Alternative mRNA Splicing Research Articles

Quantifying binding of VEGF‐A isoforms at VEGFR2 in membranes

Vascular endothelial growth factor A (VEGF‐A) is a key mediator of angiogenesis, primarily signalling via VEGF Receptor 2 (VEGFR2) [1]. There are numerous endogenous isoforms of VEGF‐A resulting from alternative mRNA splicing. We previously used bioluminescence resonance energy transfer (BRET) to quantify binding of VEGF‐A isoforms single‐site labelled with tetramethylrhodamine (TMR) at NanoLuciferase (NanoLuc)‐tagged VEGFR2 in living cells [2,3]. However, VEGFR2 undergoes ligand‐dependent and ‐independent endocytosis, therefore we aimed to compare the pharmacology of VEGF‐A isoforms at VEGFR2 in membrane preparations. Membranes were homogenised from HEK293T cells stably expressing NanoLuc‐VEGFR2 as in [4], stored in Phosphate Buffered Saline and plated on the day of experimentation as 5μg/well in 40μl vehicle (Hanks Buffered Saline Solution/0.1% Bovine Serum Albumin; pH 7.4). For kinetic experiments, membranes were incubated with the NanoLuc substrate furimazine (10μM) for 5min, then 1–20nM fluorescent VEGF‐A was added. BRET ratios were measured every 30sec for 90min at 37°C using a BMG Pherastar. To quantify binding affinities, membranes were incubated with increasing concentrations of VEGF165a‐TMR, VEGF165b‐TMR or VEGF121a‐TMR (0.1–20nM) in the presence and absence of 100nM unlabelled VEGF. Alternatively, membranes were co‐stimulated with increasing concentrations of unlabelled VEGF‐Ax and a fixed concentration of fluorescent VEGF‐A (0.5–5nM). Following 60min stimulation at 37°C, furimazine was added and BRET ratios were measured. The kinetic binding profiles of each fluorescent VEGF‐A isoform in membranes reached a plateau at NanoLuc‐VEGFR2 and binding was maintained for 90min. This contrasted with the characteristic decrease in BRET ratio observed following 20min in intact cells [2]. Each fluorescent VEGF‐A isoform had saturable binding at NanoLuc‐VEGFR2, with a similarly low level of non‐specific binding compared to cells [3]. Derived equilibrium dissociation constants in membranes showed VEGF165a‐TMR, VEGF165b‐TMR and VEGF121a‐TMR bound NanoLuc‐VEGFR2 with nanomolar affinity. Each fluorescent VEGF‐A isoform was displaced by unlabelled VEGF‐Ax, from which nanomolar affinities were derived for VEGF‐Ax. Using NanoBRET to monitor protein‐protein interactions therefore showed that while the kinetic binding profile was strikingly different in membrane preparations compared to intact cells, derived affinity constants were identical. This quantitative approach retains the ability for receptor oligomerisation in an intact membrane bilayer, while isolating molecular ligand/receptor interactions at 37°C without the complex spatio‐temporal dynamics governing full‐length VEGFR2 in living cells.Support or Funding InformationThis project is supported by the British Pharmacological Society's AJ Clark Scholarship.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Uncoupling protein (UCP)1 and exercise induced ‘browning’ of white adipose tissue is absent at thermoneutrality

