Background:Glioblastoma(GBM)is one of the most malignant types of central nervous system tumors.Oxygen deprivation in the tumor microenvironment is thought to be an important factor in promoting GBM progression.Howeve...Background:Glioblastoma(GBM)is one of the most malignant types of central nervous system tumors.Oxygen deprivation in the tumor microenvironment is thought to be an important factor in promoting GBM progression.However,the mechanisms of hypoxia-promoted tumor progression remain elusive.Methods:Alternative splicing of diacylglycerol kinase gamma(DGKG)-Δexon13 was amplified and verified by PCR-Sanger sequencing.The functions of DGKG and DGKG-Δexon13 were analyzed by Cell counting kit-8(CCK-8),Transwell,Matrigeltranswell experiments,and in vivo orthotropic GBM animal models.Transcriptome analyses were done to find out the regulated genes.Results:In this study,we found that a new transcript DGKG-Δexon13 was generated in GBM under hypoxia via alternative splicing.Moreover,the results of CCK-8,Transwell,and Matrigel-transwell experiments showed that the proliferation,migration,and invasion abilities of U87-MG and T98G were decreased after DGKG knockdown.Compared to wild-type DGKG,DGKG-Δexon13 overexpression significantly promoted cellular proliferation,migration,and invasion abilities in GBM.Furthermore,in vivo,orthotropic GBM animal models analysis showed that the tumor volumes were much smaller in the DGKG knockdown group.However,the tumor sizes in the DGKG and DGKG-Δexon13 rescue groups were restored,especially in the DGKG-Δexon13 group.Transcriptome analysis revealed that MORC1,KLHDC7B,ATP1A2,INHBE,TMEM119,and FGD3 were altered significantly when DGKG was knocked down.IL-16,CCN2,and EFNB3 were specifically regulated by DGKG-Δexon13.Conclusions:Our study found that hypoxia-induced alternative splicing transcript DGKG-Δexon13 promotes GBM proliferation and infiltration,which might provide a new potential target for the clinical treatment and diagnosis of GBM.展开更多
OCT4 is one of the key transcription factors in maintaining the pluripotency and self-renewal of embryonic stem （ES） cells.Human OCT4 can generate two isoforms OCT4A and OCT4B by alternative splicing.OCT4B1 is a rec...OCT4 is one of the key transcription factors in maintaining the pluripotency and self-renewal of embryonic stem （ES） cells.Human OCT4 can generate two isoforms OCT4A and OCT4B by alternative splicing.OCT4B1 is a recently discovered novel OCT4 spliced variant,which has been considered as a putative marker of stemness.Compared with the OCT4B mRNA,OCT4B1 mRNA is generated by retaining intron 2 as a cryptic exon which contains a TGA stop codon in it.As a result,the protein product of OCT4B1 mRNA could be truncated.Interestingly,we present here that OCT4B1 can indirectly produce the same protein products as OCT4B.We have demonstrated that OCT4B1 mRNA can be spliced into OCT4B mRNA,and encode three protein isoforms.The splicing of OCT4B1 mRNA into OCT4B mRNA can be remarkably inhibited by the mutation of the classical splicing site.Our result suggests that OCT4B mRNA may originate from OCT4B1 mRNA by alternative splicing.展开更多
Caspases,which play key roles in cell apoptosis,undergo alternative splicing to form different splicing variants that can regulate the apoptotic process.Lepidopteran insect caspases undergo alternative splicing,althou...Caspases,which play key roles in cell apoptosis,undergo alternative splicing to form different splicing variants that can regulate the apoptotic process.Lepidopteran insect caspases undergo alternative splicing,although the functions of their splicing variants are still unclear.The Spodoptera exigua caspase-5(SeCaspase-5)gene was cloned and found to produce four different splicing variants with different gene sequences and protein functional domains,which were named SeCaspase-5a,SeCaspase-5b,SeCaspase-5c and SeCaspase-5d.Overexpression of these variants in S.exigua cells(Se-3)showed that SeCaspase-5a had a proapoptotic function,whereas SeCaspase-5b,SeCaspase-5c and SeCaspase-5d did not.Semi-qPCR analysis revealed that the expression of the SeCaspase-5 variants significantly differed during Autographa californica multiple nucleopolyhedrovirus(AcMNPV)infection.Furthermore,the SeCaspase-5 variants were constructed into the AcMNPV bacmid and transfected into Se-3 cells,which revealed that SeCaspase-5a promoted cell apoptosis and reduced virus production,whereas SeCaspase-5b,SeCaspase-5c and SeCaspase-5d did not promote cell apoptosis but instead increased virus production.Moreover,an analysis of the interactions between the SeCaspase-5 variants revealed that SeCaspase-5a directly interacted with SeCaspase-5b,SeCaspase-5c and SeCaspase-5d.Coexpression of these variants in Se-3 cells also revealed that SeCaspase-5b,SeCaspase-5c and SeCaspase-5d inhibited the proapoptotic function of SeCaspase-5a,resulting in a reduction in the percentage of apoptotic cells by about 20%.These results indicate that SeCaspase-5 undergoes alternative splicing and is involved in regulating the apoptosis induced by baculovirus infection.These findings increase our understanding of the functions of lepidopteran insect caspases and provide new insights into the mechanism of host-cell apoptosis induced by baculoviruses.展开更多
Heat stress is a major threat to maize(Zea mays L.)production worldwide.Heat shock transcription factors(HSFs)play vital roles in plant responses to heat stress.However,the molecular and genetic mechanisms underlying ...Heat stress is a major threat to maize(Zea mays L.)production worldwide.Heat shock transcription factors(HSFs)play vital roles in plant responses to heat stress.However,the molecular and genetic mechanisms underlying HSF-meditated thermotolerance in maize remain largely unexplored.In this study,we demonstrate that the alternative splicing of ZmHsf23 modulates heat stress tolerance in maize.Hsf23 produced two functional transcripts,Hsf23L and Hsf23S,which differ by the presence of a cryptic mini-exon in Hsf23L that is spliced out in Hsf23S.Both transcripts were strongly induced by heat stress.Mutants lacking Hsf23L alone(hsf23l)or both Hsf23L and Hsf23S(hsf23l23s)exhibited increased susceptibility to heat stress,whereas overexpression of Hsf23S enhanced heat stress tolerance in maize.Subsequently,we found that Hsf23S positively regulates heat stress tolerance by directly activating the transcription of three sHSP genes(Hsp16.9,Hsp17.2,and Hsp18a)and TIL1 gene.In addition,Hsf23L physically interacted with Hsf23S and enhanced the transcriptional activation of Hsf23S on the sHSPs and TIL1 promoters.Notably,genetic analysis suggested that co-overexpression of Hsf23L and Hsf23S further improves heat tolerance of the transgenic plants.Taken together,these results reveal two splicing variants of ZmHsf23 cooperatively regulate maize heat tolerance,thus highlighting potential value of ZmHsf23 in breeding heat-tolerant maize varieties.展开更多
Background:Alterations in splicing factors contribute to aberrant alternative splicing(AS),which subsequently promotes tumor progression.The splicing factor polypyrimidine tract binding protein 1(PTBP1)has been shown ...Background:Alterations in splicing factors contribute to aberrant alternative splicing(AS),which subsequently promotes tumor progression.The splicing factor polypyrimidine tract binding protein 1(PTBP1)has been shown to facilitate cancer progression by modulating oncogenic variants.However,its specific role and underlying mechanisms in hepatocellular carcinoma(HCC)remain to be elucidated.Methods:PTBP1 expression was evaluated in HCC tissues and cell lines.Subsequently,cells were transfected with vectors designed for PTBP1 overexpression or downregulation.The biological function of PTBP1 was assessed in vitro and in vivo using MTS assays,colony formation assays,transwell assays,xenograft formation,tail vein injection,and orthotopic models.Transcriptome analysis was conducted to elucidate the underlying molecular mechanisms.Results:Our findings demonstrated that PTBP1 exhibited elevated expression in HCC cell lines and tissues.Furthermore,its expression positively correlated with overall and disease-free survival rates,as well as tumor grade and stage.PTBP1 knockdown reduced HCC cell proliferation,migration,and invasion in vitro and suppressed hepatocarcinoma xenograft growth and infiltration in vivo.RNA sequencing(RNA-Seq)analysis identified the AS events associated with PTBP1.PTBP1 functionally enhanced cell proliferation,invasion,and migration by modulating the AS of the microtubule-associated protein tau(MAPT)gene and promoting oncogene expression.Notably,the dysregulation of MAPT splicing coincided with increased PTBP1 expression in HCC.Conclusions:PTBP1-guided AS of the MAPT gene enhances tumorigenicity in HCC through activation of the MAPK/ERK pathways.展开更多
