Alternative splicing of the PECTINESTERASE gene encoding a cell wall-degrading enzyme affects postharvest softening in grape

The firmness of table grape berries is a crucial quality parameter. Despite extensive research on postharvest fruit softening, its precise molecular mechanisms remain elusive. To enhance our comprehension of the underlying molecular factors, we initially identified differentially expressed genes(DEGs) by comparing the transcriptomes of folic acid(FA)-treated and water-treated(CK) berries at different time points. We then analyzed the sequences to detect alternatively spliced(AS) genes associated with postharvest softening. A total of 2,559 DEGs were identified and categorized into four subclusters based on their expression patterns, with subcluster-4 genes exhibiting higher expression in the CK group compared with the FA treatment group. There were 1,045 AS-associated genes specific to FA-treated berries and 1,042 in the CK-treated berries, respectively. Gene Ontology(GO) annotation indicated that the AS-associated genes in CK-treated berries were predominantly enriched in cell wall metabolic processes,particularly cell wall degradation processes. Through a comparison between treatment-associated AS genes and subcluster-4 DEGs, we identified eight genes, including Pectinesterase 2(VvPE2, Vitvi15g00704), which encodes a cell wall-degrading enzyme and was predicted to undergo an A3SS event. The reverse transcription polymerase chain reaction further confirmed the presence of a truncated transcript variant of VvPE2 in the FA-treated berries.Our study provides a comprehensive analysis of AS events in postharvest grape berries using transcriptome sequencing and underscores the pivotal role of VvPE2 during the postharvest storage of grape berries.

Improved genome annotation of Brassica oleracea highlights the importance of alternative splicing

Brassica oleracea has been developed into many important crops,including cabbage,kale,cauliflower,broccoli and so on.The genome and gene annotation of cabbage(cultivar JZS),a representative morphotype of B.oleracea,has been widely used as a common reference in biological research.Although its genome assembly has been updated twice,the current gene annotation still lacks information on untranslated regions(UTRs)and alternative splicing(AS).Here,we constructed a high-quality gene annotation(JZSv3)using a full-length transcriptome acquired by nanopore sequencing,yielding a total of 59452 genes and 75684 transcripts.Additionally,we re-analyzed the previously reported transcriptome data related to the development of different tissues and cold response using JZSv3 as a reference,and found that 3843 out of 11908 differentially expressed genes(DEGs)underwent AS during the development of different tissues and 309 out of 903 cold-related genes underwent AS in response to cold stress.Meanwhile,we also identified many AS genes,including BolLHCB5 and BolHSP70,that displayed distinct expression patterns within variant transcripts of the same gene,highlighting the importance of JZSv3 as a pivotal reference for AS analysis.Overall,JZSv3 provides a valuable resource for exploring gene function,especially for obtaining a deeper understanding of AS regulation mechanisms.

2,3,5,4’-Tetrahydroxystilbene-2-O-b-D-Glucoside modulates CHEK2 and CCND1 alternative splicing to inhibit MCF-7 cells proliferation

Background:In our previous study,we observed a synergistic effect of 2,3,5,4’-Tetrahydroxystilbene-2-O-b-D-glucoside combined with adriamycin to induce apoptosis in MCF-7 breast cancer cells.However,the underlying mechanisms of epigenetic modifications,such as alternative splicing,have not been explored.In this study,we aimed to investigate the mechanism by which THSG inhibits MCF-7 cell proliferation using full-length transcriptome sequencing.Methods:First,cell viability was examined using the methyl thiazolyl tetrazolium method and full-length transcriptome sequencing was performed to identify genes and pathways.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used to identify the principal pathways and targets of THSG.Flow cytometry analysis of cell cycle distribution was performed.Meanwhile,the analysis of alternative splicing and domains of the key proteins was conducted.Quantitative polymerase chain reaction and western blotting were performed for verification.Results:THSG showed significant cytotoxic activity in MCF-7 cells.Full-length transcriptome sequencing revealed differential alternative splicing with 173 upregulated and 263 downregulated genes.Further analysis identified distinct differential expression of genes(CHEK2-211 and CCND1-201)involved in the cell cycle in the THSG-treated group.Subsequently,alternative splicing types of CHEK2(mutually exclusive exon)and CCND1(intron retention).We found that THSG downregulated mRNA expression,as confirmed by quantitative polymerase chain reaction analysis.Interestingly,protein structural analysis revealed that THSG treatment led to the generation of CHK2-211,which was the result of a mutation in the amino acid residues(GLU-150,ASN-151)of the CHEK2 domain(VAL-150,GLY-151).and CyclinD1-201 were obtained when an amino acid(ASP-267)in the domain was lost in CyclinD1.Moreover,molecular docking analysis demonstrated that the domains of key proteins could bind THSG more effectively,with no difference in affinity.Western blotting confirmed that THSG inhibited the expression of CHK2 and CyclinD1.Conclusion:THSG modulated the alternative splicing of CHEK2 and CCND1 by inducing G0/G1 cell cycle arrest,consequently suppressing MCF-7 cell proliferation.

