Occurrence Of Alternative Splicing Research Articles

Replicative aging in yeast involves dynamic intron retention patterns associated with mRNA processing/export and protein ubiquitination.

Saccharomyces cerevisiae (baker's yeast) has yielded relevant insights into some of the basic mechanisms of organismal aging. Among these are genomic instability, oxidative stress, caloric restriction and mitochondrial dysfunction. Several genes are known to have an impact on the aging process, with corresponding mutants exhibiting short- or long-lived phenotypes. Research dedicated to unraveling the underlying cellular mechanisms can support the identification of conserved mechanisms of aging in other species. One of the hitherto less studied fields in yeast aging is how the organism regulates its gene expression at the transcriptional level. To our knowledge, we present the first investigation into alternative splicing, particularly intron retention, during replicative aging of S. cerevisiae. This was achieved by utilizing the IRFinder algorithm on a previously published RNA-seq data set by Janssens et al. (2015). In the present work, 44 differentially retained introns in 43 genes were identified during replicative aging. We found that genes with altered intron retention do not display significant changes in overall transcript levels. It was possible to functionally assign distinct groups of these genes to the cellular processes of mRNA processing and export (e.g., YRA1) in early and middle-aged yeast, and protein ubiquitination (e.g., UBC5) in older cells. In summary, our work uncovers a previously unexplored layer of the transcriptional program of yeast aging and, more generally, expands the knowledge on the occurrence of alternative splicing in baker's yeast.

Transcriptome analysis of turbot (Scophthalmus maximus) head kidney and liver reveals immune mechanism in response to Vibrio anguillarum infection.

The diseases triggered by Vibrio anguillarum infection have created huge economic losses to the turbot (Scophthalmus maximus) farming industry. However, the immune mechanism of turbot to V. anguillarum infection has not been deeply investigated. To better understand the immune response of turbot to V. anguillarum infection, transcriptome analysis of the head kidney and liver of turbot was performed. A total of 15,948 and 11,494 differentially expressed genes (DEGs) were obtained from the turbot head kidney and liver, respectively. Transcriptome analysis revealed that the head kidney and liver of turbot have some differences in the gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the DEGs for the different functions of these two organs. Although there are many uncertain factors in this immune process, such as the occurrence of alternative splicing (AS) events and the differences in the protein structure of the DEGs, the NFκB signalling pathway, MKK-dependent AP-1 activation, JAK-STAT signalling pathway, the signal transmission of MHC Ⅰ and a series of DEGs including HSP90 driving NLRP3 to produce inflammatory factors (IL-1β, IL-8, TNFα, etc.) were possible important immune response pathways for turbot to V. anguillarum infection. Overall, our research has conducted a preliminary exploration of the immune mechanism of turbot in response to V. anguillarum infection.

Multiple transcriptome analysis of Piwil2-induced cancer stem cells, including piRNAs, mRNAs and miRNAs reveals the mechanism of tumorigenesis and development.

Cancer stem cells play important roles in the process of tumorigenesis. Our research group obtained cancer stem cell-like cells named Piwil2-iCSCs by reprogramming human preputial fibroblasts (FBs) with the PIWIL2 gene, but the mechanism of Piwil2-iCSCs is still unclear. We sequenced the piRNAs, miRNAs and mRNAs of Piwil2-iCSCs and FBs, and analyzed the differences. Gene Ontology (GO) and, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and gene set enrichment analysis (GSEA) were performed on the differentially expressed (DE) mRNAs. In addition, we analyzed the variable shear events and fusion genes in the Piwil2-iCSCs. Target gene prediction and functional enrichment analysis were performed for the DE miRNAs. A total of 1119 DE mRNAs, 220 DE piRNAs, and 440 DE miRNAs were obtained between the Piwil2-iCSCs and FBs. Functional enrichment analysis showed that the genes with upregulated expression were mainly involved in DNA repair, mismatch repair, base excision repair, and nucleotide excision repair. Genes with downregulated expression were mainly involved in the TGF-β receptor signaling pathway, senescence and autophagy in cancer. More frequent shear events occurred in Piwil2-iCSCs and FBs, especially in intron retention (IR) events. We also identified three fusion genes MCM3AP-C21orf58, LRRFIP2-CAV3 and TMEM184B-DMC1. Enrichment analysis of DE miRNAs showed that they were associated with apoptosis, the TGF-β signaling pathway, and the stem cell regulatory signaling pathway. In particular, target gene prediction of the top three miRNAs with upregulated expression showed that they targeted SMAD, GREM1 and other genes to participate in the regulation of TGF-β and other pathways. PIWIL2-induced cancer stem cells have significantly altered levels of miRNAs, piRNAs and mRNAs.TGF-β, autophagy, apoptosis and other pathways may play an important role in stem cell development. The occurrence of alternative splicing and fusion genes may be related to the occurrence of cancer stem cells.