Background and aimThe induction of thermogenic genes in white adipose tissue (WAT) of exercise‐trained rodents is commonly seen in animals housed at standard temperatures (e.g. ~20°C) despite not being seen in exercise‐trained humans1,2. Here, we hypothesised that raising and exercise training rodents at thermoneutrality (e.g. ~28°C) thus mimicking human physiology would negate the induction of thermogenic genes typically seen at standard housing temperatures.MethodsTwelve weanling (i.e. 3 weeks old) Sprague‐Dawley male rats were housed at thermoneutrality (28°C) and fed a high‐fat diet (HFD) to induce weight gain with (Ex) or without (C) exercise training. At 12 weeks of age Ex was commenced by swim‐training (i.e. 1 hour/day, 5 days per week) for 4 weeks. Metabolic assessment was undertaken in metabolic cages during the last 48h to assess energy intake, expenditure and physical activity in addition to the acute response to administration of a β3‐agonist (Mirabegron, 0.75mg/kg/d). Then, at 16 weeks of age following an overnight fast, tissues were taken for analysis of thermogenic genes and quantitative proteomics.ResultsEx attenuated weight gain, but did not affect food intake, energy expenditure, final body weight, fat mass, plasma insulin or glucose or HOMA‐IR. There was also no increase in energy expenditure in response to administration of Mirabegron with UCP1 mRNA in IWAT remaining undetectable in EX despite an upregulation of genes considered to be classical ‘browning’ inducers i.e. PGC1a, ADRB3 and DIO2. We found 189 proteins were differentially regulated in IWAT following exercise. Among the most significant were those regulating nuclear factor kappa B signalling (LRRFIP2), the mitochondrial electron transport chain (NDUFS6) and oxidative stress (SOD3). Gene ontology analysis demonstrated these enriched 51 groups based on their biological process including ‘regulation of alternative mRNA splicing, via spliceosome’, ‘DNA damage checkpoint’ and ‘regulation of type 2 immune response’ whilst only 13 groups were enriched based on their molecular function including ‘metallodipeptidase activity’, ‘ATPase regulator activity’ and ‘pre‐mRNA binding’.ConclusionUnlike at standard housing temperatures, exercise induced ‘browning’ of IWAT was absent in animals raised at thermoneutrality corroborating human data. Proteomic analysis further highlights alternate novel pathways in adipose tissue regulated by exercise training that could have functional relevance.Support or Funding InformationBritish Heart Foundation [grant number FS/15/4/31184/]This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The splicing factor U2AF1 contributes to cancer progression through a noncanonical role in translation regulation.

Somatic mutations in the genes encoding components of the spliceosome occur frequently in human neoplasms, including myeloid dysplasias and leukemias, and less often in solid tumors. One of the affected factors, U2AF1, is involved in splice site selection, and the most common change, S34F, alters a conserved nucleic acid-binding domain, recognition of the 3' splice site, and alternative splicing of many mRNAs. However, the role that this mutation plays in oncogenesis is still unknown. Here, we uncovered a noncanonical function of U2AF1, showing that it directly binds mature mRNA in the cytoplasm and negatively regulates mRNA translation. This splicing-independent role of U2AF1 is altered by the S34F mutation, and polysome profiling indicates that the mutation affects translation of hundreds of mRNA. One functional consequence is increased synthesis of the secreted chemokine interleukin 8, which contributes to metastasis, inflammation, and cancer progression in mice and humans.

Obesity-induced MBD2_v2 expression promotes tumor-initiating triple-negative breast cancer stem cells.

Obesity is a risk factor for triple‐negative breast cancer (TNBC) incidence and poor outcomes, but the underlying molecular biology remains unknown. We previously identified in TNBC cell cultures that expression of epigenetic reader methyl‐CpG‐binding domain protein 2 (MBD2), specifically the alternative mRNA splicing variant MBD variant 2 (MBD2_v2), is dependent on reactive oxygen species (ROS) and is crucial for maintenance and expansion of cancer stem cell‐like cells (CSCs). Because obesity is coupled with inflammation and ROS, we hypothesized that obesity can fuel an increase in MBD2_v2 expression to promote the tumor‐initiating CSC phenotype in TNBC cells in vivo. Analysis of TNBC patient datasets revealed associations between high tumor MBD2_v2 expression and high relapse rates and high body mass index (BMI). Stable gene knockdown/overexpression methods were applied to TNBC cell lines to elucidate that MBD2_v2 expression is governed by ROS‐dependent expression of serine‐ and arginine‐rich splicing factor 2 (SRSF2). We employed a diet‐induced obesity (DIO) mouse model that mimics human obesity to investigate whether obesity causes increased MBD2_v2 expression and increased tumor initiation capacity in inoculated TNBC cell lines. MBD2_v2 and SRSF2 levels were increased in TNBC cell line‐derived tumors that formed more frequently in DIO mice relative to tumors in lean control mice. Stable MBD2_v2 overexpression increased the CSC fraction in culture and increased TNBC cell line tumor initiation capacity in vivo. SRSF2 knockdown resulted in decreased MBD2_v2 expression, decreased CSCs in TNBC cell cultures, and hindered tumor formation in vivo. This report describes evidence to support the conclusion that MBD2_v2 expression is induced by obesity and drives TNBC cell tumorigenicity, and thus provides molecular insights into support of the epidemiological evidence that obesity is a risk factor for TNBC. The majority of TNBC patients are obese and rising obesity rates threaten to further increase the burden of obesity‐linked cancers, which reinforces the relevance of this report.