Molecular targets and mechanisms of different aberrant alternative splicing in metastatic liver cancer

Metastasis remains a major challenge in the successful management of malignant diseases.The liver is a major site of metastatic disease and a leading cause of death from gastrointestinal malignancies such as colon,stomach,and pancreatic cancers,as well as melanoma,breast cancer,and sarcoma.As an important factor that influences the development of metastatic liver cancer,alternative splicing drives the diversity of RNA transcripts and protein subtypes,which may provide potential to broaden the target space.In particular,the dysfunction of splicing factors and abnormal expression of splicing variants are associated with the occurrence,progression,aggressiveness,and drug resistance of cancers caused by the selective splicing of specific genes.This review is the first to provide a detailed summary of the normal splicing process and alterations that occur during metastatic liver cancer.It will cover the role of alternative splicing in the mechanisms of metastatic liver cancer by examining splicing factor changes,abnormal splicing,and the contribution of hypoxia to these changes during metastasis.

Comprehensive analysis of the full-length transcripts and alternative splicing involved in clubroot resistance in Chinese cabbage

Chinese cabbage is an economically important Brassica vegetable worldwide, and clubroot, which is caused by the soilborne protist plant pathogen Plasmodiophora brassicae is regarded as a destructive disease to Brassica crops. Previous studies on the gene transcripts related to Chinese cabbage resistance to clubroot mainly employed RNA-seq technology,although it cannot provide accurate transcript assembly and structural information. In this study, PacBio RS II SMRT sequencing was used to generate full-length transcriptomes of mixed roots at 0, 2, 5, 8, 13, and 22 days after P. brassicae infection in the clubroot-resistant line DH40R. Overall, 39 376 high-quality isoforms and 26 270 open reading frames(ORFs) were identified from the SMRT sequencing data. Additionally, 426 annotated long noncoding RNAs(lncRNAs),56 transcription factor(TF) families, 1 883 genes with poly(A) sites and 1 691 alternative splicing(AS) events were identified. Furthermore, 1 201 of the genes had at least one AS event in DH40R. A comparison with RNA-seq data revealed six differentially expressed AS genes(one for disease resistance and five for defensive response) that are potentially involved in P. brassicae resistance. The results of this study provide valuable resources for basic research on clubroot resistance in Chinese cabbage.

Identification and quantitative mRNA analysis of a novel splice variant of GPIHBP1 in dairy cattle

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.

RNA m6A dynamic modification mediated by nucleuslocalized FTO is involved in follicular reserve

In eukaryotic organisms,the most common internal modification of messenger RNA(m RNA)is N6-methyladenosine(m6A).This modification can be dynamically and reversibly controlled by specific enzymes known as m6A writers and erasers.The fat-mass and obesity-associated protein(FTO)catalyzes RNA demethylation and plays a critical role in various physiological and pathological processes.Our research identified dynamic alterations in both m6A and FTO during the assembly of primordial follicles,with an inverse relationship observed for m6A levels and nuclear-localized FTO expression.Application of Fto small interfering RNA(si RNA)altered the expression of genes related to cell proliferation,hormone regulation,and cell chemotaxis,and affected RNA alternative splicing.Overexpression of the full-length Fto gene led to changes in m6A levels,alternative splicing of Cdk5,cell proliferation,cell cycle progression,and proportion of primordial follicles.Conversely,overexpression of Fto lacking a nuclear localization signal(NLS)did not significantly alter m6A levels or primordial follicle assembly.These findings suggest that FTO,localized in the nucleus but not in the cytoplasm,regulates RNA m6A demethylation and plays a role in cell proliferation,cell cycle progression,and primordial follicle assembly.These results highlight the potential of m6A and its eraser FTO as possible biomarkers and therapeutic targets.

Novel miR-490-3p/hnRNPA1-b/PKM2 axis mediates the Warburg effect and proliferation of colon cancer cells via the PI3K/AKT pathway

BACKGROUND Heterogeneous ribonucleoprotein A1(hnRNPA1)has been reported to enhance the Warburg effect and promote colon cancer(CC)cell proliferation,but the role and mechanism of the miR-490-3p/hnRNPA1-b/PKM2 axis in CC have not yet been elucidated.AIM To investigate the role and mechanism of a novel miR-490-3p/hnRNPA1-b/PKM2 axis in enhancing the Warburg effect and promoting CC cell proliferation through the PI3K/AKT pathway.METHODS Paraffin-embedded pathological sections from 220 CC patients were collected and subjected to immunohistochemical analysis to determine the expression of hnRNPA1-b.The relationship between the expression values and the clinicopathological features of the patients was investigated.Differences in mRNA expression were analyzed using quantitative real-time polymerase chain reaction,while differences in protein expression were analyzed using western blot.Cell proliferation was evaluated using the cell counting kit-8 and 5-ethynyl-2’-deoxyuridine assays,and cell cycle and apoptosis were detected using flow cytometric assays.The targeted binding of miR-490-3p to hnRNPA1-b was validated using a dual luciferase reporter assay.The Warburg effect was evaluated by glucose uptake and lactic acid production assays.RESULTS The expression of hnRNPA1-b was significantly increased in CC tissues and cells compared to normal controls(P<0.05).Immunohistochemical results demonstrated significant variations in the expression of the hnRNPA1-b antigen in different stages of CC,including stage I,II-III,and IV.Furthermore,the clinicopathologic characterization revealed a significant correlation between hnRNPA1-b expression and clinical stage as well as T classification.HnRNPA1-b was found to enhance the Warburg effect through the PI3K/AKT pathway,thereby promoting proliferation of HCT116 and SW620 cells.However,the proliferation of HCT116 and SW620 cells was inhibited when miR-490-3p targeted and bound to hnRNPA1-b,effectively blocking the Warburg effect.CONCLUSION These findings suggest that the novel miR-490-3p/hnRNPA1-b/PKM2 axis could provide a new strategy for the diagnosis and treatment of CC.

Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq

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.

Levels of v5 and v6 CD44 splice variants in serum of patients with colorectal cancer are not correlated with pT stage,histopathological grade of malignancy and clinical features

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.

Alterations of Alternative Splicing Patterns of Ser/Arg-Rich (SR) Genes in Response to Hormones and Stresses Treatments in Different Ecotypes of Rice (Oryza sativa)

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.

Genome-wide Detection and Analysis of Alternative Splicing for Nucleotide Binding Site-Leucine-Rich Repeats Sequences in Rice

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.

Expression of a novel beta adaptin subunit mRNA splice variant in human testes

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.

Three new alternative splicing variants of human cytochrome P450 2D6 mRNA in human extratumoral liver tissue

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.

Regulation of alternative splicing of Bcl-x by IL-6,GM-CSF and TPA

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.

Alternative splicing of DNA damage response genes and gastrointestinal cancers

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.

DNA methylation and alternative splicing modulate FBXW11 gene expression in Holstein bull testis and are correlated with sperm quality

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.

Effects of SNPs and alternative splicing within HGF gene on its expression patterns in Qinchuan cattle

Background： Identification of genetic variants, including SNPs（Single Nucleotide Polymorphisms）, CNVs（Copy Number Variations） and alternative splicing, within functional genes has received increasing attention in animal science research. HGF（Hepatocyte Growth Factor） is a very important growth factor that works as a mitogen or a morphogen during tissue growth, development and regeneration. However, to date, the functions of genetic variants within the bovine HGF gene, particularly their effects on m RNA expression, have not been determined well.Results： The present study aimed to perform association analysis between genetic variants and m RNA expression for the bovine HGF gene in Qinchuan cattle using various strategies, including PCR-RFLP（Restriction Fragment Length Polymorphism）, q PCR（Quantitative Real-time quantitative PCR）, TA cloning, DNA sequencing and bioinformatics analysis. A total of five SNPs were identified and only SV1 locus significantly affected HGF m RNA expression in fetal skeletal muscle（P 〈 0.05）. Heterozygous genotype individuals showed significantly higher HGF expression（P 〈 0.05）, which was significantly greater in the ＂CTCCAGGGTT＂ combined genotype than that in the＂CCCCGGGGTT＂ combined genotype（P 〈 0.05）. In addition, two alternative splicing variations, HGF-W and HGF-M,were identified, which resulted from alternative 3′ splice sites of exon 5, and HGF-W showed higher m RNA levels than HGF-M in all tissues.Conclusion： In summary, genetic variations within the HGF gene affected m RNA expression. These findings provide new insight into the molecular characteristics and functions of bovine HGF.

Identification of global alternative splicing and sex-specific splicing via comparative transcriptome analysis of gonads of Chinese tongue sole(Cynoglossus semilaevis)

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.

Alternative Splicing of OsRAD1 Defines C-Terminal Domain Essential for Protein Function in Meiosis

Alternative splicing can generate multiple mRNAs that differ in their untranslated regions or coding sequences,and these differences might affect mRNA stability or result in different protein isoforms with diverse functions and/or localizations.In this study,we isolated a sterile mutant in rice with abnormal meiosis of microspore mother cells and megaspore mother cells that carried a point mutation in OsRAD1 gene.Cloning of OsRAD1 cDNAs revealed three transcript variants,named as OsRAD1.1,OsRAD1.2 and OsRAD1.3,respectively,which were derived from alternative splicing of the last intron.Proteins derived from the three transcripts were mostly identical except the difference in the very C-terminal domain.The three transcripts exhibited similar expression patterns in various tissues,but the expression level of OsRAD1.1 was the highest.Specific knockout of OsRAD1.1 led to sterility,while knockout of OsRAD1.2 and OsRAD1.3 together did not change the plant fertility.Overexpression of OsRAD1.2 and OsRAD1.3 cDNAs in OsRAD1.1-specific mutant did not complement the plant fertility.Yeast two-hybrid assay showed that OsRAD1.1,but not OsRAD1.2 and OsRAD1.3,interacted with the three other meiosis proteins OsHUS1,OsRAD9 and OsRAD17,suggesting that the C-terminal domain of OsRAD1.1 is critical for the protein function.