Splicing factor proline and glutamine-rich is a prognostic biomarker and correlated with clinical pathologic features and immune infiltrates in hepatocellular carcinoma

Background:Hepatocellular carcinoma(HCC)is the fourth leading cause of cancer-related deaths globally.Splicing factor proline and glutamine-rich(SFPQ)is a multifunctional protein that controls various biological functions.As a potential therapeutic target and a promising prognostic indicator,the potential effects and processes of SFPQ in HCC require further investigation.Methods:The RNA sequencing data were obtained from the Gene Expression Omnibus,International Cancer Genome Consortium,and The Cancer Genome Atlas databases to analyze SFPQ expression and differentially expressed genes(DEGs).We utilized the LinkedOmics database to identify co-expressed genes.A Venn diagram was constructed to determine the overlapping genes between the DEGs and the co-expressed genes.Functional enrichment analysis was performed on the overlapping genes and DEGs.Furthermore,our study involved functional enrichment analysis,a protein-protein interaction network analysis,and an analysis of immune cell infiltration.The cBioPortal and Tumor Immune Single-cell Hub were utilized to investigate the genetic alterations of SFPQ and the single-cell transcriptome visualization of the tumor microenvironment.A ceRNA network was established with the assistance of the ENCORI website.Finally,we elucidated the clinical significance of SFPQ in HCC by employing Kaplan-Meier survival analysis,univariate and multivariate Cox regression,and prognostic nomogram models.Results:The expression of SFPQ in HCC tissues was significantly elevated compared to normal tissues.GSEA results indicated that increased expression of SFPQ was associated with pathways related to HCC.The ceRNA network,including SFPQ,hsa-miR-101-3p,AC023043.4,AC124798.1,AC145207.5,and GSEC,was constructed with the assistance of ENCORI.High SFPQ expression was related to a poor prognosis in HCC and its subtypes.Univariate and multivariate Cox regression analysis showed that elevated SFPQ expression is an independent predictive factor.Conclusions:The overexpression of SFPQ may serve as a potential prognostic biomarker,indicating a poor prognosis in HCC.

Serine and arginine rich splicing factor 1:a potential target for neuroprotection and other diseases

Alternative splicing is the process of producing variably spliced mRNAs by choosing distinct combinations of splice sites within a messenger RNA precursor.This splicing enables mRNA from a single gene to synthesize different proteins,which have different cellular properties and functions and yet arise from the same single gene.A family of splicing factors,Serine-arginine rich proteins,are needed to initiate the assembly and activation of the spliceosome.Serine and arginine rich splicing factor 1,part of the arginine/serine-rich splicing factor protein family,can either activate or inhibit the splicing of mRNAs,depending on the phosphorylation status of the protein and its interaction partners.Considering that serine and arginine rich splicing factor 1 is either an activator or an inhibitor,this protein has been studied widely to identify its various roles in different diseases.Research has found that serine and arginine rich splicing factor 1 is a key target for neuroprotection,showing its promising potential use in therapeutics for neurodegenerative disorders.Furthermore,serine and arginine rich splicing factor 1 might be used to regulate cancer development and autoimmune diseases.In this review,we highlight how serine and arginine rich splicing factor 1 has been studied concerning neuroprotection.In addition,we draw attention to how serine and arginine rich splicing factor 1 is being studied in cancer and immunological disorders,as well as how serine and arginine rich splicing factor 1 acts outside the central or peripheral nervous system.