Background： Identification of functional genes affecting milk production traits is very crucial for improving breeding efficiency in dairy cattle. Many potential candidate genes have been identified through our previ...Background： Identification of functional genes affecting milk production traits is very crucial for improving breeding efficiency in dairy cattle. Many potential candidate genes have been identified through our previous genome wide association study （GWAS）. Of these, GPIHBP1 is an important novel candidate gene for milk production traits. However, the mRNA structure of the bovine GPIHBP1 gene is not fully determined up to now. Results： In this study, we identified a novel alternatively splice transcript variant （XS） which leads to a 3] bp insertion in exon 3 and also confirmed the other four existed transcripts （X1, X2, X3 and X4） of the bovine GPIHBP1 gene. We showed that transcript X5 with a 31 bp insertion and transcript X1 with an 8 bp deletion might have tremendous effect on the protein function and structure of GPIHBP1, respectively. With semi-quantitative PCR and quantitative real-time RT-PCR, we found that the mRNA expression of GPIHBPI, GPIHBP1-X1 and GPIHBP1-X5 in mammary gland of lactating cows were much higher than that in other tissues. Conclusions： Our study reports a novel alternative splicing of GPIHBP1 in bovine for the first time and provide useful information for the further functional analyses of GPIHBP1 in dairy cattle.展开更多
Both transcription and post-transcriptional processes, such as alternative splicing, play crucial roles in controlling developmental programs in metazoans. Recently emerged RNA-seq method has brought our understanding...Both transcription and post-transcriptional processes, such as alternative splicing, play crucial roles in controlling developmental programs in metazoans. Recently emerged RNA-seq method has brought our understanding of eukaryotic transcriptomes to a new level, because it can resolve both gene expression level and alternative splicing events simultaneously. To gain a better understanding of cellular differentiation in gonads, we analyzed mRNA profiles from Drosophila testes and ovaries using RNA-seq. We identified a set of genes that have sex-specific isoforms in wild-type （WT） gonads, including several transcription factors. We found that differentiation of sperms from undifferentiated germ cells induced a dramatic downregulation of RNA splicing factors. Our data confirmed that RNA splicing events are significantly more frequent in the undifferentiated cell-enriched bag of marbles （barn） mutant testis, but downregulated upon differentiation in WT testis. Consistent with this, we showed that genes required for meiosis and terminal differentiation in WT testis were mainly regulated at the transcriptional level, but not by alternative splicing. Unexpectedly, we observed an increase in expression of all families of chromatin remodeling factors and histone modifying enzymes in the undifferentiated cell-enriched bam testis. More interestingly, chromatin regulators and histone modifying enzymes with opposite enzymatic activities are coenriched in undifferentiated cells in testis, suggesting that these cells may possess dynamic chromatin architecture. Finally, our data revealed many new features of the Drosophila gonadal transcriptomes, and will lead to a more comprehensive understanding of how differential gene expression and splicing regulate gametogenesis in Drosophila. Our data provided a foundation for the systematic study of gene expression and alternative splicing in many interesting areas of germ cell biology in Droso- phila, such as the molecular basis for sexual dimorphism and the regulation of the proliferation vs terminal differentiation programs in germline stem cell lineages. The GEO accession number for the raw and analyzed RNA-seq data is GSE16960.展开更多
Polypyrimidine tract-binding protein 1(PTBP1)plays an essential role in splicing and is expressed in almost all cell types in humans,unlike the other proteins of the PTBP family.PTBP1 mediates several cellular process...Polypyrimidine tract-binding protein 1(PTBP1)plays an essential role in splicing and is expressed in almost all cell types in humans,unlike the other proteins of the PTBP family.PTBP1 mediates several cellular processes in certain types of cells,including the growth and differentiation of neuronal cells and activation of immune cells.Its function is regulated by various molecules,including micro RNAs(mi RNAs),long non-coding RNAs(lnc RNAs),and RNA-binding proteins.PTBP1 plays roles in various diseases,particularly in some cancers,including colorectal cancer,renal cell cancer,breast cancer,and glioma.In cancers,it acts mainly as a regulator of glycolysis,apoptosis,proliferation,tumorigenesis,invasion,and migration.The role of PTBP1 in cancer has become a popular research topic in recent years,and this research has contributed greatly to the formulation of a useful therapeutic strategy for cancer.In this review,we summarize recent findings related to PTBP1 and discuss how it regulates the development of cancer cells.展开更多
AIM:This study was designed to compare the levels of v5 and v6 splice variants of CD44 evaluated using EITSA test in the serum of patients with colorectal cancer in different stages of progression of the disease estim...AIM:This study was designed to compare the levels of v5 and v6 splice variants of CD44 evaluated using EITSA test in the serum of patients with colorectal cancer in different stages of progression of the disease estimated in pT stage according to WHO score,histopathological grade of malignancy and some clinicopathological features. METHODS:The serum obtained from 114 persons with colorectal adenocarcinomas was examined using ELISA method,pT stage and grade of malignancy of the tumour were examined in formalin fixed and paraffin embedded materials obtained during operation. RESULTS:Only the level of CD44 v5 in the serum of patients before operation with G2 pT4 tumour was lower than that in other probes and the difference was statistically significant. We did not find any other correlations between the level of v5 and v6 CD44 variants and other evaluated parameters. CONCLUSION:The level of CD44 v5 and v6 estimated by ELISA test in the serum can not be used as a prognostic factor in colorectal cancer.展开更多
Ser/Arg-rich （SR） genes encode proteins that play pivotal roles in both constitutive and alternative splicing of pre-mRNA. However, not much effort has been made to investigate the alternative splicing of their own ...Ser/Arg-rich （SR） genes encode proteins that play pivotal roles in both constitutive and alternative splicing of pre-mRNA. However, not much effort has been made to investigate the alternative splicing of their own pre-mRNA. In this study, we conducted comprehensive analyses of pre-mRNA splicing for 22 SR genes in three rice （Oryza sativa L.） ecotypes indica, japonica andjavanica. Using different ecotypes we characterized the variations in expression and splicing patterns of rice SR genes in different tissues and at different developmental stages. In addition, we compared the divergence in expression and splicing patterns of SR genes from seedlings of different rice ecotypes in response to hormones application and environmental stresses. Our results revealed the complexity of alternative splicing of SR genes in rice. The splicing varies in different tissues, in different ecotypes, in response to stresses and hormones. Thus, our study suggested that SR genes were subjected to sophisticated alternative splicing although their encoding proteins were involved in the splicing process.展开更多