Distinct isoforms of Nrf1 diversely regulate different subsets of its cognate target genes

The single Nrf1 gene has capability to be differentially transcripted alongside with alternative mRNA-splicing and subsequent translation through different initiation signals so as to yield distinct lengths of polypeptide isoforms. Amongst them, three of the most representatives are Nrf1α, Nrf1β and Nrf1γ, but the putative specific contribution of each isoform to regulating ARE-driven target genes remains unknown. To address this, we have herein established three cell lines on the base of the Flp-In T-REx system, which are allowed for the tetracycline-inducibly stable expression of Nrf1α, Nrf1β and Nrf1γ. Consequently, the RNA-Sequencing results have demonstrated that a vast majority of differentially expressed genes (i.e. >90% DEGs detected) were dominantly up-regulated by Nrf1α and/or Nrf1β following induction by tetracycline. By contrast, the other DEGs regulated by Nrf1γ were far less than those regulated by Nrf1α/β (i.e. ~11% of Nrf1α and ~7% of Nrf1β). However, further transcriptomic analysis revealed that the tetracycline-induced expression of Nrf1γ significantly increased the percentage of down-regulated genes in total DEGs. These statistical data were further validated by quantitative real-time PCR. The experimental results indicate that distinct Nrf1 isoforms make diverse and even opposing contributions to regulating different subsets of target genes, such as those encoding 26S proteasomal subunits and others involved in various biological processes and functions. Collectively, Nrf1γ acts as a major dominant-negative inhibitor competitively against Nrf1α/β activity, such that a number of DEGs regulated by Nrf1α/β are counteracted by Nrf1γ.

Splicing-Dependent Subcellular Targeting of Borna Disease Virus Nucleoprotein Isoforms.

Targeting of viral proteins to specific subcellular compartments is a fundamental step for viruses to achieve successful replication in infected cells. Borna disease virus 1 (BoDV-1), a nonsegmented, negative-strand RNA virus, uniquely replicates and persists in the cell nucleus. Here, it is demonstrated that BoDV nucleoprotein (N) transcripts undergo mRNA splicing to generate truncated isoforms. In combination with alternative usage of translation initiation sites, the N gene potentially expresses at least six different isoforms, which exhibit diverse intracellular localizations, including the nucleoplasm, cytoplasm, and endoplasmic reticulum (ER), as well as intranuclear viral replication sites. Interestingly, the ER-targeting signal peptide in N is exposed by removing the intron by mRNA splicing. Furthermore, the spliced isoforms inhibit viral polymerase activity. Consistently, recombinant BoDVs lacking the N-splicing signals acquire the ability to replicate faster than wild-type virus in cultured cells, suggesting that N isoforms created by mRNA splicing negatively regulate BoDV replication. These results provided not only the mechanism of how mRNA splicing generates viral proteins that have distinct functions but also a novel strategy for replication control of RNA viruses using isoforms with different subcellular localizations.IMPORTANCE Borna disease virus (BoDV) is a highly neurotropic RNA virus that belongs to the orthobornavirus genus. A zoonotic orthobornavirus that is genetically related to BoDV has recently been identified in squirrels, thus increasing the importance of understanding the replication and pathogenesis of orthobornaviruses. BoDV replicates in the nucleus and uses alternative mRNA splicing to express viral proteins. However, it is unknown whether the virus uses splicing to create protein isoforms with different functions. The present study demonstrated that the nucleoprotein transcript undergoes splicing and produces four new isoforms in coordination with alternative usage of translation initiation codons. The spliced isoforms showed a distinct intracellular localization, including in the endoplasmic reticulum, and recombinant viruses lacking the splicing signals replicated more efficiently than the wild type. The results provided not only a new regulation of BoDV replication but also insights into how RNA viruses produce protein isoforms from small genomes.

Functional Silencing of HSD17B2 in Prostate Cancer Promotes Disease Progression.

