ObjectivesThe PTPRQ gene is essential for preserving the structure and function of stereocilia in inner ear.However,research on splicing mutations within this gene is limited.This study aims to investigate novel splic...ObjectivesThe PTPRQ gene is essential for preserving the structure and function of stereocilia in inner ear.However,research on splicing mutations within this gene is limited.This study aims to investigate novel splicing mutations in PTPRQ,clarify their molecular mechanisms,and provide new insights into the genetic factors associated with hearing loss,ultimately enhancing diagnostic accuracy.MethodClinical data and peripheral blood samples were obtained from members of a family with congenital hearing loss.Variants were identified through high-throughput sequencing and confirmed by Sanger sequencing to ensure genealogical co-segregation.The splicing effects of PTPRQ variants were evaluated using bioinformatics tools and minigene assays.ResultsWe used whole exome sequencing to identify novel double compound heterozygous splice-altering variants(c.5426+1 G>A and c.6603-3 T>G)in the PTPRQ gene with DFNB84A.We molecularly characterized these variants,and they were found to co-segregate with the disease within the family.Minigene assays and Sanger sequencing confirmed that the c.6603-3 T>G variant caused exon 43 skipping,resulting in a frameshift mutation(p.Ser2201ArgfsTer112).Further bioinformatic analysis supported these findings.ConclusionsThis study identifies a novel compound heterozygous splicing variant in the PTPRQ gene in a Chinese family with DFNB84A,expanding the known spectrum of PTPRQ mutations.These findings enhance the understanding of PTPRQ-related hearing loss and may aid in early diagnosis,prevention,and therapeutic strategies.展开更多
Alternative splicing is a cellular mechanism in eukaryotes that results in considerable diversity ofgene products. It plays an important role in several diseases and cellular signal regulation. Heat stress is a major ...Alternative splicing is a cellular mechanism in eukaryotes that results in considerable diversity ofgene products. It plays an important role in several diseases and cellular signal regulation. Heat stress is a major factor that induces immunosuppression in pigs. Little is known about the correlation between alternative splicing and heat stress in pigs. Therefore, this study aimed to clone, sequence and quantify the alternative splicing variant of toll-like receptor 4 （TLR4） in Bama miniature pigs （Sus scrofa domestica） following exposure to heat stress. The results showed that the second exon of TLR4 was spliced and 167 bp shorter in the alternative splicing variant, and the protein was putatively identified as a type of truncated membrane protein consisting of extramembrane, transmembrane and intramembrane regions lacking a signal peptide. Further, it was not a non- classical secretory protein. Five potential reference genes were screened for their potential as reliable standards to quantify the expression of TLR4 alternative spliced variants by real-time quantitative reverse-transcriptase polymerase chain reaction （qRT-PCR）. The stability of these reference genes was ranked using the geNorm and NormFinder programs, and ribosomal protein L4 （RPL4） and TATA box-binding protein （TBP） were found to be the two genes showing the most stable expression in the in vitro cultured peripheral blood mononuclear ceils （PBMCs） during heat shock. The mRNA level of the TLR4 gene （both classical and spliced） in stressed pigs increased significantly （P〈0.05）. Further, the expression levels of the alternative spliced variant of TLR4 （TLR4-ASV） showed a 2-3 folds increase in heat-stressed PBMCs as compared to control pigs. The results of the present study suggested that heat shock might modulate the host immune response by regulating the expressions of TLR4 and its alternative splicing variant.展开更多
Amino acids are the primary form of nitrogen utilization in higher plants,mainly transported by amino acid transporters.In this study,we analyzed the natural variation of amino acid transporter-like 4(OsATL4)in rice g...Amino acids are the primary form of nitrogen utilization in higher plants,mainly transported by amino acid transporters.In this study,we analyzed the natural variation of amino acid transporter-like 4(OsATL4)in rice germplasm resources,identified its spatiotemporal expression characteristics,determined its substrate transport,and validated its function using transgenic plants.We found that the promoter sequence of OsATL4 varied across 498 rice varieties.The expression level of OsATL4 was higher in japonica rice,which was negatively correlated with tiller number and grain yield.OsATL4 was highly expressed in the basal part,leaf sheath,stem,and young panicle,with its two splicing variants localized to the cell membrane.OsATL4a(the long splicing variant)had a high affinity for transporting Ser,Leu,Phe,and Thr,while OsATL4b(the short splicing variant)had a high affinity for transporting Ser,Leu,and Phe.Blocking OsATL4 promoted axillary bud outgrowth,rice tillering,and grain yield,whereas overexpression lines exhibited the opposite phenotype.Exogenous application of low concentrations of Ser promoted axillary bud outgrowth in overexpression lines,while high concentrations of Ser inhibited it.Conversely,the mutant lines showed the opposite response.Altered expression of OsATL4 might affect the expression of genes in nitrogen,auxin,and cytokinin pathways.We propose that two splicing variants of OsATL4 negatively regulate rice tillering and yield by mediating the transport of amino acids,making it a significant target for high-yield rice breeding.展开更多
Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and t...Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined. Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively, The chimera product failed to be translocated into the cell surface when expressed in ClIO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane. Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.展开更多