Molecular cloning and characterization of the ATP citrate lyase from carotenogenic yeast Phaffia rhodozyma.

ATP citrate lyase (ACL), is a key cytosolic source of acetyl-CoA for fatty acid and sterol biosynthesis and appear to be involved in carotenoid biosynthesis in yeasts. Three homologous DNA sequences encoding ACLs in Phaffia rhodozyma were isolated i.e two genes and one cDNA. The two genes were multi-intronic, with 3450-bp-coding sequences and both genes, as the cDNA, encoded identical 120.1-kDa polypeptides. Full-length amino acid sequences of these ACLs showed the two multidomains, PLN02235 and PLN02522, which are necessary for activity. The ACLs showed 82-87% similarity to putative ACLs from other basidiomycetes and 71% similarity to human ACL. The acl cDNA was used to express the heterologous ACL 6XHis-tagged which was identified using MALDI-TOF-MS. The sequenced peptides with 42.2% coverage showed 100% identity to the amino acid sequence generated in silico. The recombinant ACL purified to homogeneity showed an activity of 2 U. This is the first study to characterize a recombinant ACL from a carotenogenic yeast. The present study provides a key foundation for future studies to assess (a) the possible occurrence of alternative splicing, (b) identify the promoter(s) sequence(s) and (c) the involvement of ACL in the differential regulation of fatty acid and carotenoid biosynthesis in yeasts.

Glutamate-gated chloride channel subunit cDNA sequencing of Cochliomyia hominivorax (Diptera: Calliphoridae): cDNA variants and polymorphisms.

The New World screwworm (NWS) Cochliomyia hominivorax (Coquerel) is one of the major myiasis-causing flies that injures livestock and leads to losses of ~US$ 2.7 billions/year in the Neotropics. Ivermectin (IVM), a macrocyclic lactone (ML), is the most used preventive insecticide for this parasite and targets the glutamate-gated chloride (GLUCLα) channels. Several authors have associated altered GluClα homologues to MLs resistance in invertebrates, although studies about resistance in NWS are limited to other genes. Here, we aimed to characterise the NWS GluClα (ChGluClα) cDNA and to search for alterations associated with IVM resistance in NWS larvae from a bioassay. The open reading frame of the ChGluClα comprised 1,359 bp and encoded a sequence of 452 amino acids. The ChGluClα cDNAs of the bioassay larvae showed different sequences that could be splice variants, which agree with the occurrence of alternative splicing in GluClα homologues. In addition, we found cDNAs with premature stop codons and the K242R SNP, which occurred more frequently in the surviving larvae and was located close to mutation (L256F) involved in ML resistance. Although these alterations were in low frequency, the ChGluClα sequencing will allow further studies to find alterations in the gene of resistant natural populations.

Interrogation of alternative splicing events in duplicated genes during evolution