Steroidogenic enzymes are essential for prostate cancer development. Enzymes inactivating potent androgens were not investigated thoroughly, which leads to limited interference strategies for prostate cancer therapy. Here we characterized the clinical relevance, significance, and regulation mechanism of enzyme HSD17B2 in prostate cancer development. HSD17B2 expression was detected with patient specimens and prostate cancer cell lines. Function of HSD17B2 in steroidogenesis, androgen receptor (AR) signaling, and tumor growth was investigated with prostate cancer cell lines and a xenograft model. DNA methylation and mRNA alternative splicing were investigated to unveil the mechanisms of HSD17B2 regulation. HSD17B2 expression was reduced as prostate cancer progressed. 17βHSD2 decreased potent androgen production by converting testosterone (T) or dihydrotestosterone (DHT) to each of their upstream precursors. HSD17B2 overexpression suppressed androgen-induced cell proliferation and xenograft growth. Multiple mechanisms were involved in HSD17B2 functional silencing including DNA methylation and mRNA alternative splicing. DNA methylation decreased the HSD17B2 mRNA level. Two new catalytic-deficient isoforms, generated by alternative splicing, bound to wild-type 17βHSD2 and promoted its degradation. Splicing factors SRSF1 and SRSF5 participated in the generation of new isoforms. Our findings provide evidence of the clinical relevance, significance, and regulation of HSD17B2 in prostate cancer progression, which might provide new strategies for clinical management by targeting the functional silencing mechanisms of HSD17B2.See related commentary by Mostaghel, p. 1139.

Polypyrimidine tract binding protein 1 promotes lymphatic metastasis and proliferation of bladder cancer via alternative splicing of MEIS2 and PKM

Lymph node (LN) metastasis is the leading cause of bladder cancer-related mortality. Splicing factors facilitate cancer progression by modulating oncogenic variants, but it is unclear whether and how splicing factors regulate bladder cancer LN metastasis. In this study, Polypyrimidine tract binding protein 1 (PTBP1) expression was found to relate to bladder cancer LN metastasis, and was positively correlated with LN metastasis status, tumor stage, histological grade, and poor patient prognosis. Functional assays demonstrated that PTBP1 promoted bladder cancer cell migration, invasion, and proliferation in vitro, as well as LN metastasis and tumor growth in vivo. Mechanistic investigations revealed that PTBP1 upregulated MEIS2-L variant to promote metastasis and increased expression of PKM2 variant to enhance proliferation by modulating alternative mRNA splicing. Moreover, overexpression of MEIS2-L or PKM2 could rescue the oncogenic abilities of bladder cancer cells and the expression of MMP9 or CCND1 respectively after PTBP1 knockdown. In conclusion, our data demonstrate that PTBP1 induces bladder cancer LN metastasis and proliferation through an alternative splicing mechanism. PTBP1 may serve as a novel prognostic marker and therapeutic target for LN-metastatic bladder cancer.

Chronic exercise mitigates disease mechanisms and improves muscle function in myotonic dystrophy type 1 mice.

Myotonic dystrophy type 1 (DM1), the second most common muscular dystrophy and most prevalent adult form of muscular dystrophy, is characterized by muscle weakness, wasting and myotonia. A microsatellite repeat expansion mutation results in RNA toxicity and dysregulation of mRNA processing, which are the primary downstream causes of the disorder. Recent studies with DM1 participants demonstrate that exercise is safe, enjoyable and elicits benefits in muscle strength and function; however, the molecular mechanisms of exercise adaptation in DM1 are undefined. Our results demonstrate that 7weeks of volitional running wheel exercise in a pre-clinical DM1 mouse model resulted in significantly improved motor performance, muscle strength and endurance, as well as reduced myotonia. At the cellular level, chronic physical activity attenuated RNA toxicity, liberated Muscleblind-like 1 protein from myonuclear foci and improved mRNA alternative splicing. Myotonic dystrophy type 1 (DM1) is a trinucleotide repeat expansion neuromuscular disorder that is most prominently characterized by skeletal muscle weakness, wasting and myotonia. Chronic physical activity is safe and satisfying, and can elicit functional benefits such as improved strength and endurance in DM1 patients, but the underlying cellular basis of exercise adaptation is undefined. Our purpose was to examine the mechanisms of exercise biology in DM1. Healthy, sedentary wild-type (SED-WT) mice, as well as sedentary human skeletal actin-long repeat animals, a murine model of DM1 myopathy (SED-DM1), and DM1 mice with volitional access to a running wheel for 7weeks (EX-DM1), were utilized. Chronic exercise augmented strength and endurance in vivo and in situ in DM1 mice. These alterations coincided with normalized measures of myopathy, as well as increased mitochondrial content. Electromyography revealed a 70-85% decrease in the duration of myotonic discharges in muscles from EX-DM1 compared to SED-DM1 animals. The exercise-induced enhancements in muscle function corresponded at the molecular level with mitigated spliceopathy, specifically the processing of bridging integrator 1 and muscle-specific chloride channel (CLC-1) transcripts. CLC-1 protein content and sarcolemmal expression were lower in SED-DM1 versus SED-WT animals, but they were similar between SED-WT and EX-DM1 groups. Chronic exercise also attenuated RNA toxicity, as indicated by reduced (CUG)n foci-positive myonuclei and sequestered Muscleblind-like 1 (MBNL1). Our data indicate that chronic exercise-induced physiological improvements in DM1 occur in concert with mitigated primary downstream disease mechanisms, including RNA toxicity, MBNL1 loss-of-function, and alternative mRNA splicing.