Prostate cancer(PCa)is one of the most lethal cancers in western countries.Androgen receptor(AR)signaling pathway plays a key role in PCa progression.Despite the initial effectiveness of androgen deprivation therapy(A...Prostate cancer(PCa)is one of the most lethal cancers in western countries.Androgen receptor(AR)signaling pathway plays a key role in PCa progression.Despite the initial effectiveness of androgen deprivation therapy(ADT)for treatment of patients with advanced PCa,most of them will develop resistance to ADT and progress to metastatic castration resistant prostate cancer(mCRPC).Constitutively transcriptional activated AR splice variants(AR-Vs)have emerged as critical players in the development and progression of mCRPC.Among AR-Vs identified to date,AR-V7(a.k.a.AR3)is one of the most abundant and frequently found in both PCa cell lines and in human prostate tissues.Most of functional studies have been focused on AR-V7/AR3 and revealed its role in regulation of survival,growth,differentiation and migration in prostate cells.In this review,we will summarize our current understanding of regulation of expression and activity of AR-Vs in mCRPC.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
Glutamate is a regulated molecule in the mammalian testis. Extracellular regulation of glutamate in the body is determined largely by the expression of plasmalemmal glutamate transporters. We have examined by PCR, wes...Glutamate is a regulated molecule in the mammalian testis. Extracellular regulation of glutamate in the body is determined largely by the expression of plasmalemmal glutamate transporters. We have examined by PCR, western blotting and immunocytochemistry the expression of a panel of sodium-dependent plasmalemmal glutamate transporters in the rat testis. Proteins examined included： glutamate aspartate transporter （GLAST）, glutamate transporter 1 （GLT1）, excitatory amino acid carrier 1 （EAAC1）, excitatory amino acid transporter 4 （EAAT4） and EAAT5. We demonstrate that many of the glutamate transporters in the testis are alternately spliced. GLAST is present as exon-3- and exon-9-skipping forms. GLT1 was similarly present as the alternately spliced forms GLT1 b and GLTlc, whereas the abundant brain form （GLTla） was detectable only at the mRNA level. EAAT5 was also strongly expressed, whereas EAAC1 and EAAT4 were absent. These patterns of expression were compared with the patterns of endogenous glutamate localization and with patterns of D-aspartate accumulation, as assessed by immunocytochemistry. The presence of multiple glutamate transporters in the testis, including unusually spliced forms, suggests that glutamate homeostasis may be critical in this organ. The apparent presence of many of these transporters in the testis and sperm may indicate a need for glutamate transport by such cells.展开更多
In order to investigate the expression of cyclooxygenase-2 (COX-2) in human lower segments of myometrium obtained from women in labor and those not in labor and identify the splicing variant of COX-2, reverse transcri...In order to investigate the expression of cyclooxygenase-2 (COX-2) in human lower segments of myometrium obtained from women in labor and those not in labor and identify the splicing variant of COX-2, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of COX-2. The primers were designed and synthesized according to the sequence of rat COX-2 splice variant which was discovered firstly by us. Then the splicing variant of COX-2 in human myometrium from woman in labor was identified, cloned into vector and sequenced. The results showed that the expression of COX-2 mRNA was lower in human myometrium obtained from women who were not in labor than that in labor women and a new band of COX-2 was obtained in myometrium from labor woman. The fragment included an unspliced intron, which pitched between exons 7 and 8. It was suggested that COX-2 gene was not only expressed highly in human myometrium from woman in labor, but also produced splicing variant by alternative splicing.展开更多
Survivin variants specific real time quantitative RT-PCR was developed to analyze their expression in 53 paired cancer and para-cancerous tissues, and the expression of the wild-type survivin protein was detected by i...Survivin variants specific real time quantitative RT-PCR was developed to analyze their expression in 53 paired cancer and para-cancerous tissues, and the expression of the wild-type survivin protein was detected by immunohistochemistry. The results showed that survivin mRNA and protein were expressed in gastric cancer and para-cancerous tissues, The survivin-2B was dominantly expressed in para-cancerous tissues, whereas the survivin-△Ex3 was more frequently detected in cancer tissues. The positive rate of survivin-2a was 100% in both cancer and para-cancerous tissues, but its relative transcript expression level was not significantly increased in cancer tissues in comparison with para-cancerous tissues. The correlation analysis revealed that the expression of survivin-2a mRNA was significantly associated with that of total survivin （rs=0.4178, P=0.0018）, whereas inversely to that of survivin-△EX3 （rs=-0.4506, P=0.0007）. It was suggested that survivin-2a may act as an antagonist of survivin-△EX3. The balance between antiapoptotic survivin iso-forms and nonantiapoptotic ones may play an important role in tumorigenesis and tumor progression, Promising value is hinted to analyze survivin and its variants in tumor early diagnosis and distinguishing malignant tumors from benign ones.展开更多
