Serine-rich Domain Research Articles

Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) lncRNA Conformational Dynamics in Complex with RNA-Binding Protein with Serine-Rich Domain 1 (RNPS1) in the Pan-cancer Splicing and Gene Expression.

Alternative splicing events increase the transcriptomic and proteomic complexity in cancers. Overexpression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a highly conserved lncRNA, is widely known to promote cancer development, one mechanism for which may be through the regulation of alternative splicing and, thereby, gene expression. Its regulatory interactions with proteins have been a subject of much interest, yet little research has been carried out on the mechanisms adopted. It has been observed that MALAT1 binds to RNA-binding protein with serine-rich domain 1 (RNPS1), being colocalized in the nuclear speckles, and together, these two binding partners may regulate alternative splicing. Upregulated RNPS1 is predicted to play a key role in the pan-cancer development. Experimental tertiary structure of full-length MALAT1 is currently lacking despite the availability of the 3D structure of 3' expression and nuclear retention element. We hypothesize that the computationally modeled tertiary structures of the specific binding motifs in the M-region, E-region, and full-length structures of MALAT1 may adopt a modular structure and bind to the RNPS1 loop region of RS/P domain involved in exon skipping, interacting in a manner fully consistent with the biochemical experiments. Extensive observations using the powerful molecular dynamics (MD) simulations of MALAT1 regions bound to RNPS1 suggested that all three regions form interactive, yet stable complexes. The ranking of the MM-GBSA- and MM-PBSA-derived binding free energies between these complexes corroborated well in the MD simulations and experiments. Energy decomposition analyses suggested that arginines in the RNPS1 protein are among the major contributors toward the binding free energies as calculated by MM-GBSA present in the Amber package; while among the nucleotides, the major contributors were nucleotides with G and A nucleobases, with more contributory effect in comparison to arginines, across the bound M-region, E-region, and full-length MALAT1. This suggests that specific purines play a greater role in the complex formation, in a loop-specific manner, and the more proactive approach in complexation tilts toward MALAT1. To the best of our knowledge, our studies are the first studies taking a unique approach, utilizing the binding motifs to deduce a tertiary structure of MALAT1, toward our understanding of the lncRNA-protein interactions, stability, and binding on a structural basis. The therapeutic implications of targeting this complex formation to regulate splicing and hence, oncogenesis, is further envisaged.

Comparative proteomic analysis of regenerative mechanisms in mouse retina to identify markers for neuro-regeneration in glaucoma

The retina is part of the central nervous system (CNS). Neurons in the CNS and retinal ganglion cells lack the ability to regenerate axons spontaneously after injury. The intrinsic axonal growth regulators, their interaction and roles that enable or inhibit axon growth are still largely unknown. This study endeavored to characterize the molecular characteristics under neurodegenerative and regenerative conditions. Data-Independent Acquisition Mass Spectrometry was used to map the comprehensive proteome of the regenerative retina from 14-day-old mice (Reg-P14) and adult mice after lens injury (Reg-LI) both showing regrowing axons in vitro, untreated adult mice, and retina from adult mice subjected to two weeks of elevated intraocular pressure showing degeneration. A total of 5750 proteins were identified (false discovery rate < 1%). Proteins identified in both Reg-P14 and Reg-LI groups were correlated to thyroid hormone, Notch, Wnt, and VEGF signaling pathways. Common interactors comprising E1A binding protein P300 (EP300), CREB binding protein (CBP), calcium/calmodulin dependent protein kinase II alpha (CaMKIIα) and sirtuin 1 (SIRT1) were found in both Reg-P14 and Reg-LI retinas. Proteins identified in both regenerating and degenerative groups were correlated to thyroid hormone, Notch, mRNA surveillance and measles signaling pathways, along with PD-L1 expression and the PD-1 checkpoint pathway. Common interactors across regenerative and degenerative retinas comprising NF-kappa-B p65 subunit (RELA), RNA-binding protein with serine-rich domain 1 (RNPS1), EP300 and SIN3 transcription regulator family member A (SIN3A). The findings from our study provide the first mapping of regenerative mechanisms across postnatal, mature and degenerative mouse retinas, revealing potential biomarkers that could facilitate neuro-regeneration in glaucoma.