The neuronal microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in the brains of individuals with Alzheimer＇s disease and related neurodegenerative disord...The neuronal microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in the brains of individuals with Alzheimer＇s disease and related neurodegenerative disorders. The adult human brain expresses six isoforms of tau generated by alternative splicing of exons 2, 3, and 10 of its pre-mRNA. Exon 10 encodes the second microtubule-binding repeat of tau. Its alternative splicing produces tau isoforms with either three or four microtubule-binding repeats, termed 3R-tau and 4R- tau. In the normal adult human brain, the level of 3R-tau is approximately equal to that of 4R-tau. Several silent and intronic mutations of the tau gene associated with FTDP-17T （frontotemporal dementia with Parkinsonism linked to chromosome 17 and specifically characterized by tau pathology） only disrupt exon 10 splicing, but do not influence the primary sequence of the tau protein. Thus, abnormal exon 10 splicing is sufficient to cause neurodegeneration and dementia. Here, we review the regulation of tau exon 10 splicing by cis-elements and trans-factors and summarize all the mutations associated with FTDP-17T and related tauopathies. The findings suggest that correction of exon 10 splicing may be a potential target for tau exon 10 splicing-related tauopathies.展开更多
Aim:To identify a novel isoform of adaptin 2 beta subunit (named Ap2β-NY) and to investigate its relationship with testicular development and spermatogenesis.Methods:Using a human testis cDNA microarray,a clone (Ap2...Aim:To identify a novel isoform of adaptin 2 beta subunit (named Ap2β-NY) and to investigate its relationship with testicular development and spermatogenesis.Methods:Using a human testis cDNA microarray,a clone (Ap2β-NY), which was strongly expressed in adult testes but weakly expressed in embryo testes,was sequenced and analyzed. Using polymerase chain reaction (PCR),the tissue distribution and expression time pattern of Ap2β-NY were determined. Results:Ap2β-NY was identified and has been deposited in the GenBank (AY341427).The expression level of Ap2β-NY in the adult testis was about 3-fold higher than that in the embryo testis.PCR analysis using multi-tissue cDNA indicated that Ap2β-NY was highly expressed in the testis,spleen,thymus,prostate,ovary,blood leukocyte and brain,but not in the heart,placenta,lung,liver,skeletal muscle,kidney and pancreas.In addition,Ap2β-NY was variably expressed in the testes of patients with spermatogenesis-disturbance and spermatogenesis-arrest but not expressed in those of Sertoli-cell-only syndrome,which implied that,in the testis,Ap2β-NY was restrictively ex- pressed in germ cells.Conclusion:Ap2β-NY is an isoform of Ap2β and may be involved in regulating the process of spermatogenesis and testis development.展开更多
AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing. METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcriptio...AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing. METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced. Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of 17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA. Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced. RESULTS: One of the CYP2D6 cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4), and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples, only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6 cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6 gene were detected. The third variant was the skipped exon 3, and 153 bp was lost. CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.展开更多
Chinese tongue sole(Cynoglossus semilaevis)is an economically important marine fish species with a ZZ/ZW sex determination mechanism,which can be influenced by temperature.Alternative splicing(AS)is an important mecha...Chinese tongue sole(Cynoglossus semilaevis)is an economically important marine fish species with a ZZ/ZW sex determination mechanism,which can be influenced by temperature.Alternative splicing(AS)is an important mechanism regulating the expression of genes related to sex determination and gonadal differentiation,but has rarely been reported in fish.In this study,to explore the molecular regulatory mechanisms of sex determination and gonadal differentiation,we combined isoform and RNA sequencing(Iso-Seq and RNA-Seq)to perform transcriptome profiling of male and female gonads in C.semilaevis.In total,81883 and 32341 full-length transcripts were obtained in males and females,respectively.A total of 8279 AS genes were identified,including 2639 genes showing differential AS(DAS)between males and females.Many intersecting DAS genes and differentially expressed genes(DEGs)were enriched in the meiotic cell cycle pathway,and genes related to gonadal differentiation,such as esrrb and wt1a,were found to have sex-specific isoforms.Thus,this study revealed AS events in the gonadal transcriptomes of male and female C.semilaevis,described the characteristics of active transcription in the testes,and identified candidate genes for studying the regulatory mechanisms of AS during gonadal differentiation.展开更多
The splicing of many alternative exons in the precursor messenger RNA (pre-mRNA) is regulated by extracellular factors but the underlying molecular bases remain unclear. Here we report the differential regulation of B...The splicing of many alternative exons in the precursor messenger RNA (pre-mRNA) is regulated by extracellular factors but the underlying molecular bases remain unclear. Here we report the differential regulation of Bcl-x pre-mRNA splicing by extracellular factors and their distinct requirements for pre-mRNA elements. In K562 leukemia cells, treatment with interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF) reduced the proportion of the Bcl-xL variant mRNA while treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) had no effect. In U251 glioma cells, however, TPA efficiently increased the Bcl-xL level. These regulations were also seen for a transfected splicing reporter mini-gene. Further analyses of deletion mutants indicate that nucleotides 1-176 of the downstream intron are required for the IL-6 effect, whereas additional nucleotides 177-284 are essential for the GM-CSF effect. As for the TPA effect, only nucleotides 1-76 are required in the downstream intron. Thus, IL-6, GM-CSF and TPA differentially regulate Bcl-x splicing and require specific intronic pre-mRNA sequences for their respective effects.展开更多
Alternative splicing is a major contributor to genomic complexity and proteome diversity,yet the analysis of alternative splicing for the sequence containing nucleotide binding site and leucine-rich repeats(NBS-LRR)do...Alternative splicing is a major contributor to genomic complexity and proteome diversity,yet the analysis of alternative splicing for the sequence containing nucleotide binding site and leucine-rich repeats(NBS-LRR)domain has not been explored in rice(Oryza sativa L.).Hidden Markov model(HMM)searches were performed for NBS-LRR domain.875 NBS-LRR-encoding sequences were obtained from the Institute for Genomic Research(TIGR).All of them were used to blast Knowledge-based Oryza Molecular Biological Encyclopaedia(KOME),TIGR rice gene index(TGI),and Universal Protein Resource(UniProt)to obtain homologous full-length cDNAs(FL-cDNAs),tentative consensus sequences,and protein sequences.Alternative splicing events were detected from genomic alignment of FL-cDNAs,tentative consensus sequences,and protein sequences,which provide valuable information on splice variants of genes.These sequences were aligned to the corresponding BAC sequences using the Spidey and Sim4 programs and each of the proteins was aligned by tBLASTn.Of the 875 NBS-LRR sequences,119(13.6%)sequences had alternative splicing where multiple FL-cDNAs,TGI sequences and proteins corresponded to the same gene.71 intron retention events,20 exon skipping events,16 alternative termination events,25 alternative initiation events,12 alternative 5'splicing events,and 16 alternative 3'splicing events were identified.Most of these alternative splices were supported by two or more transcripts.The data sets are available at http://www.bioinfor.org.Furthermore,the bioinformatics analysis of splice boundaries showed that exon skipping and intron retention did not exhibit strong consensus.This implies a different regulation mechanism that guides the expression of splice isoforms.This article also presents the analysis of the effects of intron retention on proteins.The C-terminal regions of alternative proteins turned out to be more variable than the N-terminal regions.Finally,tissue distribution and protein localization of alternative splicing were explored.The largest categories of tissue distributions for alternative splicing were shoot and callus.More than one-thirds of protein localization for splice forms was plasma membrane and cytoplasm.All the NBS-LRR proteins for splice forms may have important function in disease resistance and activate downstream signaling pathways.展开更多