BackgroundGene duplication provides resources for developing novel genes and new functions while retaining the original functions. In addition, alternative splicing could increase the complexity of expression at the transcriptome and proteome level without increasing the number of gene copy in the genome. Duplication and alternative splicing are thought to work together to provide the diverse functions or expression patterns for eukaryotes. Previously, it was believed that duplication and alternative splicing were negatively correlated and probably interchangeable.ResultsWe look into the relationship between occurrence of alternative splicing and duplication at different time after duplication events. We found duplication and alternative splicing were indeed inversely correlated if only recently duplicated genes were considered, but they became positively correlated when we took those ancient duplications into account. Specifically, for slightly or moderately duplicated genes with gene families containing 2 - 7 paralogs, genes were more likely to evolve alternative splicing and had on average a greater number of alternative splicing isoforms after long-term evolution compared to singleton genes. On the other hand, those large gene families (contain at least 8 paralogs) had a lower proportion of alternative splicing, and fewer alternative splicing isoforms on average even when ancient duplicated genes were taken into consideration. We also found these duplicated genes having alternative splicing were under tighter evolutionary constraints compared to those having no alternative splicing, and had an enrichment of genes that participate in molecular transducer activities.ConclusionsWe studied the association between occurrences of alternative splicing and gene duplication. Our results implicate that there are key differences in functions and evolutionary constraints among singleton genes or duplicated genes with or without alternative splicing incidences. It implies that the gene duplication and alternative splicing may have different functional significance in the evolution of speciation diversity.

Diverging Alternative Splicing Fingerprints in the Transforming Growth Factor-β Signaling Pathway Identified in Thoracic Aortic Aneurysms

Impaired regulation of the transforming growth factor-β (TGFβ) signaling pathway has been linked to thoracic aortic aneurysm (TAA). Previous work has indicated that differential splicing is a common phenomenon, potentially influencing the function of proteins. In the present study we investigated the occurrence of differential splicing in the TGFβ pathway associated with TAA in patients with bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV). Affymetrix human exon arrays were applied to 81 intima/media tissue samples from dilated (n = 51) and nondilated (n = 30) aortas of TAV and BAV patients. To analyze the occurrence of alternative splicing in the TGFβ pathway, multivariate techniques, including principal component analysis and OPLS-DA (orthogonal partial least squares to latent structures discriminant analysis), were applied on all exons (n = 614) of the TGFβ pathway. The scores plot, based on the splice index of individual exons, showed separate clusters of patients with both dilated and nondilated aorta, thereby illustrating the potential importance of alternative splicing in TAA. In total, differential splicing was detected in 187 exons. Furthermore, the pattern of alternative splicing is clearly differs between TAV and BAV patients. Differential splicing was specific for BAV and TAV patients in 40 and 86 exons, respectively, and splicings of 61 exons were shared between the two phenotypes. The occurrence of differential splicing was demonstrated in selected genes by reverse transcription-polymerase chain reaction. In summary, alternative splicing is a common feature of TAA formation. Our results suggest that dilatation in TAV and BAV patients has different alternative splicing fingerprints in the TGFβ pathway.

Evidence of alternative splicing of the chi2 chitinase gene from Metarhizium anisopliae

Metarhizium anisopliae is a filamentous fungus used in the biological control of arthropods and produces several chitinases in order to break the host cuticle chitin fibers. Chitinase function during fungal cell development and/or infection processes is also an important aspect when analyzing the life cycle of entomopathogens. The expression profile analysis of the endochitinase chi2 gene acquired by RT-PCR experiments indicated the presence of two different transcripts, suggesting the occurrence of alternative splicing in the chi2 gene. The presence of two transcripts, characterized by the removal or retention of the second 72 bp intron, was further confirmed by DNA sequencing, Northern blot and qRT-PCR. Furthermore, we detected the synthesis of two different proteins from the transcripts by two-dimensional Western blot and mass spectrometry analyses. This is the first reported occurrence of alternative splicing in M. anisopliae.

RNA editing and alternative splicing of the insect nAChR subunit alpha6 transcript: evolutionary conservation, divergence and regulation