Uncovering chikungunya virus-encoded miRNAs and host-specific targeted genes associated with antiviral immune responses: an integrated bioinformatics approach

Chikungunya virus (CHIKV) is a single-stranded RNA virus belonging to the genus Alphavirus and is responsible for causing Chikungunya fever, a type of arboviral fever. Despite extensive research, the pathogenic mechanism of CHIKV within host cells remains unclear. In this study, an in-silico approach was used to predict that CHIKV produces micro-RNAs that target host-specific genes associated with host cellular regulatory pathways. Putative micro-RNAs of CHIKV were predicted using the miRNAFold and Vmir RNA structure web servers, and secondary structure prediction was performed using RNAfold. Host-specific target genes were then predicted, and hub genes were identified using CytoHubba and module selection through MCODE. Functional annotations of hub genes revealed their association with various pathways, including osteoclast differentiation, neuroactive ligand-receptor interaction, and mRNA surveillance. We used the freely available dataset GSE49985 to determine the level of expression of host-specific target genes and found that two genes, F-box and leucine-rich repeat protein 16 (FBXL16) and retinoic acid receptor alpha (RARA), were down-regulated, while four genes, RNA binding protein with serine-rich domain 1 (RNPS1), RNA helicase and ATPase (UPF1), neuropeptide S receptor 1 (NPSR1), and vasoactive intestinal peptide receptor 1 (VIPR1), were up-regulated. These findings provide insight into novel miRNAs and hub genes associated with CHIKV infection and suggest potential targets for therapeutic intervention. Further experimental validation of these targets could lead to the development of effective treatments for CHIKV-mediated diseases.

Emerging concepts of myosin 18A isoform mechanobiology in organismal and immune system physiology, development, and function.

Alternative and combinatorial splicing of myosin 18A (MYO18A) gene transcripts results in expression of MYO18A protein isoforms and isoform variants with different membrane and subcellular localizations, and functional properties. MYO18A proteins are members of the myosin superfamily consisting of a myosin-like motor domain, an IQ motif, and a coiled-coil domain. MYO18A isoforms, however, lack the ability to hydrolyze ATP and do not perform ATP-dependent motor activity. MYO18A isoforms are distinguished by different amino- and carboxy-terminal extensions and domains. The domain organization and functions of MYO18Aα, MYO18Aβ, and MYO18Aγ have been studied experimentally. MYO18Aα and MYO18Aβ have a common carboxy-terminal extension but differ by the presence or absence of an amino-terminal KE repeat and PDZ domain, respectively. The amino- and carboxy-terminal extensions of MYO18Aγ contain unique proline and serine-rich domains. Computationally predicted MYO18Aε and MYO18Aδ isoforms contain the carboxy-terminal serine-rich extension but differ by the presence or absence of the amino-terminal KE/PDZ extension. Additional isoform variants within each category arise by alternative utilization or inclusion/exclusion of small exons. MYO18Aα variants are expressed in somatic cells and mature immune cells, whereas MYO18Aβ variants occur mainly in myeloid and natural killer cells. MYO18Aγ expression is selective to cardiac and skeletal muscle. In the present review perspective, we discuss current and emerging concepts of the functional specialization of MYO18A proteins in membrane and cytoskeletal dynamics, cellular communication and signaling, endocytic and exocytic organelle movement, viral infection, and as the SP-R210 receptor for surfactant protein A.

RNPS1 stabilizes NAT10 protein to facilitate translation in cancer via tRNA ac4C modification

Existing studies have underscored the pivotal role of N-acetyltransferase 10 (NAT10) in various cancers. However, the outcomes of protein-protein interactions between NAT10 and its protein partners in head and neck squamous cell carcinoma (HNSCC) remain unexplored. In this study, we identified a significant upregulation of RNA-binding protein with serine-rich domain 1 (RNPS1) in HNSCC, where RNPS1 inhibits the ubiquitination degradation of NAT10 by E3 ubiquitin ligase, zinc finger SWIM domain-containing protein 6 (ZSWIM6), through direct protein interaction, thereby promoting high NAT10 expression in HNSCC. This upregulated NAT10 stability mediates the enhancement of specific tRNA ac4C modifications, subsequently boosting the translation process of genes involved in pathways such as IL-6 signaling, IL-8 signaling, and PTEN signaling that play roles in regulating HNSCC malignant progression, ultimately influencing the survival and prognosis of HNSCC patients. Additionally, we pioneered the development of TRMC-seq, leading to the discovery of novel tRNA-ac4C modification sites, thereby providing a potent sequencing tool for tRNA-ac4C research. Our findings expand the repertoire of tRNA ac4C modifications and identify a role of tRNA ac4C in the regulation of mRNA translation in HNSCC.