Alternative splicing,which is a common phenomenon in mammalian genomes,is a fundamental process of gene regulation and contributes to great protein diversity.Alternative splicing events not only occur in the normal ge...Alternative splicing,which is a common phenomenon in mammalian genomes,is a fundamental process of gene regulation and contributes to great protein diversity.Alternative splicing events not only occur in the normal gene regulation process but are also closely related to certain diseases including cancer.In this review,we briefly demonstrate the concept of alternative splicing and DNA damage and describe the association of alternative splicing and cancer pathogenesis,focusing on the potential relationship of alternative splicing,DNA damage,and gastrointestinal cancers.We will also discuss whether alternative splicing leads to genetic instability,which is considered to be a driving force for tumorigenesis.Better understanding of the role and mechanism of alternative splicing in tumorigenesis may provide new directions for future cancer studies.展开更多
Accumulating evidence supports the significance of aberrant alternative splicing(AS)events in cancer;however,genome-wide profiling of progression-free survival(PFS)-related AS events in testicular germ cell tumors(TGC...Accumulating evidence supports the significance of aberrant alternative splicing(AS)events in cancer;however,genome-wide profiling of progression-free survival(PFS)-related AS events in testicular germ cell tumors(TGCT)has not been reported.Here,we analyzed high-throughput RNA-sequencing data and percent-spliced-in values for 150 patients with TGCT.Using univariate and multivariate Cox regression analysis and a least absolute shrinkage and selection operator method,we identified the top 15 AS events most closely associated with disease progression.A risk-associated AS score(ASS)for the 15 AS events was calculated for each patient.ASS,pathological stage,and T stage were significantly associated with disease progression by univariate analysis,but only ASS and pathological stage remained significant by multivariate analysis.The ability of these variables to predict 5-year progression was assessed using receiver operating characteristic curve analysis.ASS had stronger predictive value than a combination of age,pathological stage,and T stage(area under the curve=0.899 and 0.715,respectively).Furthermore,Kaplan—Meier analysis of patients with low and high ASS demonstrated that high ASS was associated with significantly worse PFS than low ASS(P=1.46 × 10^(-7)).We also analyzed the biological functions of the PFS-related AS-related genes and found enrichment in pathways associated with DNA repair and modification.Finally,we identified a regulatory network of splicing factors with expression levels that correlated significantly with AS events in TGCT.Collectively,this study identifies a novel method for risk stratification of patients and provides insight into the molecular events underlying TGCT.展开更多
F-box and WD-40 domain protein 11(FBXW11)is an important component of the E3 ubiquitin-ligase enzyme that plays a key role in the ubiquitin-dependent regulation of spermatogenesis.In our previous research,the mRNA exp...F-box and WD-40 domain protein 11(FBXW11)is an important component of the E3 ubiquitin-ligase enzyme that plays a key role in the ubiquitin-dependent regulation of spermatogenesis.In our previous research,the mRNA expression of FBXW11 in bull sperm with high motility is significantly higher than that with low motility.In the present study,the protein expression levels of FBXW11 in bull testicular tissues with low-performance sperm quality groups were significantly higher than those in normal performance groups.The immunohistochemistry result demonstrated that FBXW11 protein was located in the periphery of Leydig cells and seminiferous tubules.Three splice variants of the FBXW11 gene,namely,FBXW11-tv1,FBXW11-tv2,and FBXW11-tv3,were identified in testicular tissues.The splicing patterns of the three variants are exon skipping.The transcript FBXW11-tv2 expressions were the highest in each sample.The low-performance groups displayed higher FBXW11-tv1 and FBXW11-tv2 transcript expressions than the normal performance groups.Two CpG islands were located within the 5’UTR and exon 1-2 region of the FBXW11 gene.Bisulfite sequencing PCR results demonstrated that the methylation levels of 11 methylation sites in the CpG island 2 from−99 to−43 in the normal performance groups were significantly lower than those in the low-performance groups.Pearson correlation analysis suggested that the CpG island 2 methylation level was negatively correlated with sperm motility and the transcript FBXW11-tv2 expression level.Our data revealed that alternative splicing and DNA methylation jointly regulated FBXW11 gene expression and were correlated with sperm quality traits during spermatogenesis in Holsteins.展开更多
Plant adaptation to drought stress is essential for plant survival and crop yield.Recently,harnessing drought memory,which is induced by repeated stress and recovery cycles,was suggested as a means to improve drought ...Plant adaptation to drought stress is essential for plant survival and crop yield.Recently,harnessing drought memory,which is induced by repeated stress and recovery cycles,was suggested as a means to improve drought resistance at the transcriptional level.However,the genetic mechanism underlying drought memory is unclear.Here,we carried out a quantitative analysis of alternative splicing(AS)events in rice memory under drought stress,generating 12 transcriptome datasets.Notably,we identified exon skipping(ES)as the predominant AS type(>80%)in differential alternative splicing(DAS)in response to drought stress.Applying our analysis pipeline to investigate DAS events following drought stress in six other plant species revealed variable ES frequencies ranging from 9.94%to 60.70%depending on the species,suggesting that the relative frequency of DAS types in plants is likely to be speciesspecific.The dinucleotide sequence at AS splice sites in rice following drought stress was preferentially GC-AG and AT-AC.Since U12-type splicing uses the AFAC site,this suggests that drought stress may increase U12-type splicing,and thus increase ES frequency.We hypothesize that multiple isofbrms derived from exon skipping may be induced by drought stress in rice.We also identified 20 transcription factors and three highly connected hub genes with potential roles in drought memory that may be good targets for plant breeding.展开更多
基金funded by Guizhou Province Science and Technology Plan Project Qiankehe Foundation-ZK[2023]General 360,362Science and Technology Fund project of Guizhou Provincial Health Commission(gzwkj-2022-09,gzwkj-2023-035)+1 种基金National Natural Science Foundation Cultivation Project of Guizhou Medical University(21NSFCP14,gyfynsfc-2022-25)The PhD Scientific Research Launch Fund Project of the Affiliated Hospital of Guizhou Medical University(gyfybsky-2022-02).
