Rice Transcriptome Analysis Reveals Nitrogen Starvation Modulates Differential Alternative Splicing and Transcript Usage in Various Metabolism-Related Genes.
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ABSTRACT:
SUBMITTER: Chaudhary S
PROVIDER: S-EPMC8066416 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:At the heart of an innate immune response lies a tightly regulated gene expression program. This precise regulation is crucial because small changes can shift the balance from protective to destructive immunity. Here we identify a frequently used alternative splice site in the gene oligoadenylate synthetase 1g (Oas1g), a key component of the 2-5A antiviral system. Usage of this splice site leads to the generation of a transcript subject to decay, and removal of the site leads to increased expression of Oas1g and an improved antiviral response. However, removal of the splice site also leads to an increase in apoptotic cell death, suggesting this splicing event exists as a compromise between the pathogen protective benefits and collateral damage associated with OAS1g activity. Across the innate immune response, we show that a multitude of alternative splicing events predicted to lead to decay exist, and thus have the potential to play a significant role in the regulation of gene expression in innate immunity.
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Project description:BackgroundThe latest generation of Affymetrix microarrays are designed to interrogate expression over the entire length of every locus, thus giving the opportunity to study alternative splicing genome-wide. The Exon 1.0 ST (sense target) platform, with versions for Human, Mouse and Rat, is designed primarily to probe every known or predicted exon. The smaller Gene 1.0 ST array is designed as an expression microarray but still interrogates expression with probes along the full length of each well-characterized transcript. We explore the possibility of using the Gene 1.0 ST platform to identify differential splicing events.ResultsWe propose a strategy to score differential splicing by using the auxiliary information from fitting the statistical model, RMA (robust multichip analysis). RMA partitions the probe-level data into probe effects and expression levels, operating robustly so that if a small number of probes behave differently than the rest, they are downweighted in the fitting step. We argue that adjacent poorly fitting probes for a given sample can be evidence of differential splicing and have designed a statistic to search for this behaviour. Using a public tissue panel dataset, we show many examples of tissue-specific alternative splicing. Furthermore, we show that evidence for putative alternative splicing has a strong correspondence between the Gene 1.0 ST and Exon 1.0 ST platforms.ConclusionWe propose a new approach, FIRMAGene, to search for differentially spliced genes using the Gene 1.0 ST platform. Such an analysis complements the search for differential expression. We validate the method by illustrating several known examples and we note some of the challenges in interpreting the probe-level data.Software implementing our methods is freely available as an R package.
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Project description:What at the genomic level underlies organism complexity? Although several genomic features have been associated with organism complexity, in the case of alternative splicing, which has long been proposed to explain the variation in complexity, no such link has been established. Here, we analyzed over 39 million expressed sequence tags available for 47 eukaryotic species with fully sequenced genomes to obtain a comparable index of alternative splicing estimates, which corrects for the distorting effect of a variable number of transcripts per species--an important obstacle for comparative studies of alternative splicing. We find that alternative splicing has steadily increased over the last 1,400 My of eukaryotic evolution and is strongly associated with organism complexity, assayed as the number of cell types. Importantly, this association is not explained as a by-product of covariance between alternative splicing with other variables previously linked to complexity including gene content, protein length, proteome disorder, and protein interactivity. In addition, we found no evidence to suggest that the relationship of alternative splicing to cell type number is explained by drift due to reduced N(e) in more complex species. Taken together, our results firmly establish alternative splicing as a significant predictor of organism complexity and are, in principle, consistent with an important role of transcript diversification through alternative splicing as a means of determining a genome's functional information capacity.
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Project description:Studies of differential gene expression have identified several molecular signatures and pathways associated with Parkinson's disease (PD). The role of isoform switches and differential transcript usage (DTU) remains, however, unexplored. Here, we report the first genome-wide study of DTU in PD. We performed RNA sequencing following ribosomal RNA depletion in prefrontal cortex samples of 49 individuals from two independent case-control cohorts. DTU was assessed using two transcript-count based approaches, implemented in the DRIMSeq and DEXSeq tools. Multiple PD-associated DTU events were detected in each cohort, of which 23 DTU events in 19 genes replicated across both patient cohorts. For several of these, including THEM5, SLC16A1 and BCHE, DTU was predicted to have substantial functional consequences, such as altered subcellular localization or switching to non-protein coding isoforms. Furthermore, genes with PD-associated DTU were enriched in functional pathways previously linked to PD, including reactive oxygen species generation and protein homeostasis. Importantly, the vast majority of genes exhibiting DTU were not differentially expressed at the gene-level and were therefore not identified by conventional differential gene expression analysis. Our findings provide the first insight into the DTU landscape of PD and identify novel disease-associated genes. Moreover, we show that DTU may have important functional consequences in the PD brain, since it is predicted to alter the functional composition of the proteome. Based on these results, we propose that DTU analysis is an essential complement to differential gene expression studies in order to provide a more accurate and complete picture of disease-associated transcriptomic alterations.