The regulatory mechanisms of IRF7 mediated by the type I IFN signalling pathway against Streptococcus iniae in yellowfin seabream, Acanthopagrus latus (Hottuyn, 1782)

Interferon regulatory factor 7 (IRF7) regulates type I interferon (IFN) genes via combining to the ISRE region in the immune response against bacteria. Streptococcus iniae is one of the dominant pathogenic bacteria of yellowfin seabream, Acanthopagrus latus. However, the regulatory mechanisms of A. latus IRF7 (AlIRF7) mediated by the type I IFN signalling pathway against S. iniae was ambiguously. In the present study, IRF7, and two IFNa3s (IFNa3 and IFNa3-like) were authenticated from A. latus. The total length of AlIRF7 cDNA is 2142 bp, containing a 1314 bp open reading frame (ORF) encoding an inferred 437 amino acids (aa). Three typical regions, a serine-rich domain (SRD), a DNA-binding domain (DBD), and an IRF association domain (IAD), are conserved in AlIRF7. Furthermore, AlIRF7 is fundamentally expressed in various kinds of organs, with high levels in the spleen and liver. Additionally, S. iniae challenge promoted AlIRF7 expression in the spleen, liver, kidney, and brain. AlIRF7 is confirmed to be located at the nucleus and cytoplasm by overexpression of AlIRF7. Moreover, truncation mutation analyses shows that the regions, −821 bp to +192 bp and −928 bp to +196 bp, were known as core promoters from AlIFNa3 and AlIFNa3-like, respectively. The point mutation analyses and electrophoretic mobile shift assay (EMSA) verified that AlIFNa3 and AlIFNa3-like transcriptions are depended on the M2/5 and M2/3/4 binding sites with AlIRF7 regulation, respectively. Additionally, an overexpression experiment showed that AlIRF7 can dramatically decrease the mRNA levels of two AlIFNa3s and interferon signalling molecules. These results suggest that two IFNa3s may mediate the regulation of AlIRF7 in the immune responses of A. latus against S. iniae infection.

Serine-rich domain of RNPS1 functions in activation of alternative splicing.

RNA-binding protein with serine-rich domain 1 (RNPS1) gets deposited on the mRNA during the process of splicing and concomitantly associates with the exon junction complex (EJC). RNPS1 participates in post-transcriptional gene regulation, including constitutive and alternative splicing, transcriptional regulation and nonsense-mediated mRNA decay. In this study, we found that the tethering of RNPS1 or its isolated serine-rich domain (S domain) causes exon inclusion of an HIV-1 splicing substrate. In contrast, overexpressing the RRM domain of RNPS1 acts in a dominant negative manner and leads to the exon skipping of endogenous apoptotic pre-mRNAs (Bcl-X and MCL-1). Further, tethering of core EJC proteins, eIF4A3, MAGOH, or Y14, does not lead to exon inclusion of an HIV substrate. Together, our results demonstrate how RNPS1 and its domains are differentially involved in alternative splicing activity.

The first discovery of Tc1 transposons in yeast.

Identification of transposons without close homologs is still a difficult task. IS630/Tc1/mariner transposons, classified into a superfamily, are probably the most widespread DNA transposons in nature. Tc1/mariner transposons have been discovered in animals, plants, and filamentous fungi, however, not in yeast. In the present study, we report the discovery of two intact Tc1 transposons in yeast and filamentous fungi, respectively. The first one, named Tc1-OP1 (DD40E), represents Tc1 transposons in Ogataea parapolymorpha. The second one, named Tc1-MP1 (DD34E), represents Tc1 transposons in the Rhizopodaceae and Mucoraceae families. As a homolog of Tc1-OP1 and Tc1-MP1, IS630-AB1 (DD34E) was discovered as an IS630 transposon in Acinetobacter spp. Tc1-OP1 is not only the first reported Tc1 transposon in yeast, but also the first reported nonclassical Tc1 transposon. Tc1-OP1 is the largest of IS630/Tc1/mariner transposons reported to date and significantly different from others. Notably, Tc1-OP1 encodes a serine-rich domain and a transposase, extending the current knowledge of Tc1 transposons. The phylogenetic relationships of Tc1-OP1, Tc1-MP1 and IS630-AB1 indicated that these transposons had evolved from a common ancestor. Tc1-OP1, Tc1-MP1 and IS630-AB1 can be used as reference sequences to facilitate the identification of IS630/Tc1/mariner transposons. More Tc1/mariner transposons will be identified in yeast, following our discovery.