Mettl3 Regulates Osteogenic Differentiation and Alternative Splicing of Vegfa in Bone Marrow Mesenchymal Stem Cells

Bone mesenchymal stem cells (BMSCs) can be a useful cell resource for developing biological treatment strategies for bone repair and regeneration, and their therapeutic applications hinge on an understanding of their physiological characteristics. N6-methyl-adenosine (m6A) is the most prevalent internal chemical modification of mRNAs and has recently been reported to play important roles in cell lineage differentiation and development. However, little is known about the role of m6A modification in the cell differentiation of BMSCs. To address this issue, we investigated the expression of N6-adenosine methyltransferases (Mettl3 and Mettl14) and demethylases (Fto and Alkbh5) and found that Mettl3 was upregulated in BMSCs undergoing osteogenic induction. Furthermore, we knocked down Mettl3 and demonstrated that Mettl3 knockdown decreased the expression of bone formation-related genes, such as Runx2 and Osterix. The alkaline phosphatase (ALP) activity and the formation of mineralized nodules also decreased after Mettl3 knockdown. RNA sequencing analysis revealed that a vast number of genes affected by Mettl3 knockdown were associated with osteogenic differentiation and bone mineralization. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed that the phosphatidylinositol 3-kinase/AKT (PI3K-Akt) signaling pathway appeared to be one of the most enriched pathways, and Western blotting results showed that Akt phosphorylation was significantly reduced after Mettl3 knockdown. Mettl3 has been reported to play an important role in regulating alternative splicing of mRNA in previous research. In this study, we found that Mettl3 knockdown not only reduced the expression of Vegfa but also decreased the level of its splice variants, vegfa-164 and vegfa-188, in Mettl3-deficient BMSCs. These findings might contribute to novel progress in understanding the role of epitranscriptomic regulation in the osteogenic differentiation of BMSCs and provide a promising perspective for new therapeutic strategies for bone regeneration.

RNA Dysregulation in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease and is characterized by the degeneration of upper and lower motor neurons. It has become increasingly clear that RNA dysregulation is a key contributor to ALS pathogenesis. The major ALS genes SOD1, TARDBP, FUS, and C9orf72 are involved in aspects of RNA metabolism processes such as mRNA transcription, alternative splicing, RNA transport, mRNA stabilization, and miRNA biogenesis. In this review, we highlight the current understanding of RNA dysregulation in ALS pathogenesis involving these major ALS genes and discuss the potential of therapeutic strategies targeting disease RNAs for treating ALS.

Statistical Methods To Explore The Phenotypic Consequences Of Gene Regulation

BackgroundOver the last 15 years, enormous advances have been made in the technology to interrogate the genome, which in turn has allowed us to discover thousands of genetic variants robustly associated with complex traits including psychiatric disorders. However, most of these variants are not located in coding regions making their interpretation challenging. It is generally accepted that an important mechanism that mediate the genotype phenotype relationship is the regulation of gene expression levels as well as alternative splicing of mRNAs. MethodsWe will describe a method (PrediXcan) to integrate large scale of genome wide association studies (GWAS) with reference transcriptome studies in order to investigate the underlying mechanism behind these discoveries. PrediXcan imputes the transcriptome and computes the correlation between the genetically determined component and the phenotype to prioritize genes that are likely to be causal. ResultsWe will show the results of applying PrediXcan to several large scale GWAS studies using prediction models of 44 human tissues. We find that most associations are tissue specific and that because of sharing between tissues, we increase our chances to discover the mediating genes when we scan across a broad set of tissues. ConclusionsTo take full advantage of the large amounts of genomic data that are being generated, methods that integrate multiple sources of high throughput data are needed. Here we present PrediXcan and its extension that addresses this need. Application to a broad set of phenotypes allows us to explore the phenotypic consequences of gene regulatory variation.