Expression and Purification of Serine/Arginine-Rich Splicing Factor 1 from Escherichia coli Expression System

Serine/arginine-rich splicing factor 1(SRSF1), as a prototype member of the highly conserved serine/arginine family of RNA binding proteins, plays an important role in mRNA alternative splicing, stabilization, nuclear export, and translation. Here, the expression system was established to purify full-length human SRSF1 from Escherichia coli(E. coli). The SRSF1 coding sequence was amplified by polymerase chain reaction(PCR) and inserted into the pET-28 a-ppSUMO vector with His-tag to construct a recombinant plasmid His-SUMO-SRSF1. Then the plasmid was transformed into BL21(DE3) competent cells for expression. After purification by affinity chromatography and cleavage of His-SUMO moiety, a highly purified SRSF1 with a molecular weight of around 28 kg/mol was obtained. The protein was analyzed by sizing chromatography and it was found that SRSF1 would form a polymer structure in the solution. According to Expasy bioinformatics analysis, SRSF1 is extremely unstable. Purification of full-length SRSF1 protein provides an opportunity to study mRNA splicing in vitro.

lncR-GAS5 upregulates the splicing factor SRSF10 to impair endothelial autophagy,leading to atherogenesis

Long noncoding RNAs(lncRNAs)play a critical role in the regulation of atherosclerosis.Here,we investigated the role of the lncRNA growth arrest-specific 5(lncR-GAS5)in atherogenesis.We found that the enforced expression of lncR-GAS5 contributed to the development of atherosclerosis,which presented as increased plaque size and reduced collagen content.Moreover,impaired autophagy was observed,as shown by a decreased LC3II/LC3I protein ratio and an elevated P62 level in lncR-GAS5-overexpressing human aortic endothelial cells.By contrast,lncR-GAS5 knockdown promoted autophagy.Moreover,serine/arginine-rich splicing factor 10(SRSF10)knockdown increased the LC3II/LC3I ratio and decreased the P62 level,thus enhancing the formation of autophagic vacuoles,autolysosomes,and autophagosomes.Mechanistically,lncR-GAS5 regulated the downstream splicing factor SRSF10 to impair autophagy in the endothelium,which was reversed by the knockdown of SRSF10.Further results revealed that overexpression of the lncR-GAS5-targeted gene miR-193-5p promoted autophagy and autophagic vacuole accumulation by repressing its direct target gene,SRSF10.Notably,miR-193-5p overexpression decreased plaque size and increased collagen content.Altogether,these findings demonstrate that lncR-GAS5 partially contributes to atherogenesis and plaque instability by impairing endothelial autophagy.In conclusion,lncR-GAS5 overexpression arrested endothelial autophagy through the miR-193-5p/SRSF10 signaling pathway.Thus,miR-193-5p/SRSF10 may serve as a novel treatment target for atherosclerosis.

Splicing factor SF3B3,a NS5-binding protein,restricts ZIKV infection by targeting GCH1

Zika virus(ZIKV),a positive-sense single-stranded RNA virus,causes congenital ZIKV syndrome in children and Guillain-Barre Syndrome(GBS)in adults.ZIKV expresses nonstructural protein 5(NS5),a large protein that is essential for viral replication.ZIKV NS5 confers the ability to evade interferon(IFN)signalling;however,the exact mechanism remains unclear.In this study,we employed affinity pull-down and liquid chromatography-tandem mass spectrometry(LC-MS/MS)analyses and found that splicing factor 3b subunit 3(SF3B3)is associated with the NS5-Flag pull-down complex through interaction with NS5.Functional assays showed that SF3B3 overexpression inhibited ZIKV replication by promoting IFN-stimulated gene(ISG)expression whereas silencing of SF3B3 inhibited expression of ISGs to promote ZIKV replication.GTP cyclohydrolase I(GCH1)is the first and ratelimiting enzyme in tetrahydrobiopterin(BH4)biosynthesis.NS5 upregulates the expression of GCH1 during ZIKV infection.And GCH1 marginally promoted ZIKV replication via the IFN pathway.Additionally,GCH1 expression is related to the regulation of SF3B3.Overexpression of the SF3B3 protein effectively reduced GCH1 protein levels,whereas SF3B3 knockdown increased its levels.These findings indicated that ZIKV NS5 binding protein SF3B3 contributed to the host immune response against ZIKV replication by modulating the expression of GCH1.