Objective Ovarian cancer(OC)is one of the most common and most lethal gynecological malignancies.OC has an age-dependent incidence and occurs more commonly in females older than 50 years old.Most OC patients are diagn...Objective Ovarian cancer(OC)is one of the most common and most lethal gynecological malignancies.OC has an age-dependent incidence and occurs more commonly in females older than 50 years old.Most OC patients are diagnosed at an advanced stage and have a poor prognosis.Germline mutations in the BRCA1 DNA repair associated gene(BRCA1)and the BRCA2 DNA repair associated gene(BRCA2)account for 20%–25%of epithelial ovarian cancer(EOC).BRCA1 germline mutations are more common in Chinese EOC patients.Methods This study reported a three-generation Han-Chinese family containing four EOC patients and a rectal adenocarcinoma patient.Whole-exome sequencing was performed on two EOC patients and an unaffected individual.Variant validation was also performed in all available members by Sanger sequencing.Results A heterozygous splice site variant,c.4358-2A>G in the BRCA1 gene,was identified.Bioinformatic analysis showed that the variant may change the splicing machinery.Conclusion The BRCA1 splice site variant,c.4358-2A>G was identified as the likely genetic cause for EOC,and may also be associated with the increased risk of rectal adenocarcinoma in the family.The findings were beneficial for genetic counseling,helpful for cancer prevention in other family members,and may facilitate therapy decision-making in the future to reduce cancer lethality.展开更多
Background:In 2021,the American College of Medical Genetics and Genomics(ACMG)recommended reporting actionable genotypes in 73 genes associated with diseases for which preventive or therapeutic measures are available....Background:In 2021,the American College of Medical Genetics and Genomics(ACMG)recommended reporting actionable genotypes in 73 genes associated with diseases for which preventive or therapeutic measures are available.Evaluations of the association of actionable genotypes in these genes with life span are currently lacking.Methods:We assessed the prevalence of coding and splice variants in genes on the ACMG Secondary Findings,version 3.0(ACMG SF v3.0),list in the genomes of 57,933 Icelanders.We assigned pathogenicity to all reviewed variants using reported evidence in the ClinVar database,the frequency of variants,and their associations with disease to create a manually curated set of actionable genotypes(variants).We assessed the relationship between these genotypes and life span and further examined the specific causes of death among carriers.展开更多
Alternative splicing(AS)serves as a fundamental regulatory mechanism in gene expression,contributing to proteomic diversity by generating an array of mRNA isoforms from precursor mRNA via distinct splice site combinat...Alternative splicing(AS)serves as a fundamental regulatory mechanism in gene expression,contributing to proteomic diversity by generating an array of mRNA isoforms from precursor mRNA via distinct splice site combinations.In light of the limited therapeutic options currently available,the exploration of AS as a target for drug development is of paramount importance.This review offers an exhaustive analysis of the biological functions and underlying molecular mechanisms associated with various AS-induced splice variants,RNA-binding proteins,and cis-elements,highlighting their significance as clinical biomarkers.We place particular emphasis on the current therapeutic applications of AS in an array of lung diseases,including but not limited to lung cancer,cystic fibrosis,silicosis,acute respiratory distress syndrome,pneumonia,asthma,chronic obstructive pulmonary diseases,pulmonary arterial hypertension,and idiopathic pulmonary fibrosis.The review delves into the role of AS events in the diagnosis and treatment of lung diseases,focusing on the regulatory influence of splicing factors and RNA-binding proteins,while also enumerating the mutated components implicated in AS misregulation.Consequently,a comprehensive understanding of the intricate mechanisms governing these splicing events could potentially offer novel avenues for the development of splicing-targeted therapeutics and diagnostic tools for the prevention and treatment of lung diseases.展开更多
Medline/Pubmed articles relevant to this topic were considered using the search terms ?-microseminoprotein, MSMB, prostate secretory protein of 94 amino acids and PSP94. Full articles were retrieved when the abstract ...Medline/Pubmed articles relevant to this topic were considered using the search terms ?-microseminoprotein, MSMB, prostate secretory protein of 94 amino acids and PSP94. Full articles were retrieved when the abstract was considered relevant. In addition, other data related to this topic including our own are discussed. Summary of fi ndings-?-microseminoprotein(MSMB) is increasingly being considered as a marker for prostatecancer, as reduced levels have been associated with the disease. Here we review various aspects of this protein including its biological and physiological variants, binding proteins and immune modulation; its importance as a marker for biochemical recurrence of prostate cancer; prostate cancer related splice variants and its therapeutic utility. Two of the most important properties of MSMB are related to anticancer functions and immune modulation. Predominant expression of two(short and full-length) splice variants of MSMB has been observed from normal prostate and several other tissues. In benign prostate hyperplasia the short isoform is dominant, constituting 98% of this isoform, whereas in prostate cancer 96% constitute the fulllength isoform. The MSMB promoter single nucleotide polymorphism rs10993994 with the C allele functions as an activated cyclic adenosine monophosphate response element binding protein binding site. This C variant of rs10993994 could be responsible for the production of splice variants under variable conditions. MSMB has binding motifs to a few known proteins including immunoglobulin G and several Cysteine-rich secretory proteins family proteins. MSMB bound to these proteins is considered as immune modulating. Use of MSMB as a urinary marker for detecting aggressive prostate cancers that could resist radiation and surgical treatments, seems possible, but needs further investigation. The ratio of MSMB splice variants could also be a possible approach in understanding prostate cancers, with higher ratios indicating severe disease.展开更多