文摘Background:Glioblastoma(GBM)is one of the most malignant types of central nervous system tumors.Oxygen deprivation in the tumor microenvironment is thought to be an important factor in promoting GBM progression.However,the mechanisms of hypoxia-promoted tumor progression remain elusive.Methods:Alternative splicing of diacylglycerol kinase gamma(DGKG)-Δexon13 was amplified and verified by PCR-Sanger sequencing.The functions of DGKG and DGKG-Δexon13 were analyzed by Cell counting kit-8(CCK-8),Transwell,Matrigeltranswell experiments,and in vivo orthotropic GBM animal models.Transcriptome analyses were done to find out the regulated genes.Results:In this study,we found that a new transcript DGKG-Δexon13 was generated in GBM under hypoxia via alternative splicing.Moreover,the results of CCK-8,Transwell,and Matrigel-transwell experiments showed that the proliferation,migration,and invasion abilities of U87-MG and T98G were decreased after DGKG knockdown.Compared to wild-type DGKG,DGKG-Δexon13 overexpression significantly promoted cellular proliferation,migration,and invasion abilities in GBM.Furthermore,in vivo,orthotropic GBM animal models analysis showed that the tumor volumes were much smaller in the DGKG knockdown group.However,the tumor sizes in the DGKG and DGKG-Δexon13 rescue groups were restored,especially in the DGKG-Δexon13 group.Transcriptome analysis revealed that MORC1,KLHDC7B,ATP1A2,INHBE,TMEM119,and FGD3 were altered significantly when DGKG was knocked down.IL-16,CCN2,and EFNB3 were specifically regulated by DGKG-Δexon13.Conclusions:Our study found that hypoxia-induced alternative splicing transcript DGKG-Δexon13 promotes GBM proliferation and infiltration,which might provide a new potential target for the clinical treatment and diagnosis of GBM.
基金supported by the National Basic Research Program of China (973 Program) (No 2006CB943601)the National Natural Science Foundation of China (NSFC) (No 90919042)
文摘OCT4 is one of the key transcription factors in maintaining the pluripotency and self-renewal of embryonic stem （ES） cells.Human OCT4 can generate two isoforms OCT4A and OCT4B by alternative splicing.OCT4B1 is a recently discovered novel OCT4 spliced variant,which has been considered as a putative marker of stemness.Compared with the OCT4B mRNA,OCT4B1 mRNA is generated by retaining intron 2 as a cryptic exon which contains a TGA stop codon in it.As a result,the protein product of OCT4B1 mRNA could be truncated.Interestingly,we present here that OCT4B1 can indirectly produce the same protein products as OCT4B.We have demonstrated that OCT4B1 mRNA can be spliced into OCT4B mRNA,and encode three protein isoforms.The splicing of OCT4B1 mRNA into OCT4B mRNA can be remarkably inhibited by the mutation of the classical splicing site.Our result suggests that OCT4B mRNA may originate from OCT4B1 mRNA by alternative splicing.
基金funded by the National Natural Science Foundation of China(32202393)the Natural Science Foundation of Shandong Province,China(ZR2021QC190)+1 种基金the Science and Technology Benefiting the People Demonstration Project of Qingdao,China(24-1-8-xdny-10-nsh)the Qingdao Agricultural University High-level Talent Fund,China(663/1120101)。
文摘Caspases,which play key roles in cell apoptosis,undergo alternative splicing to form different splicing variants that can regulate the apoptotic process.Lepidopteran insect caspases undergo alternative splicing,although the functions of their splicing variants are still unclear.The Spodoptera exigua caspase-5(SeCaspase-5)gene was cloned and found to produce four different splicing variants with different gene sequences and protein functional domains,which were named SeCaspase-5a,SeCaspase-5b,SeCaspase-5c and SeCaspase-5d.Overexpression of these variants in S.exigua cells(Se-3)showed that SeCaspase-5a had a proapoptotic function,whereas SeCaspase-5b,SeCaspase-5c and SeCaspase-5d did not.Semi-qPCR analysis revealed that the expression of the SeCaspase-5 variants significantly differed during Autographa californica multiple nucleopolyhedrovirus(AcMNPV)infection.Furthermore,the SeCaspase-5 variants were constructed into the AcMNPV bacmid and transfected into Se-3 cells,which revealed that SeCaspase-5a promoted cell apoptosis and reduced virus production,whereas SeCaspase-5b,SeCaspase-5c and SeCaspase-5d did not promote cell apoptosis but instead increased virus production.Moreover,an analysis of the interactions between the SeCaspase-5 variants revealed that SeCaspase-5a directly interacted with SeCaspase-5b,SeCaspase-5c and SeCaspase-5d.Coexpression of these variants in Se-3 cells also revealed that SeCaspase-5b,SeCaspase-5c and SeCaspase-5d inhibited the proapoptotic function of SeCaspase-5a,resulting in a reduction in the percentage of apoptotic cells by about 20%.These results indicate that SeCaspase-5 undergoes alternative splicing and is involved in regulating the apoptosis induced by baculovirus infection.These findings increase our understanding of the functions of lepidopteran insect caspases and provide new insights into the mechanism of host-cell apoptosis induced by baculoviruses.
基金supported by the National Key Research and Development Program of China(2021YFF1000301)the National Natural Science Foundation of China(31771805)。
文摘Heat stress is a major threat to maize(Zea mays L.)production worldwide.Heat shock transcription factors(HSFs)play vital roles in plant responses to heat stress.However,the molecular and genetic mechanisms underlying HSF-meditated thermotolerance in maize remain largely unexplored.In this study,we demonstrate that the alternative splicing of ZmHsf23 modulates heat stress tolerance in maize.Hsf23 produced two functional transcripts,Hsf23L and Hsf23S,which differ by the presence of a cryptic mini-exon in Hsf23L that is spliced out in Hsf23S.Both transcripts were strongly induced by heat stress.Mutants lacking Hsf23L alone(hsf23l)or both Hsf23L and Hsf23S(hsf23l23s)exhibited increased susceptibility to heat stress,whereas overexpression of Hsf23S enhanced heat stress tolerance in maize.Subsequently,we found that Hsf23S positively regulates heat stress tolerance by directly activating the transcription of three sHSP genes(Hsp16.9,Hsp17.2,and Hsp18a)and TIL1 gene.In addition,Hsf23L physically interacted with Hsf23S and enhanced the transcriptional activation of Hsf23S on the sHSPs and TIL1 promoters.Notably,genetic analysis suggested that co-overexpression of Hsf23L and Hsf23S further improves heat tolerance of the transgenic plants.Taken together,these results reveal two splicing variants of ZmHsf23 cooperatively regulate maize heat tolerance,thus highlighting potential value of ZmHsf23 in breeding heat-tolerant maize varieties.
基金supported by the National Natural Science Foundation of China(Nos.81972771,82173062)the Key Areas Project of Education Department of Guangdong Province(No.2021ZDZX2017)+3 种基金the Tertiary Education Scientific Research Project of Guangzhou Municipal Education Bureau(No.202235387)the Guangzhou Science and Technology Project of Guangzhou Municipal Science and Technology Bureau(No.2023A03J0428)the Natural Science Foundation of Guangdong Province,China(No.2024A1515013082)the Guangdong Basic and Applied Basic Research 21 Foundation(No.2021A1515010403).