Nuclearly Encoded Splicing Factors Implicated in RNA Splicing in Higher Plant Organelles

Plant organelles arose from two independent endosymbiosis events. Throughout evolutionary history, tight control of chloroplasts and mitochondria has been gained by the nucleus, which regulates most steps of organelle genome expression and metabolism. In particular, RNA maturation, including RNA splicing, is highly dependent on nuclearly encoded splicing factors. Most introns in organelles are group II introns, whose catalytic mechanism closely resembles that of the nuclear spliceosome. Plant group II introns have lost the ability to self-splice in vivo and require nuclearly encoded proteins as cofactors. Since the first splicing factor was identified in chloroplasts more than 10 years ago, many other proteins have been shown to be involved in splicing of one or more introns in chloroplasts or mitochondria. These new proteins belong to a variety of different families of RNA binding proteins and provide new insights into ribonucleoprotein complexes and RNA splicing machineries in organelles. In this review, we describe how splicing factors, encoded by the nucleus and targeted to the organelles, take part in post-transcriptional steps in higher plant organelle gene expression. We go on to discuss the potential for these factors to regulate organelle gene expression.

The Splicing Factor PRP31 Is Involved in Transcriptional Gene Silencing and Stress Response in Arabidopsis

Although DNA methylation is known to play an important role in the silencing of transposable elements （TEs） and introduced transgenes, the mechanisms that generate DNA methylation-independent transcrip- tional silencing are poorly understood. Previous studies suggest that RNA-directed DNA methylation （RdDM） is required for the silencing of the RD29A-LUC transgene in the Arabidopsis rosl mutant back- ground with defective DNA demethylase. Loss of function of ARGONAUTE 4 （AGO4） gene, which encodes a core RdDM component, partially released the silencing of RD29A-LUC in the rosl/ago4 double mutant plants. A forward genetic screen was performed to identify the mutants with elevated RD29A-LUC trans- gene expression in the rosl/ago4 mutant background. We identified a mutation in the homologous gene of PRP31, which encodes a conserved pre-mRNA splicing factor that regulates the formation of the U4/ U6.U5 snRNP complex in fungi and animals. We previously demonstrated that the splicing factors ZOP1 and STA1 contribute to transcriptional gene silencing. Here, we reveal that Arabidopsis PRP31 associates with ZOP1, STA1, and several other splicing-related proteins, suggesting that these splicing factors are both physically and functionally connected. We show that Arabidopsis PRP31 participates in transcrip- tional gene silencing. Moreover, we report that PRP31, STA1, and ZOP1 are required for development and stress response. Under cold stress, PRP31 is not only necessary for pre-mRNA splicing but also for regulation of cold-responsive gene expression. Our results suggest that the splicing machinery has multiple functions including pre-mRNA splicing, gene regulation, transcriptional gene silencing, and stress response.

Deregulation of splicing factors and breast cancer development

It is well known that many genes implicated in the development and progression of breast cancer undergo aberrant alternative splicing events to produce proteinswithpro-cancerproperties.These changes in alternative splicingcan arise frommutations or single-nucleotide polymorphisms(SNPs)within the DNA sequences of cancer-related genes,which can strongly affect the activity of splicing factors and influence the splice site choice.However,it is important to note that absence of mutations is not sufficient to prevent misleading choices in splice site selection.There is nowincreasing evidence to demonstrate that the expression profile of ten splicing factors(including SRs and hnRNPs)and eight RNA-binding proteins changes in breast cancer cells compared with normal cells.These modifications strongly influence the alternative splicing pattern of many cancer-related genes despite the absence of any detrimental mutations within their DNA sequences.Thus,a comprehensive assessment of the splicing factor status in breast cancer is important to provide insights into the mechanisms that lead to breast cancer development and metastasis.Whilst most studies focus on mutations that affect alternative splicing in cancer-related genes,this review focuses on splicing factors and RNA-binding proteins that are themselves deregulated in breast cancer and implicated in cancer-related alternative splicing events.