Networks of mRNA Processing and Alternative Splicing Regulation in Health and Disease.

mRNA processing events introduce an intricate layer of complexity into gene expression processes, supporting a tremendous level of diversification of the genome's coding and regulatory potential, particularly in vertebrate species. The recent development of massive parallel sequencing methods and their adaptation to the identification and quantification of different RNA species and the dynamics of mRNA metabolism and processing has generated an unprecedented view over the regulatory networks that are established at this level, which contribute to sustain developmental, tissue specific or disease specific gene expression programs. In this chapter, we provide an overview of the recent evolution of transcriptome profiling methods and the surprising insights that have emerged in recent years regarding distinct mRNA processingevents - from the 5' end to the 3' end of the molecule.

Pluripotent Stem Cell-Based Drug Screening Reveals Cardiac Glycosides as Modulators of Myotonic Dystrophy Type 1

SummaryThere is currently no treatment for myotonic dystrophy type 1 (DM1), the most frequent myopathy of genetic origin. This progressive neuromuscular disease is caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors, resulting in alternative splicing misregulation. By combining human mutated pluripotent stem cells and phenotypic drug screening, we revealed that cardiac glycosides act as modulators for both upstream nuclear aggregations of DMPK mRNAs and several downstream alternative mRNA splicing defects. However, these occurred at different drug concentration ranges. Similar biological effects were recorded in a DM1 mouse model. At the mechanistic level, we demonstrated that this effect was calcium dependent and was synergic with inhibition of the ERK pathway. These results further underscore the value of stem-cell-based assays for drug discovery in monogenic diseases.

Endonuclease G modulates the alternative splicing of deoxyribonuclease 1 mRNA in human CD4+ T lymphocytes and prevents the progression of apoptosis

Apoptotic endonucleases act cooperatively to fragment DNA and ensure the irreversibility of apoptosis. However, very little is known regarding the potential regulatory links between endonucleases. Deoxyribonuclease 1 (DNase I) inactivation is caused by alternative splicing (AS) of DNase I pre-mRNA skipping exon 4, which occurs in response to EndoG overexpression in cells. The current study aimed to determine the role of EndoG in the regulation of DNase I mRNA AS and the modulation of its enzymatic activity. A strong correlation was identified between the EndoG expression levels and DNase I splice variants in human lymphocytes. EndoG overexpression in CD4+ T cells down-regulated the mRNA levels of the active full-length DNase I variant and up-regulated the levels of the non-active spliced variant, which acts in a dominant-negative fashion. DNase I AS was induced by the translocation of EndoG from mitochondria into nuclei during the development of apoptosis. The DNase I spliced variant was induced by recombinant EndoG or by incubation with EndoG-digested cellular RNA in an in vitro system with isolated cell nuclei. Using antisense DNA oligonucleotides, we identified a 72-base segment that spans the adjacent segments of exon 4 and intron 4 and appears to be responsible for the AS. DNase I-positive CD4+ T cells overexpressing EndoG demonstrated decreased progression towards bleomycin-induced apoptosis. Therefore, EndoG is an endonuclease with the unique ability to inactivate another endonuclease, DNase I, and to modulate the development of apoptosis.

Systematic Identification of Survival-Associated Alternative mRNA Splicing for Pancreatic Ductal Adenocarcinoma