Thyroid peroxidase （TPO） is the key enzyme involved in thyroid hormone synthesis. Several mutations in the TPO gene may affect the normal growth and development of mammals. In this study, the TPO gene was mapped, it...Thyroid peroxidase （TPO） is the key enzyme involved in thyroid hormone synthesis. Several mutations in the TPO gene may affect the normal growth and development of mammals. In this study, the TPO gene was mapped, its expression level was determined in thyroid grand at the age of 1, 20, 45, 60, 90, 120 and 150 days for Jinhua pig, the alternative splicing form was searched and the single nucleotide polymorphism （SNP, A/G642） of TPO gene was analyzed. The results showed that the porcine TPO was mapped to 3q22-27, the expres- sion level of TPO was relatively stable among the various ages and two novel transcript variants in porcine TPO gene were found： the splicing variant TPO-2 lacked exon 8, while TPO-3 lacked exon 8 and exon 14, 15, 16. Moreover, we found that the SNP of A/G642in the fourteenth exon of TPO gene was significantly associated with ham weight （P 〈 0.05）. Our results provided important basis on the regulation and metabolism of the thyroid gland in animals.展开更多
Disease-recapitulating animal models are valuable tools in preclinical development for the study of compounds.In the case of fibrotic pulmonary diseases such as idiopathic pulmonary fibrosis(IPF),the bleomycin model o...Disease-recapitulating animal models are valuable tools in preclinical development for the study of compounds.In the case of fibrotic pulmonary diseases such as idiopathic pulmonary fibrosis(IPF),the bleomycin model of lung injury in the mouse is widely used.To evaluate bleomycin-induced changes in the lung,we employed a quantitative,multimodal approach.Using in vivo microcomputed tomography(μCT),we demonstrated radiographic changes associated with disease progression in aeration levels of the lung parenchyma.There exists an unmet need for a quantitative,high-resolution imaging probe to detect pulmonary fibrosis,particularly that can differentiate between inflammatory and fibrotic components of the disease.Matrix remodeling and overexpression of extracellular matrix(ECM)proteins such as collagen and fibronectin are hallmarks of organ fibrosis.A splice variant of fibronectin containing extra domain A(FnEDA)is of particular interest in fibrosis due to its high level of expression in diseased tissue,which is confirmed here using immunohistochemistry(IHC)in mouse and human lungs.An antibody against FnEDA was evaluated for use as an imaging tool,particularly by using in vivo single-photon emission computed tomography(SPECT)and ex vivo near-infrared(NIR)fluorescence imaging.These data were further corroborated with histological tissue staining and fibrosis quantitation based on a Modified Ashcroft(MA)score and a digital image analysis of whole slide lung tissue sections.The fusion of these different approaches represents a robust integrated workflow combining anatomical and molecular imaging technologies to enable the visualization and quantitation of disease activity and treatment response with an inhibitor of the TGFβsignaling pathway.展开更多
Interferon regulatory factor 3(IRF3),one member of the IRF family,plays a central role in induction of type I interferon(IFN)and regulation of apoptosis.Controlled activity of IRF3 is essential for its functions.Durin...Interferon regulatory factor 3(IRF3),one member of the IRF family,plays a central role in induction of type I interferon(IFN)and regulation of apoptosis.Controlled activity of IRF3 is essential for its functions.During reverse transcription(RT)-PCR to clone the full-length open reading frame(ORF)of IRF3,we cloned a full-length ORF encoding an isoform of IRF3,termed as IRF3-CL,and has a unique carboxyl-terminus of 125 amino acids.IRF3-CL is ubiquitously expressed in distinct cell lines.Overexpression of IRF3-CL inhibits Sendai virus(SeV)-triggered induction of IFN-β and SeV-induced and inhibitor of NF-kB kinase-e(IKKε)-mediated nuclear translocation of IRF3.When IKKε is overexpressed,IRF3-CL is associated with IRF3.These results suggest that IRF3-CL,the alternative splicing isoform of IRF-3,may function as a negative regulator of IRF3.展开更多
As confusion mounts over RNA isoforms involved in phenotypic plasticity,aberrant CpG methylation-mediated disruption of alternative splicing is increasingly recognized as a driver of intratumor heterogeneity(ITH).Prot...As confusion mounts over RNA isoforms involved in phenotypic plasticity,aberrant CpG methylation-mediated disruption of alternative splicing is increasingly recognized as a driver of intratumor heterogeneity(ITH).Protease serine 3(PRSS3),possessing four splice variants(PRSS3-SVs;PRSS3-V1—V4),is an indispensable trypsin that shows paradoxical effects on cancer development.Here,we found that PRSS3 transcripts and their isoforms were divergently expressed in lung cancer,exhibiting opposing functions and clinical outcomes,namely,oncogenic PRSS3-V1 and PRSS3-V2 versus tumorsuppressive PRSS3-V3,by targeting different downstream genes.We identified an intragenic CpG island(iCpGI)in PRSS3.Hypermethylation of iCpGI was mediated by UHRF1/DNMT1 complex interference with the binding of myeloid zinc finger 1(MZF1)to regulate PRSS3 transcription.The garlic-derived compound diallyl trisulfide cooperated with 5-aza-2'-deoxycytidine to exert antitumor effects in lung adenocarcinoma cells through site-specific iCpGI demethylation specifically allowing MZF1 to upregulate PRSS3-V3 expression.Epigenetic silencing of PRSS3-V3 via i CpGI methylation(iCpGIm)in BALF and tumor tissues was associated with early clinical progression in patients with lung cancer but not in those with squamous cell carcinoma or inflammatory disease.Thus,UHRF1/DNMT1—MZF1 axismodulated site-specific iCpGIm regulates divergent expression of PRSS3-SVs,conferring nongenetic functional ITH,with implications for early detection of lung cancer and targeted therapies.展开更多
基金supported in part by the Natural Science Foundation of Shandong Province(no.ZR2022QH373,ZR2022QH292 and ZR2023MH2474).