Cilia regeneration requires an RNA splicing factor from the ciliary base

Cilia are microtubule-based organelles projected from most eukaryotic cell surfaces performing cell motility and signaling.Several previously recognized non-ciliary proteins play crucial roles in cilium formation and function.Here,we provide additional evidence that the Caenorhabditis elegans RNA splicing factor PRP-8/PRPF8 regulates ciliogen-esis and regeneration from the ciliary base.Live imaging of GFP knock-in animals reveals that the endogenous PRP-8 localizes in the nuclei and the ciliary base.A weak loss-of-function allele of prp-8 affects ciliary structure but with little impact on RNA splicing.Conditional degradation of PRP-8 within ciliated sensory neurons showed its direct and spe-cific roles in cilium formation.Notably,the penetrance of ciliary defects correlates with the reduction of PRP-8 at the ciliary base but not nuclei,and sensory neurons regenerated cilia accompanying PRP-8 recovery from the ciliary base rather than the nuclei.We suggest that PRP-8 at the ciliary base contributes to cilium formation and regeneration.

Effects of B[a]P-UVA Co-Exposure on Epigenetic Marks of Isolated Skin Cells and Impact of an Arundo donax L. Extract of These Changes

Background: Extrinsic aging results from environmental stressors such as UVR or pollutants. While the effects of single pollutants are better understood, those of their combination remain poorly scrutinized. Objective: Building on a study showing downregulation of several processes upon co-exposure to B[a]P and UVA, we investigated changes induced by epigenetic marks. Materials and Methods: Human primary fibroblasts and HaCaT cells were exposed to B[a]P and UVA. After 24 hours, exposed and unexposed cells were compared to assess DNA methylation. Focusing on HaCaT, multiplex assays enabled quantifying histone H3 modifications and evaluating four splicing factors (SRSF1, SRSF3, SFPQ, and SF3B1) by immunohistochemical labeling. The expression of keratinocyte-/fibroblast-relevant genes was assessed by RT-qPCR. Finally, the impact of an Arundo donax L. extract added 24 hours before B[a]P-UVA co-exposure was analyzed. Results: Exposure to B[a]P-UVA raised DNA methylation (HaCaT: ×3.6, fibroblasts: ×1.9), an increase prevented by the extract. In HaCaT cells, B[a]P-UVA increases the frequency of S10P (+38%). When exposure was preceded by extract treatment, the frequency of several methylations was impacted. B[a]P-UVA only induced the expression of SRSF1 and SFPQ in HaCaT (+46% and +34%). Treatment with the extract abolished this effect. Co-exposure increases the expression of inflammation-related genes (IL-1α, IL-1β) in HaCaT cells and decreases those of AQP3, KRT15, and SOD2. The extract has little effect on these changes. In primary fibroblasts, exposure to B[a]P-UVA lowered the expression of LOXL2, LUM, and TGFBR2 (−38%, −59%, and −51%, respectively), and the extract did not affect these modifications. Conclusion: Within 24 hours, a single B[a]P-UVA co-exposure changes epigenetic marks of skin cells but has only mild effects on gene expression. An Arundo donax L. extract can prevent part of the epigenetic marks’ changes and could stimulate the expression of some genes in primary fibroblasts.

Impairment of pre-mRNA splicing in liver disease: Mechanisms and consequences

Pre-mRNA splicing is an essential step in the process of gene expression in eukaryotes and consists of the removal ofintrons and the linking of exons to generate mature mRNAs. This is a highly regulated mechanism that allows the alternative usage of exons, the retention ofintronic sequences and the generation of exonic sequences of variable length. Most human genes undergo splicing events, and disruptions of this process have been associated with a variety of diseases, including cancer. Hepatocellular carcinoma (HCC) is a molecularly heterogeneous type of tumor that usually develops in a cirrhotic liver. Alterations in pre-mRNA splicing of some genes have been observed in liver cancer, and although still scarce, the available data suggest that splicing defects may have a role in hepatocarcinogenesis. Here we briefly review the general mechanisms that regulatepre-mRNA splicing, and discuss some examples that illustrate how this process is impaired in liver tumorigenesis, and may contribute to HCC development. We believe that a more thorough examination of pre-mRNA splicing is still needed to accurately draw the molecular portrait of liver cancer. This will surely contribute to a better understanding of the disease and to the development of new effective therapies.