Background: Alternative splicing (AS) is deemed as a pivotal mechanism for post-transcriptional modification and protein diversity. Multiple studies have shown that cancer-specific AS alterations influence clinical outcome. We aimed to profile prognostic alternative mRNA splicing for pancreatic ductal adenocarcinoma (PDAC). Methods: RNA-sequencing data and clinical information of PDAC patients were downloaded from TCGA database, and the percent-spliced-in (PSI) data of AS was obtained from the SpliceSeq database. We selected overall survival (OS)-associated AS events using univariate Cox regression analysis. Gene functional enrichment analysis demonstrated the pathways enriched by survival-associated AS. Then, prognostic AS signatures were constructed for OS and chemoresistance prediction using the least absolute shrinkage and selection operator (LASSO) method. We also analyzed splicing factors (SFs) underlying regulation mechanisms by Pearson correlation and then built corresponding regulatory networks. Findings: After selection, 10354 AS events in 140 PDAC patients were profiled. We detected 677 OS-related AS events in 485 genes from Cox model. Computational algorithm results showed that RNA splicing-related pathways may be the potential mechanisms regulating poor prognosis. The AS signatures revealed high performance in predicting PDAC survival and gemcitabine chemoresistance. The area under of the ROC curve for AS-based predictor, constructed with significant survival-associated AS events, was 0.889 at 2000 days of OS. We identified prognostic SFs to build the AS regulatory network, which may be the underlying mechanism of AS events. Interpretation: Genome-wide prognostic AS signature was profiled in PDAC. We created AS-based models for OS and gemcitabine resistance, and revealed the regulatory splicing correlation networks. Funding Statement: This work was supported by grants from the Incubating Program for Clinical Research and Innovation of Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University (Grant No. PYMDT-001), Science and Technology Commission of Shanghai Municipality (Grant No. 16441906903), Three-year action plan for Shin Kang of Shanghai (16CR4005A). Declaration of Interests: The authors declare no potential conflicts of interest.

The Pleiotropic Immunomodulatory Functions of IL-33 and Its Implications in Tumor Immunity.

Interleukin-33 (IL-33) is a IL-1 family member of cytokines exerting pleiotropic activities. In the steady-state, IL-33 is expressed in the nucleus of epithelial, endothelial, and fibroblast-like cells acting as a nuclear protein. In response to tissue damage, infections or necrosis IL-33 is released in the extracellular space, where it functions as an alarmin for the immune system. Its specific receptor ST2 is expressed by a variety of immune cell types, resulting in the stimulation of a wide range of immune reactions. Recent evidences suggest that different IL-33 isoforms exist, in virtue of proteolytic cleavage or alternative mRNA splicing, with potentially different biological activity and functions. Although initially studied in the context of allergy, infection, and inflammation, over the past decade IL-33 has gained much attention in cancer immunology. Increasing evidences indicate that IL-33 may have opposing functions, promoting, or dampening tumor immunity, depending on the tumor type, site of expression, and local concentration. In this review we will cover the biological functions of IL-33 on various immune cell subsets (e.g., T cells, NK, Treg cells, ILC2, eosinophils, neutrophils, basophils, mast cells, DCs, and macrophages) that affect anti-tumor immune responses in experimental and clinical cancers. We will also discuss the possible implications of diverse IL-33 mutations and isoforms in the anti-tumor activity of the cytokine and as possible clinical biomarkers.

PTBP1 contributes to spermatogenesis through regulation of proliferation in spermatogonia.

Polypyrimidine tract-binding protein 1 (PTBP1) is a highly conserved RNA-binding protein that is a well-known regulator of alternative splicing. Testicular tissue is one of the richest tissues with respect to the number of alternative splicing mRNA isoforms, but the molecular role(s) of PTBP1 in the regulation of these isoforms during spermatogenesis is still unclear. Here, we developed a germ cell–specific Ptbp1 conditional knockout (cKO) mouse model by using the Cre-loxP system to investigate the role of PTBP1 in spermatogenesis. Testis weight in Ptbp1 cKO mice was comparable to that in age-matched controls until 3 weeks of age; at ≥ 2 months old, testis weight was significantly lighter in cKO mice than in age-matched controls. Sperm count in Ptbp1 cKO mice at 2 months old was comparable to that in controls, whereas sperm count significantly decreased at 6 months old. Seminiferous tubules that exhibited degeneration in spermatogenic function were more evident in the 2-month-old Ptbp1 cKO mice than in controls. In addition, the early neonatal proliferation of spermatogonia, during postnatal days 1–5, was significantly retarded in Ptbp1 cKO mice compared with that in controls. An in vitro spermatogonia culture model (germline stem cells) revealed that hydroxytamoxifen-induced deletion of PTBP1 from germline stem cells caused severe proliferation arrest accompanied by an increase of apoptotic cell death. These data suggest that PTBP1 contributes to spermatogenesis through regulation of spermatogonia proliferation.