文摘ObjectivesThe PTPRQ gene is essential for preserving the structure and function of stereocilia in inner ear.However,research on splicing mutations within this gene is limited.This study aims to investigate novel splicing mutations in PTPRQ,clarify their molecular mechanisms,and provide new insights into the genetic factors associated with hearing loss,ultimately enhancing diagnostic accuracy.MethodClinical data and peripheral blood samples were obtained from members of a family with congenital hearing loss.Variants were identified through high-throughput sequencing and confirmed by Sanger sequencing to ensure genealogical co-segregation.The splicing effects of PTPRQ variants were evaluated using bioinformatics tools and minigene assays.ResultsWe used whole exome sequencing to identify novel double compound heterozygous splice-altering variants(c.5426+1 G>A and c.6603-3 T>G)in the PTPRQ gene with DFNB84A.We molecularly characterized these variants,and they were found to co-segregate with the disease within the family.Minigene assays and Sanger sequencing confirmed that the c.6603-3 T>G variant caused exon 43 skipping,resulting in a frameshift mutation(p.Ser2201ArgfsTer112).Further bioinformatic analysis supported these findings.ConclusionsThis study identifies a novel compound heterozygous splicing variant in the PTPRQ gene in a Chinese family with DFNB84A,expanding the known spectrum of PTPRQ mutations.These findings enhance the understanding of PTPRQ-related hearing loss and may aid in early diagnosis,prevention,and therapeutic strategies.
基金supported by grants from the National Natural Science Foundation of China (31101862)the China Postdoctor Science Foundation and Guangdong Ocean University Doctor Seed Grant, China (0712107)
文摘Alternative splicing is a cellular mechanism in eukaryotes that results in considerable diversity ofgene products. It plays an important role in several diseases and cellular signal regulation. Heat stress is a major factor that induces immunosuppression in pigs. Little is known about the correlation between alternative splicing and heat stress in pigs. Therefore, this study aimed to clone, sequence and quantify the alternative splicing variant of toll-like receptor 4 （TLR4） in Bama miniature pigs （Sus scrofa domestica） following exposure to heat stress. The results showed that the second exon of TLR4 was spliced and 167 bp shorter in the alternative splicing variant, and the protein was putatively identified as a type of truncated membrane protein consisting of extramembrane, transmembrane and intramembrane regions lacking a signal peptide. Further, it was not a non- classical secretory protein. Five potential reference genes were screened for their potential as reliable standards to quantify the expression of TLR4 alternative spliced variants by real-time quantitative reverse-transcriptase polymerase chain reaction （qRT-PCR）. The stability of these reference genes was ranked using the geNorm and NormFinder programs, and ribosomal protein L4 （RPL4） and TATA box-binding protein （TBP） were found to be the two genes showing the most stable expression in the in vitro cultured peripheral blood mononuclear ceils （PBMCs） during heat shock. The mRNA level of the TLR4 gene （both classical and spliced） in stressed pigs increased significantly （P〈0.05）. Further, the expression levels of the alternative spliced variant of TLR4 （TLR4-ASV） showed a 2-3 folds increase in heat-stressed PBMCs as compared to control pigs. The results of the present study suggested that heat shock might modulate the host immune response by regulating the expressions of TLR4 and its alternative splicing variant.
基金supported by the National Natural Science Foundation of China (32060064/32260498)the Guizhou Provincial Excellent Young Talents Project of Science and Technology (Qiankehepingtairencai-YQK (2023)002)+6 种基金the Guizhou Provincial Science and Technology Projects (Qiankehejichu-ZK (2021)General 128Qiankehejichu-ZK (2022)Key 008Qiankehechengguo (2024)General 116Qiankehepingtairencai-BQW (2024)001,qiankehepingtai-YWZ (2024)004)the Key Laboratory of Molecular Breeding for Grain and Oil Crops in Guizhou Province (Qiankehezhongyindi (2023)008)the Key Laboratory of Functional Agriculture of Guizhou Provincial Department of Education (Qianjiaoji (2023)007)the Qiandongnan Science and Technology Support Project (Qiandongnan Kehe Support (2023)06).
文摘Amino acids are the primary form of nitrogen utilization in higher plants,mainly transported by amino acid transporters.In this study,we analyzed the natural variation of amino acid transporter-like 4(OsATL4)in rice germplasm resources,identified its spatiotemporal expression characteristics,determined its substrate transport,and validated its function using transgenic plants.We found that the promoter sequence of OsATL4 varied across 498 rice varieties.The expression level of OsATL4 was higher in japonica rice,which was negatively correlated with tiller number and grain yield.OsATL4 was highly expressed in the basal part,leaf sheath,stem,and young panicle,with its two splicing variants localized to the cell membrane.OsATL4a(the long splicing variant)had a high affinity for transporting Ser,Leu,Phe,and Thr,while OsATL4b(the short splicing variant)had a high affinity for transporting Ser,Leu,and Phe.Blocking OsATL4 promoted axillary bud outgrowth,rice tillering,and grain yield,whereas overexpression lines exhibited the opposite phenotype.Exogenous application of low concentrations of Ser promoted axillary bud outgrowth in overexpression lines,while high concentrations of Ser inhibited it.Conversely,the mutant lines showed the opposite response.Altered expression of OsATL4 might affect the expression of genes in nitrogen,auxin,and cytokinin pathways.We propose that two splicing variants of OsATL4 negatively regulate rice tillering and yield by mediating the transport of amino acids,making it a significant target for high-yield rice breeding.
基金The study was supported by National Basic Science Key Infrastructure Development Program (973 program), National Ministry of Science and Technology (Grant No: G19990559) and in part by E-institutes fund of Shanghai Municipal Education Commission. (Grant No:E03003)
文摘Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined. Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively, The chimera product failed to be translocated into the cell surface when expressed in ClIO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane. Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.