文摘Background:Alterations in splicing factors contribute to aberrant alternative splicing(AS),which subsequently promotes tumor progression.The splicing factor polypyrimidine tract binding protein 1(PTBP1)has been shown to facilitate cancer progression by modulating oncogenic variants.However,its specific role and underlying mechanisms in hepatocellular carcinoma(HCC)remain to be elucidated.Methods:PTBP1 expression was evaluated in HCC tissues and cell lines.Subsequently,cells were transfected with vectors designed for PTBP1 overexpression or downregulation.The biological function of PTBP1 was assessed in vitro and in vivo using MTS assays,colony formation assays,transwell assays,xenograft formation,tail vein injection,and orthotopic models.Transcriptome analysis was conducted to elucidate the underlying molecular mechanisms.Results:Our findings demonstrated that PTBP1 exhibited elevated expression in HCC cell lines and tissues.Furthermore,its expression positively correlated with overall and disease-free survival rates,as well as tumor grade and stage.PTBP1 knockdown reduced HCC cell proliferation,migration,and invasion in vitro and suppressed hepatocarcinoma xenograft growth and infiltration in vivo.RNA sequencing(RNA-Seq)analysis identified the AS events associated with PTBP1.PTBP1 functionally enhanced cell proliferation,invasion,and migration by modulating the AS of the microtubule-associated protein tau(MAPT)gene and promoting oncogene expression.Notably,the dysregulation of MAPT splicing coincided with increased PTBP1 expression in HCC.Conclusions:PTBP1-guided AS of the MAPT gene enhances tumorigenicity in HCC through activation of the MAPK/ERK pathways.
基金financially supported by the National Natural Science Foundations of China[31201772]Chinese Universities Scientific Fund [2014JD021]+2 种基金the 948 Program of the Ministry of Agriculture of China [2011-G2A(3)]the National High Technology Research and Development Program of China[863 Program 2011AA100302]the Program for Changjiang Scholars and Innovative Research Team in University(IRT1191)
文摘Background： Identification of functional genes affecting milk production traits is very crucial for improving breeding efficiency in dairy cattle. Many potential candidate genes have been identified through our previous genome wide association study （GWAS）. Of these, GPIHBP1 is an important novel candidate gene for milk production traits. However, the mRNA structure of the bovine GPIHBP1 gene is not fully determined up to now. Results： In this study, we identified a novel alternatively splice transcript variant （XS） which leads to a 3] bp insertion in exon 3 and also confirmed the other four existed transcripts （X1, X2, X3 and X4） of the bovine GPIHBP1 gene. We showed that transcript X5 with a 31 bp insertion and transcript X1 with an 8 bp deletion might have tremendous effect on the protein function and structure of GPIHBP1, respectively. With semi-quantitative PCR and quantitative real-time RT-PCR, we found that the mRNA expression of GPIHBPI, GPIHBP1-X1 and GPIHBP1-X5 in mammary gland of lactating cows were much higher than that in other tissues. Conclusions： Our study reports a novel alternative splicing of GPIHBP1 in bovine for the first time and provide useful information for the further functional analyses of GPIHBP1 in dairy cattle.
文摘Both transcription and post-transcriptional processes, such as alternative splicing, play crucial roles in controlling developmental programs in metazoans. Recently emerged RNA-seq method has brought our understanding of eukaryotic transcriptomes to a new level, because it can resolve both gene expression level and alternative splicing events simultaneously. To gain a better understanding of cellular differentiation in gonads, we analyzed mRNA profiles from Drosophila testes and ovaries using RNA-seq. We identified a set of genes that have sex-specific isoforms in wild-type （WT） gonads, including several transcription factors. We found that differentiation of sperms from undifferentiated germ cells induced a dramatic downregulation of RNA splicing factors. Our data confirmed that RNA splicing events are significantly more frequent in the undifferentiated cell-enriched bag of marbles （barn） mutant testis, but downregulated upon differentiation in WT testis. Consistent with this, we showed that genes required for meiosis and terminal differentiation in WT testis were mainly regulated at the transcriptional level, but not by alternative splicing. Unexpectedly, we observed an increase in expression of all families of chromatin remodeling factors and histone modifying enzymes in the undifferentiated cell-enriched bam testis. More interestingly, chromatin regulators and histone modifying enzymes with opposite enzymatic activities are coenriched in undifferentiated cells in testis, suggesting that these cells may possess dynamic chromatin architecture. Finally, our data revealed many new features of the Drosophila gonadal transcriptomes, and will lead to a more comprehensive understanding of how differential gene expression and splicing regulate gametogenesis in Drosophila. Our data provided a foundation for the systematic study of gene expression and alternative splicing in many interesting areas of germ cell biology in Droso- phila, such as the molecular basis for sexual dimorphism and the regulation of the proliferation vs terminal differentiation programs in germline stem cell lineages. The GEO accession number for the raw and analyzed RNA-seq data is GSE16960.
基金Project supported by the National Natural Science Foundation of China(Nos.81773179,81272972,and 81472355)the Program for New Century Excellent Talents in University(No.NCET-10-0790)+2 种基金the Hunan Provincial Science and Technology Department(Nos.2016JC 2049 and 2014FJ6006)the Hunan Provincial Natural Science Foundation of China(No.2016JJ2172)the Undergraduate Training Programs for Innovation and Entrepreneurship(Nos.201810533368,GS201910533474,and GS201910533236),China.
文摘Polypyrimidine tract-binding protein 1(PTBP1)plays an essential role in splicing and is expressed in almost all cell types in humans,unlike the other proteins of the PTBP family.PTBP1 mediates several cellular processes in certain types of cells,including the growth and differentiation of neuronal cells and activation of immune cells.Its function is regulated by various molecules,including micro RNAs(mi RNAs),long non-coding RNAs(lnc RNAs),and RNA-binding proteins.PTBP1 plays roles in various diseases,particularly in some cancers,including colorectal cancer,renal cell cancer,breast cancer,and glioma.In cancers,it acts mainly as a regulator of glycolysis,apoptosis,proliferation,tumorigenesis,invasion,and migration.The role of PTBP1 in cancer has become a popular research topic in recent years,and this research has contributed greatly to the formulation of a useful therapeutic strategy for cancer.In this review,we summarize recent findings related to PTBP1 and discuss how it regulates the development of cancer cells.
文摘AIM:This study was designed to compare the levels of v5 and v6 splice variants of CD44 evaluated using EITSA test in the serum of patients with colorectal cancer in different stages of progression of the disease estimated in pT stage according to WHO score,histopathological grade of malignancy and some clinicopathological features. METHODS:The serum obtained from 114 persons with colorectal adenocarcinomas was examined using ELISA method,pT stage and grade of malignancy of the tumour were examined in formalin fixed and paraffin embedded materials obtained during operation. RESULTS:Only the level of CD44 v5 in the serum of patients before operation with G2 pT4 tumour was lower than that in other probes and the difference was statistically significant. We did not find any other correlations between the level of v5 and v6 CD44 variants and other evaluated parameters. CONCLUSION:The level of CD44 v5 and v6 estimated by ELISA test in the serum can not be used as a prognostic factor in colorectal cancer.