NOVA1 promotes SMN2 exon 7 splicing by binding the UCAC motif and increases SMN protein expression

Spinal muscular atrophy(SMA)is a rare hereditary neuromuscular disease with a high lethality rate in infants.Variants in the homologous genes survival of motor neuron(SMN)1 and SMN2 have been reported to be SMA pathogenic factors.Previous studies showed that a highinclusion rate of SMN2 exon 7 increased SMN expression,which in turn reduced the severity of SMA.The inclusion rate of SMN2 exon 7 was higher in neural tissues than in non-neural tissues.Neuro-oncological ventral antigen(NOVA)is a splicing factor that is specifically and highly expressed in neurons.It plays a key role in nervous system development and in the induction of nervous system diseases.Howeve r,it remains unclear whether this splicing factor affects SMA.In this study,we analyzed the inclusion of SMN2 exon 7 in different tissues in a mouse model of SMA(genotype smn^(-/-)SMN2^(2 tg/0))and litter mate controls(genotype smn^(+/-)SMN2^(2 tg/0)).We found that inclusion level of SMN2 exon 7 was high in the brain and spinal co rd tissue,and that NOVA1 was also highly expressed in nervous system tissues.In addition,SMN2 exon 7 and NOVA1 were expressed synchronously in the central nervous system.We further investigated the effects of NOVA1 on disease and found that the number of neurons in the anterior horn of spinal cord decreased in the mouse model of SMA during postnatal days 1-7,and that NOVA1 expression levels in motor neurons decreased simultaneously as spinal muscular atrophy developed.We also found that in vitro expression of NOVA1 increased the inclusion of SMN2 exon 7 and expression ofthe SMN2 protein in the U87 MG cell line,whereas the opposite was observed when NOVA1 was knocked down.Finally,point mutation and RNA pull-down showed that the UCAC motif in SMN2 exon 7 plays a critical role in NOVA1 binding and promoting the inclusion of exon 7.Moreove r,CA was more essential for the inclusion of exon 7 than the order of Y residues in the motif.Collectively,these findings indicate that NOVA1 intera cts with the UCAC motif in exon 7 of SMN2,there by enhancing inclusion of exon 7 in SMN2,which in turn increases expression of the SMN protein.

Dysfunctional gene splicing in glucose metabolism may contribute to Alzheimer’s disease

The glucose metabolism is crucial for sustained brain activity as it provides energy and is a carbon source for multiple biomacromolecules;glucose metabolism decreases dramatically in Alzheimer’s disease(AD)and may be a fundamental cause for its development.Recent studies reveal that the alternative splicing events of certain genes effectively regulate several processes in glucose metabolism including insulin receptor,insulin-degrading enzyme,pyruvate kinase M,receptor for advanced glycation endproducts,and others,thereby,influencing glucose uptake,glycolysis,and advanced glycation end-products-mediated signaling pathways.Indeed,the discovery of aberrant alternative splicing that changes the proteomic diversity and protein activity in glucose metabolism has been pivotal in our understanding of AD development.In this review,we summarize the alternative splicing events of the glucose metabolism-related genes in AD pathology and highlight the crucial regulatory roles of splicing factors in the alternative splicing process.We also discuss the emerging therapeutic approaches for targeting splicing factors for AD treatment.