Molecular characteristics, expression analysis and antiviral activity against RSIV of red sea bream IRF3 and IRF7

Interferon regulatory factor (IRF) 3 and IRF7 are important innate immune-related genes that induce type I interferon (IFN). In this study, PmIRF3 and PmIRF7 were identified from red sea bream (Pagrus major) infected by red sea bream iridovirus (RSIV), and expression and functional analyses were performed. PmIRF3 and PmIRF7 consisted of 465 and 436 amino acids, respectively, and contained a well-conserved IRF domain, IRF3 domain, and serine-rich domain (SRD). PmIRF3 and PmIRF7 are ubiquitously expressed in the liver and gills in normal fish, and with a high expression being recorded in kidney and spleen during RSIV infection. In P. major fin cells (PMF cells), pAcGFP-PmIRF3 and pAcGFP-PmIRF7 were localized to the cytoplasm, and overexpression of PmIRF3 and PmIRF7 using the pcDNA3.1 vector induced type I IFN. In addition, in the experiment in vitro, PMF cells overexpressed with PmIRF3 or PmIRF7 exhibited antiviral activity against RSIV and significantly reduced viral genome copies and virus titer. Therefore, PmIRF3 and PmIRF7 may have antiviral activity through the induction of type I IFN against RSIV infection of red sea bream.

RNPS1 functions as an oncogenic splicing factor in cervical cancer cells.

Numerous recent studies suggest that cancer-specific splicing alteration is a critical contributor to the pathogenesis of cancer. RNA-binding protein with serine-rich domain 1, RNPS1, is an essential regulator of the splicing process. However, the defined role of RNPS1 in tumorigenesis still remains elusive. We report here that the expression of RNPS1 is higher in cervical carcinoma samples from The Cancer Genome Atlas (TCGA-cervical squamous cell carcinoma and endocervical adenocarcinoma) compared to the normal tissues. Consistently, the expression of RNPS1 was high in cervical cancer cells compared to a normal cell line. This study shows for the first time that RNPS1 promotes cell proliferation and colony-forming ability of cervical cancer cells. Importantly, RNPS1 positively regulates migration-invasion of cervical cancer cells. Intriguingly, depletion of RNPS1 increases the chemosensitivity against the chemotherapeutic drug doxorubicin in cervical cancer cells. Further, we characterized the genome-wide isoform switching stimulated by RNPS1 in cervical cancer cell. Mechanistically, RNA-sequencing analysis showed that RNPS1 regulates the generation of tumor-associated isoforms of key genes, particularly Rac1b, RhoA, MDM4, and WDR1, through alternative splicing. RNPS1 regulates the splicing of Rac1 pre-mRNA via a specific alternative splicing switch and promotes the formation of its tumorigenic splice variant, Rac1b. While the transcriptional regulation of RhoA has been well studied, the role of alternative splicing in RhoA upregulation in cancer cells is largely unknown. Here, we have shown that the knockdown of RNPS1 in cervical cancer cells leads to the skipping of exons encoding the RAS domain of RhoA, consequently causing decreased expression of RhoA. Collectively, we conclude that the gain of RNPS1 expression may be associated with tumor progression in cervical carcinoma. RNPS1-mediated alternative splicing favors an active Rac1b/RhoA signaling axis that could contribute to cervical cancer cell invasion and metastasis. Thus, our work unveils a novel role of RNPS1 in the development of cervical cancer.

Histological, antioxidant, apoptotic and transcriptomic responses under cold stress and the mitigation of blue wavelength light of zebrafish eyes

As global climate changes, severely temperature variations have significant impacts on survival and development of fish. While the potential effects of light wavelength on cold shock and associated mechanisms remain largely unknown in fish. Here, zebrafish (Danio rerio) were pre-exposed to white LEDs (Light emitting diodes) at an irradiance of 0.9 W/m2 and blue LEDs (LDB, peak at 450 nm, 0.9 W/m2) for 2 weeks, and then exposed to 26 ℃ or 11 ℃ for 48 h, respectively. Cold shock led to low survival rate. Cold shock altered retinal structure, increased the number of apoptotic cells and Caspase-3 activity, inhibited superoxide dismutase (SOD) and catalase (CAT) activities, up-regulated mRNA expression of nrf2 (NF-E2-related factor 2), p53 (Tumor suppressor), casp3 (Caspase-3) and casp9 (Caspase-9), and down-regulated cat (Catalase) expression in fish eyes. These results demonstrated that acute cold exposure induced oxidative stress and apoptosis in zebrafish eyes, which may lead to mortality. However, cold shock in combination with LDB apparently mitigated these negative effects, which might be involved in the up-regulation of antioxidant response and down-regulation of apoptotic responses at transcriptional and translational levels. Furthermore, cold shock also caused dysregulation of genome-wide gene expression involved in circadian rhythm, phototransduction and il-17 signaling pathway, indicating that cold shock disturbed phototransduction cascade and circadian rhythm signals and caused inflammatory responses. Ten key genes involved in circadian rhythm, phototransduction, cell cycle arrest, RNA processing or inflammatory responses were identified, including muc5d (Mucin 5d), rnps1 (RNA-binding protein with serine-rich domain 1), si:dkey-243i1.1, opn1mw1 (Medium-wave-sensitive opsin 3), gadd45ba (Growth arrest and DNA damage-inducible protein), cebpd (CCAAT/enhancer binding protein delta), btg2 (Anti-proliferative cofactor), si:dkey-242g16.2, nr1d1 (Nuclear receptor subfamily 1 group D member 1) and zgc:122979, which may play an important role in the protection of LDB against cold shock. Finally, our study suggested the relationship between spectrum and cold stress and demonstrated LDB could protect fish against the negative effect of cold stress in the eyes.