BackgroundRNA editing and alternative splicing play an important role in expanding protein diversity and this is well illustrated in studies of nicotinic acetylcholine receptors (nAChRs).ResultsHere, we compare the RNA editing and alternative splicing of the nAChR alpha6 subunit genes from different insects spanning ~300 million years of evolution– Drosophila melanogaster, Anopheles gambiae, Bombyx mori, Tribolium castaneum and Apis mellifera. The conserved and species-specific A-to-I RNA editing occurred across all species except A. gambiae, which displayed extraordinarily short flanking intronic sequences. Interestingly, some A-to-I editing sites were a genomically encoded G in other species. A combination of the experimental data and computational analysis of orthologous alpha6 genes from different species indicated that RNA editing and alternative splicing predated at least the radiation of insect orders spanning ~300 million years of evolution; however, they might have been lost in some species during subsequent evolution. The occurrence of alternative splicing was found to be regulated in distinct modes and, in some cases, even correlated with RNA editing.ConclusionOn the basis of comparative analysis of orthologous nAChR alpha6 genes from different insects spanning ~300 million years of evolution, we have documented the existence, evolutionary conservation and divergence, and also regulation of RNA editing and alternative splicing. Phylogenetic analysis of RNA editing and alternative splicing, which can create a multitude of functionally distinct protein isoforms, might have a crucial role in the evolution of complex organisms beyond nucleotide and protein sequences.

Identification of alternatively spliced dab1 isoforms in zebrafish

We have investigated the genomic organization, the occurrence of alternative splicing and the differential expression of the zebrafish disabled1 (dab1) gene. Dab1 is a key effector of the Reelin pathway, which regulates neuronal migration during brain development in vertebrates. The coding region of the zebrafish dab1 gene spans over 600 kb of genomic DNA and is composed of 15 exons. Alternative splicing in a region enriched for tyrosine residues generates at least three different isoforms. These isoforms are developmentally regulated and show differential tissue expression. Comparison with mouse and human data shows an overall conservation of the genomic organization with different alternative splicing events generating species-specific isoforms. Because these alternative splicing events give rise to isoforms with different numbers of phosphorylateable tyrosines, we speculate that alternative splicing of the dab1 gene in zebrafish and in other vertebrates regulates the nature of the cellular response to the Reelin signal.

Isolation and molecular characterization of the rat MR1 homologue, a non-MHC-linked class I-related gene.

We isolated and analyzed a new rat gene which is homologous to the recently described human major histocompatibility complex class I-related gene MR1. The deduced amino acid sequence of the rat Mr1 gene shows conserved cysteine residues typical of class I genes as well as conserved beta2-microglobulin and CD8 contact sites. Analysis of partial DNA sequences and restriction fragment length polymorphism patterns of several inbred rat strains indicate that Mr1 is not polymorphic. Mr1 is a single-copy gene, which could be mapped to rat chromosome 13 by co-segregation analysis of Mr1 and a microsatellite marker in the renin (Ren) gene in double-backcross hybrids. The recombination frequency between both genes was determined to be 14.7% (4.9-31.1, 95% confidence limits). Expression analysis revealed various Mr1 transcripts in each organ tested and occurrence of alternative splicing.

A novel type of glutathione peroxidase: expression and regulation during wound repair.

We have previously identified and cloned a novel keratinocyte growth factor (KGF)-regulated gene in human keratinocytes that encodes the human homologue of a bovine non-selenium glutathione peroxidase (GPx). To gain insight into the regulation of this gene in vivo, we isolated the murine homologue from a mouse skin cDNA library. In vitro transcription/translation demonstrated that the cDNA encodes a 27 kDa protein. Furthermore, we amplified by PCR a partial cDNA that most likely corresponds to a related gene. RNase protection analysis revealed tissue-specific expression of both genes and the occurrence of alternative splicing or RNA editing of at least one of the primary transcripts. Similar to that of KGF, expression of GPx was strongly induced after cutaneous injury, and each isoform displayed unique kinetics of expression during the repair process. In situ hybridization studies demonstrated high levels of GPx mRNA in keratinocytes of the hyperproliferative epithelium at the wound edge. Since these cells express functional KGF receptors, induction of GPx expression by KGF might also occur in vivo. These data suggest a role for GPx in the protection of epithelial cells against oxidative stress, particularly during the inflammatory phase of wound repair.