基金This work was supported by NIH(CA106504,CA169524)and DOD(W81XWH-15-1-0612)grants to YQThe authors apologize for not being able to cite many important studies related to this subject due to limited space.
文摘Prostate cancer(PCa)is one of the most lethal cancers in western countries.Androgen receptor(AR)signaling pathway plays a key role in PCa progression.Despite the initial effectiveness of androgen deprivation therapy(ADT)for treatment of patients with advanced PCa,most of them will develop resistance to ADT and progress to metastatic castration resistant prostate cancer(mCRPC).Constitutively transcriptional activated AR splice variants(AR-Vs)have emerged as critical players in the development and progression of mCRPC.Among AR-Vs identified to date,AR-V7(a.k.a.AR3)is one of the most abundant and frequently found in both PCa cell lines and in human prostate tissues.Most of functional studies have been focused on AR-V7/AR3 and revealed its role in regulation of survival,growth,differentiation and migration in prostate cells.In this review,we will summarize our current understanding of regulation of expression and activity of AR-Vs in mCRPC.
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Glutamate is a regulated molecule in the mammalian testis. Extracellular regulation of glutamate in the body is determined largely by the expression of plasmalemmal glutamate transporters. We have examined by PCR, western blotting and immunocytochemistry the expression of a panel of sodium-dependent plasmalemmal glutamate transporters in the rat testis. Proteins examined included： glutamate aspartate transporter （GLAST）, glutamate transporter 1 （GLT1）, excitatory amino acid carrier 1 （EAAC1）, excitatory amino acid transporter 4 （EAAT4） and EAAT5. We demonstrate that many of the glutamate transporters in the testis are alternately spliced. GLAST is present as exon-3- and exon-9-skipping forms. GLT1 was similarly present as the alternately spliced forms GLT1 b and GLTlc, whereas the abundant brain form （GLTla） was detectable only at the mRNA level. EAAT5 was also strongly expressed, whereas EAAC1 and EAAT4 were absent. These patterns of expression were compared with the patterns of endogenous glutamate localization and with patterns of D-aspartate accumulation, as assessed by immunocytochemistry. The presence of multiple glutamate transporters in the testis, including unusually spliced forms, suggests that glutamate homeostasis may be critical in this organ. The apparent presence of many of these transporters in the testis and sperm may indicate a need for glutamate transport by such cells.
基金This project was supported by grants from National Natu ral Sciences Foundation of China ( No. 30070929,30271363).
文摘In order to investigate the expression of cyclooxygenase-2 (COX-2) in human lower segments of myometrium obtained from women in labor and those not in labor and identify the splicing variant of COX-2, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of COX-2. The primers were designed and synthesized according to the sequence of rat COX-2 splice variant which was discovered firstly by us. Then the splicing variant of COX-2 in human myometrium from woman in labor was identified, cloned into vector and sequenced. The results showed that the expression of COX-2 mRNA was lower in human myometrium obtained from women who were not in labor than that in labor women and a new band of COX-2 was obtained in myometrium from labor woman. The fragment included an unspliced intron, which pitched between exons 7 and 8. It was suggested that COX-2 gene was not only expressed highly in human myometrium from woman in labor, but also produced splicing variant by alternative splicing.
文摘Survivin variants specific real time quantitative RT-PCR was developed to analyze their expression in 53 paired cancer and para-cancerous tissues, and the expression of the wild-type survivin protein was detected by immunohistochemistry. The results showed that survivin mRNA and protein were expressed in gastric cancer and para-cancerous tissues, The survivin-2B was dominantly expressed in para-cancerous tissues, whereas the survivin-△Ex3 was more frequently detected in cancer tissues. The positive rate of survivin-2a was 100% in both cancer and para-cancerous tissues, but its relative transcript expression level was not significantly increased in cancer tissues in comparison with para-cancerous tissues. The correlation analysis revealed that the expression of survivin-2a mRNA was significantly associated with that of total survivin （rs=0.4178, P=0.0018）, whereas inversely to that of survivin-△EX3 （rs=-0.4506, P=0.0007）. It was suggested that survivin-2a may act as an antagonist of survivin-△EX3. The balance between antiapoptotic survivin iso-forms and nonantiapoptotic ones may play an important role in tumorigenesis and tumor progression, Promising value is hinted to analyze survivin and its variants in tumor early diagnosis and distinguishing malignant tumors from benign ones.
基金the National Natural Science Foundation of China(No.81800219 and No.81873686)Natural Science Foundation of Hunan Province(No.2019JJ50927,No.2020JJ3057 and No.2020JJ4830)the Lotus Scholars Program of Hunan Province,and the Wisdom Accumulation and Talent Cultivation Project of the Third Xiangya Hospital of Central South University(No.YX202109).
文摘Objective Ovarian cancer(OC)is one of the most common and most lethal gynecological malignancies.OC has an age-dependent incidence and occurs more commonly in females older than 50 years old.Most OC patients are diagnosed at an advanced stage and have a poor prognosis.Germline mutations in the BRCA1 DNA repair associated gene(BRCA1)and the BRCA2 DNA repair associated gene(BRCA2)account for 20%–25%of epithelial ovarian cancer(EOC).BRCA1 germline mutations are more common in Chinese EOC patients.Methods This study reported a three-generation Han-Chinese family containing four EOC patients and a rectal adenocarcinoma patient.Whole-exome sequencing was performed on two EOC patients and an unaffected individual.Variant validation was also performed in all available members by Sanger sequencing.Results A heterozygous splice site variant,c.4358-2A>G in the BRCA1 gene,was identified.Bioinformatic analysis showed that the variant may change the splicing machinery.Conclusion The BRCA1 splice site variant,c.4358-2A>G was identified as the likely genetic cause for EOC,and may also be associated with the increased risk of rectal adenocarcinoma in the family.The findings were beneficial for genetic counseling,helpful for cancer prevention in other family members,and may facilitate therapy decision-making in the future to reduce cancer lethality.