Molecular cloning, expression and generation of a polyclonal antibody specific for RNPS1.

RNA-binding protein with serine-rich domain 1 (RNPS1) is a member of a splicing-dependent mega Dalton protein complex or exon junction complex (EJC). During splicing, RNPS1 acts as a protector of global transcriptome integrity by suppressing the usage of cryptic splice sites. Additionally, RNPS1 functions in almost all stages of mRNA metabolism, including constitutive splicing, alternative splicing, translation and nonsense-mediated mRNA decay (NMD). The aim of the present study was to generate a highly specific polyclonal antibody against human RNPS1. A plasmid, pHis-TEV-RNPS1, has been constructed to overexpress recombinant RNPS1 (22-305 amino acids) by cloning the nucleotide sequence downstream of an N-terminal His-tag in the parent plasmid pHis-TEV. The recombinant plasmid was then transformed into Rosetta and expression was induced using IPTG. The His-tagged RNPS1 protein was purified using Ni-NTA affinity chromatography. The rabbit antiserum was then obtained by immunizing rabbits with the purified recombinant RNPS1 protein. The antiserum was further purified by antigen-immunoaffinity chromatography. The sensitivity and the specificity of the polyclonal antibody were assessed by enzyme-linked immunosorbent assay (ELISA) and knockdown assay. ELISA demonstrated that the antibody has a high binding affinity for RNPS1 and the usable titre is 1:2000. The antibody detected RNPS1 in human, mouse cell lines and rat tissue in Western blot. Importantly, the antibody efficiently detected the decrease in RNPS1 expression in siRNA induced knockdown assay, indicating the specificity of the antibody. The polyclonal antibody against RNPS1 will be a useful tool for performing further functional studies on RNPS1.

Mouse Model of Loeys-Dietz Syndrome Shows Elevated Susceptibility to Periodontitis via Alterations in Transforming Growth Factor-Beta Signaling.

Loeys–Dietz syndrome (LDS) is a syndromic connective tissue disorder caused by a heterozygous missense mutation in genes that encode transforming growth factor (TGF)-β receptor (TGFBR) 1 and 2. We encountered a patient with LDS, who had severe periodontal tissue destruction indicative of aggressive periodontitis. The patient had a missense mutation in the glycine and serine-rich domain of TGFBR1 exon 3. This G-to-T mutation at base 563 converted glycine to valine. We established an LDS model knock-in mouse that recapitulated the LDS phenotype. Homozygosity of the mutation caused embryonic lethality and heterozygous knock-in mice showed distorted and ruptured elastic fibers in the aorta at 24 weeks of age and died earlier than wildtype (WT) mice. We stimulated mouse embryonic fibroblasts (MEFs) from the knock-in mouse with TGF-β and examined their responses. The knock-in MEFs showed downregulated Serpine 1 mRNA expression and phosphorylation of Smad2 to TGF-β compared with WT MEFs. To clarify the influence of TGF-β signaling abnormalities on the pathogenesis or progression of periodontitis, we performed pathomolecular analysis of the knock-in mouse. There were no structural differences in periodontal tissues between WT and LDS model mice at 6 or 24 weeks of age. Micro-computed tomography revealed no significant difference in alveolar bone resorption between WT and knock-in mice at 6 or 24 weeks of age. However, TGF-β-related gene expression was increased significantly in periodontal tissues of the knock-in mouse compared with WT mice. Next, we assessed a mouse periodontitis model in which periodontal bone loss was induced by oral inoculation with the bacterial strain Porphyromonas gingivalis W83. After inoculation, we collected alveolar bone and carried out morphometric analysis. P. gingivalis-induced alveolar bone loss was significantly greater in LDS model mice than in WT mice. Peritoneal macrophages isolated from Tgfbr1G188V/+ mice showed upregulation of inflammatory cytokine mRNA expression induced by P. gingivalis lipopolysaccharide compared with WT macrophages. In this study, we established an LDS mouse model and demonstrated that LDS model mice had elevated susceptibility to P. gingivalis-induced periodontitis, probably through TGF-β signal dysfunction. This suggests that TGF-β signaling abnormalities accelerate the pathogenesis or progression of periodontitis.

