved in viral transcription and reactivation from latency and may serve as a therapeutic target in the HIV cure efforts.IMPORTANCE The reason why HIV cannot be cured by current therapy is because of viral persistence in resting T cells. One approach to permanent HIV remission that has received less attention is the so-called "block and lock" approach. The idea behind this approach is that the virus could be permanently disabled in patients if viral genome or surrounding chromatin could be altered to silence the virus, thus enabling patients to stop therapy. In this work, we have identified splicing factor 3B subunit 1 (SF3B1) as a potential target for this approach. SF3B1 interacts with the viral protein Tat, which is critical for viral transcription. Inhibition of SF3B1 prevents HIV transcription and reactivation from latency. Since there are preclinical inhibitors for this protein, our findings could pave the way to silence HIV transcription, potentially leading to prolonged or permanent remission.

SRSF6 is upregulated in asthmatic horses and involved in the MYH11 SMB expression.

Smooth muscle has a central role in bronchospasm‐induced airway obstruction in asthma. Alternative mRNA splicing of the smooth muscle myosin heavy chain (myh11) gene produces four different isoforms, one of which (SMB) is characterized by the inclusion of the exon5b, which doubles the smooth muscle cells contraction velocity. Deciphering the regulation of the expression levels of the SMB isoform would represent a major step for the understanding of the triggers and pathways leading to airway smooth muscle contraction in asthma. Our objective was therefore, to study the splicing regulation mechanisms of the exon5b in airway smooth muscle cells. Bioinformatics analysis was performed to identify the cis‐regulatory elements present in the exon5b using HSF finder 3 tool. The expression of the corresponding serine/arginine rich protein (SR) genes thus identified was evaluated by quantitative RT‐PCR (qPCR). SRSF1, SRSF6, and hnRNPA1 cis‐acting elements were identified by in silico analysis of the exon5b sequence as splicing regulator candidates. QPCR analyses showed that SRSF1 and SRSF6 are upregulated in ASM cells from asthmatic horses in exacerbation (n = 5) compared to controls (n = 5). The inhibition of the identified splicing factors by small interfering RNA allowed identifying the regulation of the SMB isoform by SRSF6. Our results implicate for the first time the upregulation of SRSF6 and SRSF1 in the asthmatic ASM cells and indicate that SRSF6 induces the exon5b inclusion. This study provides an important first step for the understanding of the triggers and pathways leading to ASM hypercontraction and identifies a possible new target for asthma.

NOVA1 regulates hTERT splicing and cell growth in non-small cell lung cancer

Alternative splicing is dysregulated in cancer and the reactivation of telomerase involves the splicing of TERT transcripts to produce full-length (FL) TERT. Knowledge about the splicing factors that enhance or silence FL hTERT is lacking. We identified splicing factors that reduced telomerase activity and shortened telomeres using a siRNA minigene reporter screen and a lung cancer cell bioinformatics approach. A lead candidate, NOVA1, when knocked down resulted in a shift in hTERT splicing to non-catalytic isoforms, reduced telomerase activity, and progressive telomere shortening. NOVA1 knockdown also significantly altered cancer cell growth in vitro and in xenografts. Genome engineering experiments reveal that NOVA1 promotes the inclusion of exons in the reverse transcriptase domain of hTERT resulting in the production of FL hTERT transcripts. Utilizing hTERT splicing as a model splicing event in cancer may provide new insights into potentially targetable dysregulated splicing factors in cancer.

Abstract 3406: Comprehensive genomic profiling of hematologic malignancies identifies recurrent somatic splicing factor mutations in non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM)