文摘Background:In 2021,the American College of Medical Genetics and Genomics(ACMG)recommended reporting actionable genotypes in 73 genes associated with diseases for which preventive or therapeutic measures are available.Evaluations of the association of actionable genotypes in these genes with life span are currently lacking.Methods:We assessed the prevalence of coding and splice variants in genes on the ACMG Secondary Findings,version 3.0(ACMG SF v3.0),list in the genomes of 57,933 Icelanders.We assigned pathogenicity to all reviewed variants using reported evidence in the ClinVar database,the frequency of variants,and their associations with disease to create a manually curated set of actionable genotypes(variants).We assessed the relationship between these genotypes and life span and further examined the specific causes of death among carriers.
基金supported by the National Natural Science Foundation of China(Grant Nos.82200070,32171127,and U24A20645)the Scientific Fund Project of Heilongjiang Province(JQ2022H001,China)+2 种基金the Harbin Medical University Graduate Research and Practice Innovation Project(Grant No.YJSCX2023-37HYD,China)the Innovation and Development Joint Fund of the Natural Science Foundation of Chongqing(Grant No.CSTB2023NSCQ-LMX0037,China)the CAMS Innovation Fund for Medical Sciences(CIFMS,2019-I2M-5-078,China).
文摘Alternative splicing(AS)serves as a fundamental regulatory mechanism in gene expression,contributing to proteomic diversity by generating an array of mRNA isoforms from precursor mRNA via distinct splice site combinations.In light of the limited therapeutic options currently available,the exploration of AS as a target for drug development is of paramount importance.This review offers an exhaustive analysis of the biological functions and underlying molecular mechanisms associated with various AS-induced splice variants,RNA-binding proteins,and cis-elements,highlighting their significance as clinical biomarkers.We place particular emphasis on the current therapeutic applications of AS in an array of lung diseases,including but not limited to lung cancer,cystic fibrosis,silicosis,acute respiratory distress syndrome,pneumonia,asthma,chronic obstructive pulmonary diseases,pulmonary arterial hypertension,and idiopathic pulmonary fibrosis.The review delves into the role of AS events in the diagnosis and treatment of lung diseases,focusing on the regulatory influence of splicing factors and RNA-binding proteins,while also enumerating the mutated components implicated in AS misregulation.Consequently,a comprehensive understanding of the intricate mechanisms governing these splicing events could potentially offer novel avenues for the development of splicing-targeted therapeutics and diagnostic tools for the prevention and treatment of lung diseases.
基金the Auckland Medical Research Foundation, New Zealand and Goodfellow Trust, Urology Department, Auckland Hospital, New Zealand for funding the patient recruitment related to this communication.
文摘Medline/Pubmed articles relevant to this topic were considered using the search terms ?-microseminoprotein, MSMB, prostate secretory protein of 94 amino acids and PSP94. Full articles were retrieved when the abstract was considered relevant. In addition, other data related to this topic including our own are discussed. Summary of fi ndings-?-microseminoprotein(MSMB) is increasingly being considered as a marker for prostatecancer, as reduced levels have been associated with the disease. Here we review various aspects of this protein including its biological and physiological variants, binding proteins and immune modulation; its importance as a marker for biochemical recurrence of prostate cancer; prostate cancer related splice variants and its therapeutic utility. Two of the most important properties of MSMB are related to anticancer functions and immune modulation. Predominant expression of two(short and full-length) splice variants of MSMB has been observed from normal prostate and several other tissues. In benign prostate hyperplasia the short isoform is dominant, constituting 98% of this isoform, whereas in prostate cancer 96% constitute the fulllength isoform. The MSMB promoter single nucleotide polymorphism rs10993994 with the C allele functions as an activated cyclic adenosine monophosphate response element binding protein binding site. This C variant of rs10993994 could be responsible for the production of splice variants under variable conditions. MSMB has binding motifs to a few known proteins including immunoglobulin G and several Cysteine-rich secretory proteins family proteins. MSMB bound to these proteins is considered as immune modulating. Use of MSMB as a urinary marker for detecting aggressive prostate cancers that could resist radiation and surgical treatments, seems possible, but needs further investigation. The ratio of MSMB splice variants could also be a possible approach in understanding prostate cancers, with higher ratios indicating severe disease.
基金supported by National Basic Research Program of China (973 Program) (No. 2006CB102100)Major Agriculture Research Project of Zhejiang Province (No. 2005C12005-2)National Natural Science Foun-dation of China (No. 30972078)
文摘Thyroid peroxidase （TPO） is the key enzyme involved in thyroid hormone synthesis. Several mutations in the TPO gene may affect the normal growth and development of mammals. In this study, the TPO gene was mapped, its expression level was determined in thyroid grand at the age of 1, 20, 45, 60, 90, 120 and 150 days for Jinhua pig, the alternative splicing form was searched and the single nucleotide polymorphism （SNP, A/G642） of TPO gene was analyzed. The results showed that the porcine TPO was mapped to 3q22-27, the expres- sion level of TPO was relatively stable among the various ages and two novel transcript variants in porcine TPO gene were found： the splicing variant TPO-2 lacked exon 8, while TPO-3 lacked exon 8 and exon 14, 15, 16. Moreover, we found that the SNP of A/G642in the fourteenth exon of TPO gene was significantly associated with ham weight （P 〈 0.05）. Our results provided important basis on the regulation and metabolism of the thyroid gland in animals.