Alternative splicing in breast cancer

Alternative splicing allows a gene to produce multiple proteins and is the main source of human proteome diversity.Aberrant regulation of alternative splicing produces proteins with different structures and can lead to altered protein function,inactivation,or deleterious variants,which can have severe effects on normal cellular function,leading to many diseases,including cancer.Alternative splicing abnormalities are often found in diseases,so alternative splicing is highly correlated with diseases and treatment.According to relevant statistics,breast cancer is an important factor that threatens women’s lives and health.A common sign of tumors in humans is abnormal alternative splicing.An increasing amount of evidence shows that abnormal alternative splicing plays an important role in the occurrence and development of breast cancer.At the same time,in the related treatment of breast cancer,alternative splicing can lead to the failure of existing treatment methods and induce drug resistance.Currently,targeted therapy is still the key strategy for breast cancer treatment.In this review,we discuss the five classifications of alternative splicing and aberrant alternative splicing in breast cancer.The role of alternative splicing in the drug resistance of breast cancer is also discussed.Evidence has shown that aberrant alternative splicing can lead to drug resistance to current therapies.Because splicing factors are involved in many aspects of cancer,splicing factors may provide a new strategy for cancer treatment and a powerful tool for progress in the fight against breast cancer.Antisense oligonucleotides form a double-stranded structure by complementary pairing with mRNA,which prevents the binding of mRNA and ribosomes,thus inhibiting protein synthesis.Therefore,after finding effective targets,antisense oligonucleotides can be applied in gene therapy,drug research and development,biological research,and other fields to improve therapeutic effects.

RNA splicing alterations in lung cancer pathogenesis and therapy

RNA splicing alterations are widespread and play critical roles in cancer pathogenesis and therapy.Lung cancer is highly heterogeneous and causes the most cancer-related deaths worldwide.Large-scale multi-omics studies have not only characterized the mutational landscapes but also discovered a plethora of transcriptional and post-transcriptional changes in lung cancer.Such resources have greatly facilitated the development of new diagnostic markers and therapeutic options over the past two decades.Intriguingly,altered RNA splicing has emerged as an important molecular feature and therapeutic target of lung cancer.In this review,we provide a brief overview of splicing dysregulation in lung cancer and summarize the recent progress on key splicing events and splicing factors that contribute to lung cancer pathogenesis.Moreover,we describe the general strategies targeting splicing alterations in lung cancer and highlight the potential of combining splicing modulation with currently approved therapies to combat this deadly disease.This review provides new mechanistic and therapeutic insights into splicing dysregulation in cancer.

New insights into pathogen‑mediated modulation of host RNA splicing

Alternative splicing(AS)regulation of pre-mRNA has been proven to be one of the fundamental layers of plant immune system.How pathogens disrupt plant AS process to suppress plant immunity by secreted effectors remain poorly understood.In the recent study,Gui et al.revealed that a previously identified effector PSR1 of Phytophthora interferes with host RNA splicing machinery to modulate small RNA biogenesis,leading to compromised plant immu-nity.The study provided a novel insight into the importance of AS process during pathogen-host interactions.

Specific Regulation of m^(6)A by SRSF7 Promotes the Progression of Glioblastoma

Serine/arginine-rich splicing factor 7(SRSF7),a known splicing factor,has been revealed to play oncogenic roles in multiple cancers.However,the mechanisms underlying its oncogenic roles have not been well addressed.Here,based on N6-methyladenosine(m^(6)A)co-methylation network analysis across diverse cell lines,we find that the gene expression of SRSF7 is positively correlated with glioblastoma(GBM)cell-specific m^(6)A methylation.We then indicate that SRSF7 is a novel m^(6)A regulator,which specifically facilitates the m^(6)A methylation near its binding sites on the mRNAs involved in cell proliferation and migration,through recruiting the methyltransferase complex.Moreover,SRSF7 promotes the proliferation and migration of GBM cells largely dependent on the presence of the m^(6)A methyltransferase.The two m^(6)A sites on the mRNA for PDZ-binding kinase(PBK)are regulated by SRSF7 and partially mediate the effects of SRSF7 in GBM cells through recognition by insulin-like growth factor 2 mRNA-binding protein 2(IGF2BP2).Together,our discovery reveals a novel role of SRSF7 in regulating m^(6)A and validates the presence and functional importance of temporal-and spatial-specific regulation of m^(6)A mediated by RNA-binding proteins(RBPs).