基金supported by the National Basic Research Program of China (2011CB100401)the National Science Fund of China for Distinguished Young Scientists(30825030)+1 种基金the National Natural Science Foundation of China (30970260,30770466 and 30971752)the Key Project from Chongqing Local Government,China(2010AA1019)
文摘Ser/Arg-rich （SR） genes encode proteins that play pivotal roles in both constitutive and alternative splicing of pre-mRNA. However, not much effort has been made to investigate the alternative splicing of their own pre-mRNA. In this study, we conducted comprehensive analyses of pre-mRNA splicing for 22 SR genes in three rice （Oryza sativa L.） ecotypes indica, japonica andjavanica. Using different ecotypes we characterized the variations in expression and splicing patterns of rice SR genes in different tissues and at different developmental stages. In addition, we compared the divergence in expression and splicing patterns of SR genes from seedlings of different rice ecotypes in response to hormones application and environmental stresses. Our results revealed the complexity of alternative splicing of SR genes in rice. The splicing varies in different tissues, in different ecotypes, in response to stresses and hormones. Thus, our study suggested that SR genes were subjected to sophisticated alternative splicing although their encoding proteins were involved in the splicing process.
基金supported by Nantong UniversityNew York State Institute for Basic Research in Developmental Disabilities+3 种基金by grants from the National Natural Science Foundation of China(81030059 and 81170317)the Basic Research Program of Education Department of Jiangsu Province,China(10KJA310040)the U.S.Alzheimer’s Association(Grant IIRG-10-173154)the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)of China
文摘The neuronal microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in the brains of individuals with Alzheimer＇s disease and related neurodegenerative disorders. The adult human brain expresses six isoforms of tau generated by alternative splicing of exons 2, 3, and 10 of its pre-mRNA. Exon 10 encodes the second microtubule-binding repeat of tau. Its alternative splicing produces tau isoforms with either three or four microtubule-binding repeats, termed 3R-tau and 4R- tau. In the normal adult human brain, the level of 3R-tau is approximately equal to that of 4R-tau. Several silent and intronic mutations of the tau gene associated with FTDP-17T （frontotemporal dementia with Parkinsonism linked to chromosome 17 and specifically characterized by tau pathology） only disrupt exon 10 splicing, but do not influence the primary sequence of the tau protein. Thus, abnormal exon 10 splicing is sufficient to cause neurodegeneration and dementia. Here, we review the regulation of tau exon 10 splicing by cis-elements and trans-factors and summarize all the mutations associated with FTDP-17T and related tauopathies. The findings suggest that correction of exon 10 splicing may be a potential target for tau exon 10 splicing-related tauopathies.
文摘Aim:To identify a novel isoform of adaptin 2 beta subunit (named Ap2β-NY) and to investigate its relationship with testicular development and spermatogenesis.Methods:Using a human testis cDNA microarray,a clone (Ap2β-NY), which was strongly expressed in adult testes but weakly expressed in embryo testes,was sequenced and analyzed. Using polymerase chain reaction (PCR),the tissue distribution and expression time pattern of Ap2β-NY were determined. Results:Ap2β-NY was identified and has been deposited in the GenBank (AY341427).The expression level of Ap2β-NY in the adult testis was about 3-fold higher than that in the embryo testis.PCR analysis using multi-tissue cDNA indicated that Ap2β-NY was highly expressed in the testis,spleen,thymus,prostate,ovary,blood leukocyte and brain,but not in the heart,placenta,lung,liver,skeletal muscle,kidney and pancreas.In addition,Ap2β-NY was variably expressed in the testes of patients with spermatogenesis-disturbance and spermatogenesis-arrest but not expressed in those of Sertoli-cell-only syndrome,which implied that,in the testis,Ap2β-NY was restrictively ex- pressed in germ cells.Conclusion:Ap2β-NY is an isoform of Ap2β and may be involved in regulating the process of spermatogenesis and testis development.
基金Supported by the National Key Basic Research and Development Program of China,No.2002CB512901National Natural Science Foundation of China,No.39770868 and Natural Science Foundation of Zhejiang Province,No.397490
文摘AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing. METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced. Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of 17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA. Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced. RESULTS: One of the CYP2D6 cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4), and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples, only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6 cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6 gene were detected. The third variant was the skipped exon 3, and 153 bp was lost. CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.
基金supported by the National Natural Science Foundation of China(31722058,31802275,and 31472269)National Key R&D Program of China(2018YFD0900301)+6 种基金AoShan Talents Cultivation Program Supported by Qingdao National Laboratory for Marine Science and Technology(2017ASTCP-ES06)Taishan Scholar Project Fund of Shandong of China to C.W.S.National Ten-Thousands Talents Special Support Program to C.W.S.Central Public-Interest Scientific Institution Basal Research Fund,CAFS(2020TD19)Central Public-interest Scientific Institution Basal Research Fund,YSFRI,CAFS(20603022021018)China Agriculture Research System(CARS-47-G03)Guangdong South China Sea Key Laboratory of Aquaculture for Aquatic Economic Animals,Guangdong Ocean University(KFKT2019ZD03)。
文摘Chinese tongue sole(Cynoglossus semilaevis)is an economically important marine fish species with a ZZ/ZW sex determination mechanism,which can be influenced by temperature.Alternative splicing(AS)is an important mechanism regulating the expression of genes related to sex determination and gonadal differentiation,but has rarely been reported in fish.In this study,to explore the molecular regulatory mechanisms of sex determination and gonadal differentiation,we combined isoform and RNA sequencing(Iso-Seq and RNA-Seq)to perform transcriptome profiling of male and female gonads in C.semilaevis.In total,81883 and 32341 full-length transcripts were obtained in males and females,respectively.A total of 8279 AS genes were identified,including 2639 genes showing differential AS(DAS)between males and females.Many intersecting DAS genes and differentially expressed genes(DEGs)were enriched in the meiotic cell cycle pathway,and genes related to gonadal differentiation,such as esrrb and wt1a,were found to have sex-specific isoforms.Thus,this study revealed AS events in the gonadal transcriptomes of male and female C.semilaevis,described the characteristics of active transcription in the testes,and identified candidate genes for studying the regulatory mechanisms of AS during gonadal differentiation.
文摘The splicing of many alternative exons in the precursor messenger RNA (pre-mRNA) is regulated by extracellular factors but the underlying molecular bases remain unclear. Here we report the differential regulation of Bcl-x pre-mRNA splicing by extracellular factors and their distinct requirements for pre-mRNA elements. In K562 leukemia cells, treatment with interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF) reduced the proportion of the Bcl-xL variant mRNA while treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) had no effect. In U251 glioma cells, however, TPA efficiently increased the Bcl-xL level. These regulations were also seen for a transfected splicing reporter mini-gene. Further analyses of deletion mutants indicate that nucleotides 1-176 of the downstream intron are required for the IL-6 effect, whereas additional nucleotides 177-284 are essential for the GM-CSF effect. As for the TPA effect, only nucleotides 1-76 are required in the downstream intron. Thus, IL-6, GM-CSF and TPA differentially regulate Bcl-x splicing and require specific intronic pre-mRNA sequences for their respective effects.
基金This work was supported by Natural Sciences Foundation of Guangdong Province(No.0409078)Natural Sciences Foundation from the Education Department of Guangdong Province(No.z02051).