基金financial support for the experiments were provided by AbbVie.
文摘Disease-recapitulating animal models are valuable tools in preclinical development for the study of compounds.In the case of fibrotic pulmonary diseases such as idiopathic pulmonary fibrosis(IPF),the bleomycin model of lung injury in the mouse is widely used.To evaluate bleomycin-induced changes in the lung,we employed a quantitative,multimodal approach.Using in vivo microcomputed tomography(μCT),we demonstrated radiographic changes associated with disease progression in aeration levels of the lung parenchyma.There exists an unmet need for a quantitative,high-resolution imaging probe to detect pulmonary fibrosis,particularly that can differentiate between inflammatory and fibrotic components of the disease.Matrix remodeling and overexpression of extracellular matrix(ECM)proteins such as collagen and fibronectin are hallmarks of organ fibrosis.A splice variant of fibronectin containing extra domain A(FnEDA)is of particular interest in fibrosis due to its high level of expression in diseased tissue,which is confirmed here using immunohistochemistry(IHC)in mouse and human lungs.An antibody against FnEDA was evaluated for use as an imaging tool,particularly by using in vivo single-photon emission computed tomography(SPECT)and ex vivo near-infrared(NIR)fluorescence imaging.These data were further corroborated with histological tissue staining and fibrosis quantitation based on a Modified Ashcroft(MA)score and a digital image analysis of whole slide lung tissue sections.The fusion of these different approaches represents a robust integrated workflow combining anatomical and molecular imaging technologies to enable the visualization and quantitation of disease activity and treatment response with an inhibitor of the TGFβsignaling pathway.
基金This work was supported by grants from the China National Human Liver Proteomics Project(2004BA711A19)the China National High-Tech 863 Program(2006AA 02A310)+1 种基金the Natural Science Foundation of Hubei Province of China(2009CDB012)the Educational Commission of Hubei Province of China(D20091004).
文摘Interferon regulatory factor 3(IRF3),one member of the IRF family,plays a central role in induction of type I interferon(IFN)and regulation of apoptosis.Controlled activity of IRF3 is essential for its functions.During reverse transcription(RT)-PCR to clone the full-length open reading frame(ORF)of IRF3,we cloned a full-length ORF encoding an isoform of IRF3,termed as IRF3-CL,and has a unique carboxyl-terminus of 125 amino acids.IRF3-CL is ubiquitously expressed in distinct cell lines.Overexpression of IRF3-CL inhibits Sendai virus(SeV)-triggered induction of IFN-β and SeV-induced and inhibitor of NF-kB kinase-e(IKKε)-mediated nuclear translocation of IRF3.When IKKε is overexpressed,IRF3-CL is associated with IRF3.These results suggest that IRF3-CL,the alternative splicing isoform of IRF-3,may function as a negative regulator of IRF3.
基金National Natural Science Foundation of China(NSFC grant No.32200462,China)National Natural Science Foundation of China(NSFC grant No.81872021,China)+4 种基金Beijing Municipal Administration of Hospitals Youth Program(grant No.QMS20221603,China)R&D Program of Beijing Municipal Education Commission(grant No.KM202110025004,China)Beijing Natural Science Foundation of China(BJSFC No.7214242,China)Beijing Municipal Administration of Hospitals Incubating Program(grant No.PX2021063,China)Intramural Research Funding Program from Beijing Tuberculosis and Thoracic Tumor Research Institute/Beijing Chest Hospital。
文摘As confusion mounts over RNA isoforms involved in phenotypic plasticity,aberrant CpG methylation-mediated disruption of alternative splicing is increasingly recognized as a driver of intratumor heterogeneity(ITH).Protease serine 3(PRSS3),possessing four splice variants(PRSS3-SVs;PRSS3-V1—V4),is an indispensable trypsin that shows paradoxical effects on cancer development.Here,we found that PRSS3 transcripts and their isoforms were divergently expressed in lung cancer,exhibiting opposing functions and clinical outcomes,namely,oncogenic PRSS3-V1 and PRSS3-V2 versus tumorsuppressive PRSS3-V3,by targeting different downstream genes.We identified an intragenic CpG island(iCpGI)in PRSS3.Hypermethylation of iCpGI was mediated by UHRF1/DNMT1 complex interference with the binding of myeloid zinc finger 1(MZF1)to regulate PRSS3 transcription.The garlic-derived compound diallyl trisulfide cooperated with 5-aza-2'-deoxycytidine to exert antitumor effects in lung adenocarcinoma cells through site-specific iCpGI demethylation specifically allowing MZF1 to upregulate PRSS3-V3 expression.Epigenetic silencing of PRSS3-V3 via i CpGI methylation(iCpGIm)in BALF and tumor tissues was associated with early clinical progression in patients with lung cancer but not in those with squamous cell carcinoma or inflammatory disease.Thus,UHRF1/DNMT1—MZF1 axismodulated site-specific iCpGIm regulates divergent expression of PRSS3-SVs,conferring nongenetic functional ITH,with implications for early detection of lung cancer and targeted therapies.