SRSF10: an atypical splicing regulator with critical roles in stress response, organ development, and viral replication

Serine/arginine splicing factor 10 (SRSF10) is a member of the family of mammalian splicing regulators known as SR proteins. Like several of its SR siblings, the SRSF10 protein is composed of an RNA binding domain (RRM) and of arginine and serine-rich auxiliary domains (RS) that guide interactions with other proteins. The phosphorylation status of SRSF10 is of paramount importance for its activity and is subjected to changes during mitosis, heat-shock, and DNA damage. SRSF10 overexpression has functional consequences in a growing list of cancers. By controlling the alternative splicing of specific transcripts, SRSF10 has also been implicated in glucose, fat, and cholesterol metabolism, in the development of the embryonic heart, and in neurological processes. SRSF10 is also important for the proper expression and processing of HIV-1 and other viral transcripts. We discuss how SRSF10 could become a potentially appealing therapeutic target to combat cancer and viral infections.

Abstract PO088: Sensitivity of cancer cells to oncolytic viruses is defined by IWS1 phosphorylation dependent epigenetic regulation of U2AF2 splicing and nucleocytoplasmic export of type I IFN transcripts

Abstract We have previously shown that loss of IWS1 phosphorylation promotes the alternative RNA splicing of U2 Associated-Factor 2 (U2AF2), resulting in transcripts lacking exon 2. This exon encodes part of the Serine-Rich (SR) domain of U2AF65, which is responsible for its binding with pre-mRNA Processing Factor 19 (Prp19). Here, we show that whereas both U2AF65 isoforms bind cytoplasmic accumulation response elements (CAR-E) of intronless mRNAs, the loading of Prp19 occurs only in exon 2-containing U2AF65, in cells expressing phosphorylated IWS1, promoting their nucleocytoplasmic export. Furthermore, this Prp19 loading is RNA Pol II dependent. Given that IFNA1 and IFNB1 are among the target genes, the expression of IFNα and IFNβ was decreased in cells deficient in IWS1 phosphorylation, and their sensitivity to the oncolytic VSV and Reovirus virus infection was increased accordingly. More importantly, treatment of the lung adenocarcinoma cells with the pan-AKT inhibitor, MK2206 phenocopied the loss of IWS1 phosphorylation and showed increased sensitivity to oncolytic viral infection. These data identify a novel mechanism by which the AKT/p-IWS1 axis, via the epigenetic regulation of alternative RNA splicing, contribute to the sensitivity to oncolytic viruses. Citation Format: Georgios I. Laliotis, Adam D. Kenney, Evangelia Chavdoula, Arturo Orlacchio, Vollter Anastas, Alessandro La Ferlita, Abdul Kaba, Lalit Sehgal, Vincenzo Coppola, Jacob S. Yount, Philip N. Tsichlis. Sensitivity of cancer cells to oncolytic viruses is defined by IWS1 phosphorylation dependent epigenetic regulation of U2AF2 splicing and nucleocytoplasmic export of type I IFN transcripts [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO088.

Abstract PO010: The inhibition of IWS1 phosphorylation promotes genomic instability, the cGAS/STING pathway activation and PD-L1 levels, through the U2AF2 alternative RNA splicing and Sororin expression

Abstract We have previously shown that the loss of IWS1 phosphorylation promotes the alternative RNA splicing of U2 Associated-Factor 2 (U2AF2), resulting in transcripts lacking exon 2 in lung adenocarcinoma cells. This exon encodes part of the U2AF65 Serine-Rich (SR) Domain, which is required for its binding with pre-mRNA Processing factor 19 (Prp19). We have also shown that inhibition of the pathway results in the downregulation of cell cycle division associated 5 (CDCA5), and its protein product Sororin, a phosphorylation target and regulator of ERK and member of the cohesin complex, leading to G2/M phase arrest, sister-chromatid cohesion defects, impaired cell proliferation and tumor growth in mouse xenografts models. Here, we show that the loss of IWS1 results in genomic instability and accumulation of cytosolic dsDNA, an effect rescued by the expression of Sororin. The dsDNA is censored by cyclic GMP-AMP synthetase (cGAS), activating the STING/TBK1 pathway. The latter leads to increased expression of Interferon Regulatory Factor-3 (IRF-3) targets along with PD-L1. More importantly, IWS1 phosphorylation, U2AF2 RNA splicing pattern and Sororin expression negatively correlate with the cGAS/STING pathway and PD-L1 expression in lung adenocarcinoma patients. These results highlight the role of IWS1 phosphorylation-dependent RNA splicing in governing genomic stability, and proposes this axis as a novel drug target for a synergy with PD-L1/PD-1 blockade in lung adenocarcinoma patients. Citation Format: Georgios I. Laliotis, Evangelia Chavdoula, Abdul Kaba, Alessandro La Ferlita, Vollter Anastas, Arturo Orlacchio, Lalit Sehgal, David P. Carbone, Vincenzo Coppola, Philip N. Tsichlis. The inhibition of IWS1 phosphorylation promotes genomic instability, the cGAS/STING pathway activation and PD-L1 levels, through the U2AF2 alternative RNA splicing and Sororin expression [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO010.