Alternative Splicing in COL1A1 mRNA Leads to a Partial Null Allele and Two In-frame Forms with Structural Defects in Non-lethal Osteogenesis Imperfecta

We have identified a novel multiexon genomic deletion in one COL1A1 collagen allele that results in three alternative forms of mutant mRNA. This mutation occurs in a 9-year-old girl and her father, both affected with severe type III osteogenesis imperfecta (OI). We previously reported detection of a mismatch in their alpha1(I) amino acids 558-861 region by RNA/RNA hybrid analysis (Grange, D. K., Gottesman, G. S., Lewis, M. B., and Marini, J. C. (1990) Nucleic Acids Res. 18, 4227-4236). Single Strand Conformational Polymorphism further localized the mRNA mutation to the amino acids 579-679 coding region. At the gene level, polymerase chain reaction (PCR) amplification of patient leukocyte DNA from the exon 33-38 region yielded the normal 1004-base pair (bp) fragment and an additional 442-bp fragment. Sequencing of the shorter genomic PCR product confirmed the presence of a 562-bp deletion, extending from the last 3 nucleotides (nt) of exon 34 to 156 nt from the 3'-end of intron 36. The genomic deletion was also detected in the clinically normal grandmother, who was confirmed to be a mosaic carrier. PCR amplification and RNase protection experiments were used to investigate the mRNA structure and occurrence of alternative splicing. One form of the mutant cDNA has a deletion with end points that are identical to the genomic deletion. This results in a combination deletion/insertion, with a deletion of amino acids 603-639 followed by an insertion of 156 nt from the 3'-end of intron 36. In addition, we found two alternatively spliced forms. One form uses a cryptic donor site in exon 34 and the exon 37 acceptor. The second form uses the normal exon 32 splice donor and exon 37 acceptor. Use of the cryptic donor results in a coding sequence that is out-of-frame. Both the retained intron form and the use of the exon 32 donor site result in coding sequences that are in-frame. This is the first report of a collagen defect in OI with alternative splicing generating both in-frame and out-of-frame forms of mRNA. Although the in-frame forms constitute more than 60% of the mRNA from the mutant allele, no mutant protein chain was identified. Collagen produced by cultured OI osteoblasts showed a significant increase in the relative amount of type III collagen but no mutant alpha1(I) chain.

The Gene Structure and Organization of Mouse PG-M, a Large Chondroitin Sulfate Proteoglycan: GENOMIC BACKGROUND FOR THE GENERATION OF MULTIPLE PG-M TRANSCRIPTS

We previously showed not only the presence of multiple RNA transcripts of different sizes encoding the core protein of mouse PG-M, but also their tissue-dependent expression. Major causes for the multiple forms were found to be due to alternative usage of the two different chondroitin sulfate attachment domains (alpha and beta). In this study, genomic DNA analysis has revealed that these domains are encoded by two large exons, exon VII (2880 base pairs) and exon VIII (5229 base pairs). The splice sites of these two exons were consistent with the occurrence of alternative splicing without frameshift. Furthermore, the mouse PG-M gene was shown to have four distinct polyadenylation signals and three candidates for the transcription initiation site as well. These genomic structural variations may contribute to the multiplicity of PG-M transcripts. Northern hybridization analysis showed that at least three different transcripts were generated by different usage of the distinct polyadenylation signals.

Developmentally regulated alternative splicing generates a complex array of Drosophila para sodium channel isoforms

The para locus encodes the predominant class of sodium channels expressed in Drosophila neurons. Previous sequence analysis of para cDNAs indicated the occurrence of alternative splicing at several sites within the open reading frame. Here we report a detailed analysis of this alternative splicing and its regulation during development. We have used a combination of RNA-PCR and sequence analysis to examine a 1.7 kilobase region of the para mRNA that encompasses the previously reported sites of alternative splicing. Five sites of alternative splicing were identified; 48 different splice variants could be generated by the differential exon usage observed. The number of splice forms and their relative frequency in vivo were characterized in RNA samples of both embryos and adults. The range of splice types was found to be much more diverse in adults than in embryos; of a total of 19 different combinations of alternative exons, 11 splice types were found in embryos and 18 in adults. Usage of some individual alternative exons changed during development; a newly identified exon, which is found in one of two forms either 24 or 30 base pairs long, was present in about 85% of para transcripts from embryos but only 7% of those in adults. These data suggest that a wide variety of subtly distinct Na channel isoforms are present in Drosophila, and that these may provide a range of voltage-gated sodium channel functions. Although multiple sodium channel genes have already been described in both Drosophila and mammalian systems, this study provides a clear indication that sodium channel variability may be much greater than previously thought.