文摘Alternative splicing is a major contributor to genomic complexity and proteome diversity,yet the analysis of alternative splicing for the sequence containing nucleotide binding site and leucine-rich repeats(NBS-LRR)domain has not been explored in rice(Oryza sativa L.).Hidden Markov model(HMM)searches were performed for NBS-LRR domain.875 NBS-LRR-encoding sequences were obtained from the Institute for Genomic Research(TIGR).All of them were used to blast Knowledge-based Oryza Molecular Biological Encyclopaedia(KOME),TIGR rice gene index(TGI),and Universal Protein Resource(UniProt)to obtain homologous full-length cDNAs(FL-cDNAs),tentative consensus sequences,and protein sequences.Alternative splicing events were detected from genomic alignment of FL-cDNAs,tentative consensus sequences,and protein sequences,which provide valuable information on splice variants of genes.These sequences were aligned to the corresponding BAC sequences using the Spidey and Sim4 programs and each of the proteins was aligned by tBLASTn.Of the 875 NBS-LRR sequences,119(13.6%)sequences had alternative splicing where multiple FL-cDNAs,TGI sequences and proteins corresponded to the same gene.71 intron retention events,20 exon skipping events,16 alternative termination events,25 alternative initiation events,12 alternative 5'splicing events,and 16 alternative 3'splicing events were identified.Most of these alternative splices were supported by two or more transcripts.The data sets are available at http://www.bioinfor.org.Furthermore,the bioinformatics analysis of splice boundaries showed that exon skipping and intron retention did not exhibit strong consensus.This implies a different regulation mechanism that guides the expression of splice isoforms.This article also presents the analysis of the effects of intron retention on proteins.The C-terminal regions of alternative proteins turned out to be more variable than the N-terminal regions.Finally,tissue distribution and protein localization of alternative splicing were explored.The largest categories of tissue distributions for alternative splicing were shoot and callus.More than one-thirds of protein localization for splice forms was plasma membrane and cytoplasm.All the NBS-LRR proteins for splice forms may have important function in disease resistance and activate downstream signaling pathways.
文摘Alternative splicing,which is a common phenomenon in mammalian genomes,is a fundamental process of gene regulation and contributes to great protein diversity.Alternative splicing events not only occur in the normal gene regulation process but are also closely related to certain diseases including cancer.In this review,we briefly demonstrate the concept of alternative splicing and DNA damage and describe the association of alternative splicing and cancer pathogenesis,focusing on the potential relationship of alternative splicing,DNA damage,and gastrointestinal cancers.We will also discuss whether alternative splicing leads to genetic instability,which is considered to be a driving force for tumorigenesis.Better understanding of the role and mechanism of alternative splicing in tumorigenesis may provide new directions for future cancer studies.
文摘Accumulating evidence supports the significance of aberrant alternative splicing(AS)events in cancer;however,genome-wide profiling of progression-free survival(PFS)-related AS events in testicular germ cell tumors(TGCT)has not been reported.Here,we analyzed high-throughput RNA-sequencing data and percent-spliced-in values for 150 patients with TGCT.Using univariate and multivariate Cox regression analysis and a least absolute shrinkage and selection operator method,we identified the top 15 AS events most closely associated with disease progression.A risk-associated AS score(ASS)for the 15 AS events was calculated for each patient.ASS,pathological stage,and T stage were significantly associated with disease progression by univariate analysis,but only ASS and pathological stage remained significant by multivariate analysis.The ability of these variables to predict 5-year progression was assessed using receiver operating characteristic curve analysis.ASS had stronger predictive value than a combination of age,pathological stage,and T stage(area under the curve=0.899 and 0.715,respectively).Furthermore,Kaplan—Meier analysis of patients with low and high ASS demonstrated that high ASS was associated with significantly worse PFS than low ASS(P=1.46 × 10^(-7)).We also analyzed the biological functions of the PFS-related AS-related genes and found enrichment in pathways associated with DNA repair and modification.Finally,we identified a regulatory network of splicing factors with expression levels that correlated significantly with AS events in TGCT.Collectively,this study identifies a novel method for risk stratification of patients and provides insight into the molecular events underlying TGCT.
基金supported by the Major Project of National Transgene in China(2018ZX08007001-002)the National Natural Science Foundation of China(31671286,31672397,31771374)+2 种基金Shandong Provincial Natural Science Foundation for Distinguished Young Scholars of China(JQ201709)the Program of National Cow Industrial Technology System of China(CARS-37)the Shandong Provincial Key Research and Development Program of China(2017GNC10120).
文摘F-box and WD-40 domain protein 11(FBXW11)is an important component of the E3 ubiquitin-ligase enzyme that plays a key role in the ubiquitin-dependent regulation of spermatogenesis.In our previous research,the mRNA expression of FBXW11 in bull sperm with high motility is significantly higher than that with low motility.In the present study,the protein expression levels of FBXW11 in bull testicular tissues with low-performance sperm quality groups were significantly higher than those in normal performance groups.The immunohistochemistry result demonstrated that FBXW11 protein was located in the periphery of Leydig cells and seminiferous tubules.Three splice variants of the FBXW11 gene,namely,FBXW11-tv1,FBXW11-tv2,and FBXW11-tv3,were identified in testicular tissues.The splicing patterns of the three variants are exon skipping.The transcript FBXW11-tv2 expressions were the highest in each sample.The low-performance groups displayed higher FBXW11-tv1 and FBXW11-tv2 transcript expressions than the normal performance groups.Two CpG islands were located within the 5’UTR and exon 1-2 region of the FBXW11 gene.Bisulfite sequencing PCR results demonstrated that the methylation levels of 11 methylation sites in the CpG island 2 from−99 to−43 in the normal performance groups were significantly lower than those in the low-performance groups.Pearson correlation analysis suggested that the CpG island 2 methylation level was negatively correlated with sperm motility and the transcript FBXW11-tv2 expression level.Our data revealed that alternative splicing and DNA methylation jointly regulated FBXW11 gene expression and were correlated with sperm quality traits during spermatogenesis in Holsteins.
基金This study was funded by National Natural Science Foundation of China(31571311 to CZ and 31971410 to LL)the CAS Pioneer Hundred Talents Program(CZ and LL).
文摘Plant adaptation to drought stress is essential for plant survival and crop yield.Recently,harnessing drought memory,which is induced by repeated stress and recovery cycles,was suggested as a means to improve drought resistance at the transcriptional level.However,the genetic mechanism underlying drought memory is unclear.Here,we carried out a quantitative analysis of alternative splicing(AS)events in rice memory under drought stress,generating 12 transcriptome datasets.Notably,we identified exon skipping(ES)as the predominant AS type(>80%)in differential alternative splicing(DAS)in response to drought stress.Applying our analysis pipeline to investigate DAS events following drought stress in six other plant species revealed variable ES frequencies ranging from 9.94%to 60.70%depending on the species,suggesting that the relative frequency of DAS types in plants is likely to be speciesspecific.The dinucleotide sequence at AS splice sites in rice following drought stress was preferentially GC-AG and AT-AC.Since U12-type splicing uses the AFAC site,this suggests that drought stress may increase U12-type splicing,and thus increase ES frequency.We hypothesize that multiple isofbrms derived from exon skipping may be induced by drought stress in rice.We also identified 20 transcription factors and three highly connected hub genes with potential roles in drought memory that may be good targets for plant breeding.