Identification and functional characterization of three irf7 transcript variants in obscure puffer (Takifugu obscurus)

Interferon regulatory factor 7 (IRF7) is a key mediator in regulating the type Ι IFN response. Although irf7 has been identified in more than twenty fish species, alternative splicing has not been found in teleost irf7. Alternative splicing is an important mechanism expanding the transcriptomic and proteomic diversity, and has been found in several IRF family members. Here, three alternative splicing variants of irf7 were identified and characterized in obscure puffer. The first splicing transcript (Toirf7v1) was predicted to encode 428 amino acids with a DNA-binding domain (DBD), an interaction-associated domain (IAD) and a serine-rich domain (SRD). Toirf7v2 encoded 430 amino acids caused by the intron retention, and contained the whole conserved domains. Toirf7v3 encoded a truncated protein with 337 amino acids resulting from the alternative 5′ splice-site selection, and lacked part of IAD domain and the entire SRD domain. Functional studies demonstrated that all of the three isoforms could activate the expression of type I IFN and IFN-stimulated genes (ISGs). Nevertheless, the two variants (Toirf7v2 and Toirf7v3) exhibited much less ability to induce transcription of IFN and ISGs compared to the Toirf7v1. Our findings suggest that these splicing variants may have distinct roles in the regulation of immune response. These results will be beneficial to understand the functional characteristics of irf7 variants in fish.

RNA-binding protein with serine-rich domain 1 regulates microsatellite instability of uterine corpus endometrial adenocarcinoma

OBJECTIVE:To determine the role of RNA-binding protein with serine-rich domain 1 (RNPS1) in uterine corpus endometrial carcinoma (UCEC), the role of RNPS1 knockdown in UCEC development in vitro and in vivo, and the relationship between RNPS1 and mismatch repair (MMR) in UCEC.METHODS:We predicted the potential function of RNPS1 using bioinformatics systems. The expression of RNPS1 in tissues and cell lines was analyzed by western blotting and immunohistochemistry. The expression of RNPS1 in MMR was assessed using bioinformatics and western blotting. The proliferation and apoptosis of UCEC cells were assessed under RNPS1 knockdown conditions, and RNPS1 regulation in MMR was detected by suppressing Notch signaling. Associations between RNPS1 and gene mutations in UCEC and prognosis were analyzed.RESULTS:The RNPS1 level was higher in UCEC tumors than in normal tissues and tumors or RL952 cells. Prognostic outcomes were worse when UCEC showed abundant RNPS1 expression. Lentiviral RNPS1 knockdown weakened tumor cell proliferation and suppressed biomarker expression, reduced the tumor volume, promoted apoptosis in vitro and in vivo, and inhibited UCEC development. Increased MutS homolog 2 (MSH2) and MutS homolog 6 (MSH6) levels in MMR after RNPS1 knockdown were reversed by inhibiting Notch signaling. Furthermore, RNPS1 was associated with mutations in NAA11, C2orf57, NUPR1, and other genes involved in UCEC prognosis.CONCLUSION: RNPS1 may regulate the expression levels of MSH2 and MSH6 in MMR, enhancing the proliferation, development, and prognosis of UCEC through a Notch signaling pathway in UCEC. Our study offers a new method and strategy for delaying UCEC development through modulating MMR.