PTBP1 Facilitates Lymphatic Metastasis and Proliferation of Bladder Cancer Via Alternative Splicing of MEIS2 and PKM

Background: Lymph node (LN) metastasis is the leading cause of bladder cancer-related mortality. Splicing factors facilitate cancer progression by modulating oncogenic variants, but it is unclear whether and how splicing factors regulate bladder cancer LN metastasis. Methods: In this study, qRT-PCR was used to identify differential expression of splicing factors between bladder cancer with and without LN metastasis. Associations between expression of the splicing factor polypyrimidine tract binding protein 1 (PTBP1) and clinicopathologic features and prognosis of bladder cancer were analyzed. In vitro and in vivo assays were performed to explore the biological role of PTBP1 in bladder cancer cells. RNA-sequencing, RNA immunoprecipitation, and RNA pull down assays were used to investigate the molecular mechanisms of PTBP1 in bladder cancer. Findings: PTBP1 expression was found to relate to bladder cancer LN metastasis, and was positively correlated with LN metastasis status, tumor stage, histological grade, and poor patient prognosis. Functional assays demonstrated that PTBP1 promoted bladder cancer cell migration, invasion, and proliferation in vitro, as well as LN metastasis and tumor growth in vivo. Mechanistic investigations revealed that PTBP1 upregulated MEIS2-L variant to promote metastasis and increased expression of a PKM2 variant to enhance proliferation by modulating alternative mRNA splicing. Moreover, overexpression of MEIS2-L or PKM2 could rescue the oncogenic abilities of bladder cancer cells after PTBP1 knockdown. Interpretation: Our data demonstrate that PTBP1 induces bladder cancer LN metastasis and proliferation through an alternative splicing mechanism. PTBP1 may serve as a novel prognostic marker and therapeutic target for LN-metastatic bladder cancer. Funding Statement: This study was supported by the National Natural Science Foundation of China (Grant No. 81702523, 81772719, 81772728, 81572514, U1301221, 81472384), National Natural Science Foundation of Guangdong (Grant No. 2016A030313321, 2016A030313244), Science and Technology Program of Guangzhou (Grant No. 201804010041, 201604020156, 201604020177), Project Supported by Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (for Tianxin Lin) , the Fundamental Research Funds for the Central Universities (for Xu Chen, 18ykpy18), Yat-Sen Scholarship for Young Scientist (for Xu Chen), Sun Yat-sen Initiative Program for Scientific Research (for Xu Chen, YXQH201708), and National Clinical Key Specialty Construction Project for Department of Urology and Department of Oncology. Grant KLB09001 from the Key Laboratory of Malignant Tumor Gene Regulation and Target Therapy of Guangdong Higher Education Institutes, Sun-Yat-Sen University. Grant [2013]163 from Key Laboratory of Malignant Tumor Molecular Mechanism and Translational Medicine of Guangzhou Bureau of Science and Information Technology. Declaration of Interests: We declare that we have no conflicts of interest. Ethics Approval Statement: Ethical consent was approved by Sun Yat-sen University’s Committees for Ethical Review of Research involving Human Subjects

Spleen tyrosine kinase SYK(L) interacts with YY1 and coordinately suppresses SNAI2 transcription in lung cancer cells.

Spleen tyrosine kinase (SYK) is a nonreceptor tyrosine kinase with dual properties of an oncoprotein and an oncosuppressor in distinctive cell types. In solid cancers, two isoforms SYK(L) and SYK(S) of SYK were recently identified due to its alternative mRNA splicing. However, the cellular activity and the biological significance of the long isoform of SYK, SYK(L), is still not well defined in human lung cancers. Here, we describe an interaction between SYK(L) and the ubiquitously expressed transcription regulator Yin Yang 1 (YY1) in the nucleus, which suppresses the epithelial-to-mesenchymal transition (EMT) by inactivating SNAI2 (coding transcription factor SLUG) transcription. ChIP indicated that endogenous SYK(L) interacts directly with a YY1 binding cis-regulatory element in the SNAI2 promoter. Importantly, knockdown of YY1 activates SYK(L)-dependent EMT suppression in human lung cancer H1155 cells. We also found that the protein level of SYK(L) is markedly upregulated in various types of human lung cancers, and its nuclear localization is strongly correlated with clinical benefits of lung adenocarcinomas. Collectively, our data reveal a SYK(L)-dependent transcriptional regulation of EMT through SLUG as a potential biomarker for lung cancer aggressiveness.