Abstract Somatic mutations of splicing factors have been reported in multiple tumor types and have been recognized as a new hallmark of cancer. Although these mutations have been observed in myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML) and chronic lymphocytic leukemia (CLL), the frequency of these mutations in other hematological malignancies is unknown. We surveyed somatic mutations of several splicing factors (SF3B1, SRSF2, U2AF1, ZRSR2, DDX3X, ZMYM3, PCBP1 and U2AF2) in 6,235 patients across 15 hematological malignancies. 405 genes were analyzed by DNAseq at >500X coverage using FoundationOneHeme. Consistent with prior reports, we found that the hematopoietic malignancies with the most frequent splicing factor mutations were CMML (48.3%), MDS (36.9%), AML (25.3%) and CLL (22.5%). However, we also identified splicing factor mutations in NHL (13.8%) and MM (9%). In addition to mutations found across the different hematopoietic malignancies in SRSF2 (6%), SF3B1 (4.5%), U2AF1 (3.3%) and ZRSR2 (2.2%), we found DDX3X to be the fifth most frequently mutated gene at 1.6%, followed by ZMYM3 (0.8%), PCBP1 (0.5%) and U2AF2 (0.4%), indicating the importance of splicing dysregulation in hematological malignancies. Within NHL, diffuse large B cell lymphoma (DLBCL) has the highest frequency of splicing factor mutations (18.3%), and these patients exhibited increased tumor mutation burden (TMB, 13.7 vs. 10.0 mutations per Mb, P < 0.05). Among the splicing factors, the RNA helicase DDX3X is the most frequently mutated in NHL (5.2%). DDX3X is a X chromosome gene and its mutations in NHL are associated with male gender (P = 0.006). Consistent with the reported mutations in CLL and natural killer/T-cell lymphoma, the majority of mutations are loss of function or missense mutations clustered in the two helicase domains. This suggests a pathological relevance of DDX3X in lymphoid malignancies. In MM, SF3B1 and SRSF2 are two most frequently mutated genes at 3.8% and 1.9%, and patients with these mutations are associated with increased TMB (4.2 vs. 2.5, P < 0.001). Moreover, SF3B1 mutations occurred in 5.3% of samples with IGH-CCND1/2/3 or IGH-MAF/MAFB translocations, but <1% of samples with IGH-WHSC1/FGFR3 translocations. Although the most common SF3B1 mutation in hematopoietic malignancies is p.K700E, in MM the most frequent SF3B1 mutation is p.K666 (36.9% vs p.K700E 12.3%). Here, we identify splicing factor mutations in NHL and MM, including hotspot somatic mutations of SF3B1, U2AF1 and SRSF2 and loss of function or missense mutations in DDX3X. Overall, our results broaden the disease association of splicing factor mutations in hematological malignancies and strengthen their role in disease pathogenesis. Citation Format: Lee A. Albacker, Silvia Buonamici, Garrett M. Frampton, Peter Smith, Philip J. Stephens, Markus Warmuth,, Ping Zhu, Lihua Yu. Comprehensive genomic profiling of hematologic malignancies identifies recurrent somatic splicing factor mutations in non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3406.

The splicing factor transformer2 (tra2) functions in the Drosophila fat body to regulate lipid storage

Excess nutrients are stored as triglycerides mainly in the adipose tissue of an animal and these triglycerides are located in structures called lipid droplets. Previous genome-wide RNAi screens in Drosophila cells identified splicing factors as playing a role in lipid droplet formation. Our lab has recently identified the SR protein, 9G8, as an important factor in fat storage as decreasing its levels results in augmented triglyceride storage in the fat body. Previous in vitro studies have implicated 9G8 in the regulation of splicing of the sex determination gene doublesex (dsx) by binding to transformer (tra) and transformer2 (tra2); however, any function of these sex determination proteins in regulating metabolism is unknown. In this study, we have uncovered a role of tra2 to regulate fat storage in vivo. Inducing tra2dsRNA in the adult fat body resulted in an increase in triglyceride levels but had no effect on glycogen storage. Consistent with the triglyceride phenotype, tra2 knockdown flies lived longer under starvation conditions. In addition, this increase in triglycerides is due to more fat storage per cell and not an increase in the number of fat cells. Interestingly, the splicing of CPT1, an enzyme involved in the breakdown of lipids, was altered in flies with decreased tra2. The less-catalytically active isoform of CPT1 accumulated in tra2dsRNA flies suggesting a decrease in lipid breakdown, which is consistent with the increased triglyceride levels observed in these flies. Together, these results suggest a link between mRNA splicing, sex determination and lipid metabolism and may provide insight into the mechanisms underlying tissue-specific splicing and nutrient storage.

Abstract 1513: Pan-cancer analysis of alternative splicing patterns and association to genomic aberrations

Abstract Alternative splicing of pre-messenger RNA is responsible for the diversity of transcriptome and proteome, with the majority of multi exon genes producing multiple transcripts. Aberrations in regulative factors of alternative splicing are known to produce transcript isoforms associated with cancer progression and prognosis. Both mutations in the proximity of spliced regions and mutations in and regulation of previously identified splicing factor genes are known to alter splicing patterns. In this study, we utilized publicly available TCGA data to study association patterns between alternative splicing and its regulatory elements across multiple cancer tissue types. We collected evidence of splicing patterns of annotated isoforms in addition to novel patterns identified using our previously developed tools using RNA-seq data. We mined for associations between somatic mutations and changes of splicing patterns as well as genome and transcriptome level changes in splicing factors. Selective transcriptome assembly was done in loci with differentially expressed patterns and novel patterns with sufficient evidence to identify transcript isoforms and quantify isoform specific expression. In our study we compiled a catalogue of splicing patterns and their changes across multiple cancer types. Integration of this comprehensive set of quantified splicing patterns with data on proximal mutations and aberrations in splicing factors resulted in statistically relevant associations with clinical phenotypes. Our analysis enables identification of mechanisms behind splicing aberrations common to all cancers and cancer specific splicing patterns. Citation Format: Sergei Häyrynen, Matti Nykter. Pan-cancer analysis of alternative splicing patterns and association to genomic aberrations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1513.