Interferon regulatory factor 7 contributes to the host response during Vibrio harveyi infection in the golden pompano Trachinotus ovatus

Vibrio harveyi is regarded as serious pathogen for marine fishes. However, host defense mechanisms involved in V. harveyi infection remain incompletely defined. The transcription factor IFN regulatory factor 7 (IRF7) is largely associated with host defense against viral infections, and the role of IRF7 during V. harveyi infection in fish has not been well illuminated previously. In this study, IRF7 from golden pompano (Trachinotus ovatus) was characterized (TroIRF7). The TroIRF7 gene is 1323 bp, which encodes 440 amino acid residues. Multiple amino acid alignments of TroIRF7 shows 30.37%–80.18% identity with other fish IRF7s, including Epinephelus coioides (80.18%), Larimichthys crocea (79.72%), Collichthys lucidus (79.26%), Miichthys miiuy (79.26%), Channa argus (78.77%), Cynoglossus semilaevis (72.67%), and Gadus morhua (65.23%). Like other IRF7s, TroIRF7 also contains 3 conserved domains: an N-terminal DNA-binding domain (DBD), an IRF association domain (IAD), and a C-terminal serine-rich domain (SRD). In the DBD, 4–5 conserved tryptophans were observed, which is a characteristic unique to all fish IRF7 members. TroIRF7 was constitutively expressed, with high levels in gill, head kidney, spleen, skin, and intestine. V. harveyi infection-induced TroIRF7 transcripts significantly up-regulation and translocation to the nucleus. TroIRF7 overexpression promote the fish to inhibit the replication of V. harveyi. And TroIRF7 knockdown led to decreased bacterial clearance in fish tissue. Furthermore, over-expression of TroIRF7 resulted in an increased production of interferon a3 and IFN signaling molecule in the spleen, suggesting that V. harveyi activates the IRF7– IFN pathway. These results suggest that TroIRF7 is an important component of immune responses against V. harveyi infection.

Abstract PO-011: IWS1 phosphorylation promotes tumor growth and predicts poor prognosis in EGFR mutant lung adenocarcinoma patients, through the epigenetic regulation of U2AF2 RNA splicing

Abstract Our previous studies have shown that IWS1 (Interacts with Spt6) is a phosphorylation target of AKT and regulates the alternative RNA splicing of FGFR2, linking IWS1 with human Non-Small Cell Lung Cancer. To further address the role of IWS1 in alternative RNA splicing in lung cancer, we performed an RNA-seq study using lung adenocarcinoma cells in which IWS1 was knocked down or replaced by its phosphorylation site mutant. The results identified a novel, exon 2 deficient splice variant of the splicing factor U2 Associated-Factor 2 (U2AF2), whose abundance increases, upon the loss of phosphorylated IWS1. This exon encodes part of the U2AF65 Serine-Rich (SR) Domain, which is required for its binding with pre-mRNA Processing factor 19 (Prp19). Here, we show that U2AF2 exon 2 inclusion depends on phosphorylated IWS1, by promoting histone H3K36 trimethylation and the assembly of LEDGF/SRSF1 splicing complexes, in a cell-cycle specific manner. Inhibition of the pathway results in the downregulation of cell cycle division associated 5 (CDCA5), and its protein product, Sororin, a phosphorylation target of ERK and member of the cohesin complex, essential of G2/M phase progression. We also reveal the existence of a novel Sororin/ERK feedback loop controlled by the epigenetic regulation of U2AF2 RNA splicing, downstream of IWS1 phosphorylation. Given that the U2AF2 RNA splicing is regulated through the cell cycle and controls Sororin, our data unravel a novel RNA splicing pattern which is regulated through the cell cycle and feedbacks towards its regulation. Impairment of this signaling pathway leads to leading to G2/M phase arrest, impaired cell proliferation and tumor growth in mouse xenografts models, an effect more pronounced in EGFR mutant cells. Analysis of lung adenocarcinoma samples revealed strong correlations between IWS1 phosphorylation, U2AF2 RNA splicing, and Sororin/p-ERK levels, especially in EGFR, as opposed to K-RAS mutant patients. More importantly, IWS1 phosphorylation and U2AF2 RNA splicing pattern are positively correlated with tumor stage, grade and metastasis, and associated with poor survival in the same patients. This work highlights the instrumental role of the AKT/p-IWS1 axis to alternative RNA splicing in governing cell cycle progression and tumorigenesis and proposes this axis as a novel drug target in EGFR mutant lung adenocarcinoma, by concomitantly affecting the epigenetic regulation of RNA processing and oncogenic signals. Citation Format: Georgios I. Laliotis, Evangelia Chavdoula, Maria D. Paraskevopoulou, Abdul Kaba, Alessandro La Ferlita, Vollter Anastas, Arturo Orlacchio, Vasiliki Taraslia, Ioannis Vlachos, Marina Capece, Artemis Hatzigeorgiou, Dario Palmieri, Salvatore Alaimo, Christos Tsatsanis, Lalit Sehgal, David P. Carbone, Vincenzo Coppola, Philip N. Tsichlis. IWS1 phosphorylation promotes tumor growth and predicts poor prognosis in EGFR mutant lung adenocarcinoma patients, through the epigenetic regulation of U2AF2 RNA splicing [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-011.