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Project description:The mammalian fatty acid desaturase 2 (FADS2) gene codes for catalytic activity considered to be the rate limited step in long chain polyunsaturated fatty acid (LCPUFA) synthesis. FADS2 catalyzes 6-desaturation in at least five substrates and 8-desaturation in at least two substrates. However, the molecular mechanisms that regulate FADS2-mediated desaturation remain ill-defined. We report here characterization of an alternative transcript (AT1) of primate FADS2 and compare its expression to that of the classical transcript in 12 tissues of a 12 week old neonate baboon, and in human SK-N-SH neuroblastoma (NB) cells. RT-PCR analysis indicates relatively greater abundance of classical transcript than AT1 in all tissues. However, AT1 expression is highly variable, showing greater expression in liver, retina, occipital lobe, hippocampus, spleen, and ovary, than in other tissues, whereas classical transcript displayed little variability. These data suggest that FADS2 AT1 is a candidate for regulation of LCPUFA synthesis.
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Project description:Shrew-1, also called AJAP1, is a transmembrane protein associated with E-cadherin-mediated adherence junctions and a putative tumor suppressor. Apart from its interaction with ?-catenin and involvement in E-cadherin internalization, little structure or function information exists. Here we explored shrew-1 expression during postnatal differentiation of mammary gland as a model system. Immunohistological analyses with antibodies against either the extracellular or the cytoplasmic domains of shrew-1 consistently revealed the expression of full-length shrew-1 in myoepithelial cells, but only part of it in luminal cells. While shrew-1 localization remained unaltered in myoepithelial cells, nuclear localization occurred in luminal cells during lactation. Based on these observations, we identified two unknown shrew-1 transcript variants encoding N-terminally truncated proteins. The smallest shrew-1 protein lacks the extracellular domain and is most likely the only variant present in luminal cells. RNA analyses of human tissues confirmed that the novel transcript variants of shrew-1 exist in vivo and exhibit a differential tissue expression profile. We conclude that our findings are essential for the understanding and interpretation of future functional and interactome analyses of shrew-1 variants.
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Project description:Fads3 is the third member of the fatty acid desaturase gene cluster; with at least eight evolutionarily conserved alternative transcripts (AT), having no clearly established function as are known for FADS2 and FADS1. Here we present identification of a novel Fads3 transcript in mice (Fads3AT9), characterize Fads3AT9 expression in mouse tissues and evaluate correlations with metabolite profiles. Total RNA obtained from mouse tissues is reverse-transcribed into cDNA and used as template for PCR reactions. Tissue fatty acids were extracted and quantified by gas chromatography. Sequencing analysis revealed complete absence of exon 2 resulting in an open reading frame of 1239 bp, encoding a putative protein of 412 aa with loss of 37 aa compared to classical Fads3 (Fads3CS). FADS3AT9 retains all the conserved regions characteristic of front end desaturase (cytochrome b5 domain and three histidine repeats). Both Fads3CS and Fads3AT9 are ubiquitously expressed in 11 mouse tissues. Fads3AT9 abundance was greater than Fads3CS in pancreas, liver, spleen, brown adipose tissue and thymus. Fads3CS expression is low in pancreas while Fads3AT9 is over ten-fold greater abundance. The eicosanoid precursor fatty acid 20:4n - 6, the immediate desaturation product of the Fads1 coded ?5-desaturase, was highest in pancreas where Fads3CS is low. Changes in expression patterns and fatty acid profiles suggest that Fads3AT9 may play a role in the regulation and/or biosynthesis of long chain polyunsaturated fatty acids from precursors.
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Project description:BackgroundSpinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder with progressive degeneration of cerebellar Purkinje cells and selective loss of neurons in the brainstem. This neurodegenerative disorder is caused by the expansion of a polyglutamine domain in ataxin-2. Ataxin-2 is composed of 1312 amino acids, has a predicted molecular weight of 150-kDa and is widely expressed in neuronal and non-neuronal tissues. To date, the putative functions of ataxin-2 on mRNA translation and endocytosis remain ill-defined. Differential splicing with a lack of exons 10 and 21 was described in humans, and additional splicing of exon 11 in mice. In this study, we observed that the molecular size of transfected full-length wild-type ataxin-2 (22 glutamines) is different from endogenous ataxin-2 and that this variation could not be explained by the previously published splice variants alone.MethodsQuantitative immunoblots and qualitative reverse-transcriptase polymerase-chain-reaction (RT-PCR) were used to characterize isoform variants, before sequencing was employed for validation.ResultsWe report the characterization of further splice variants of ataxin-2 in different human cell lines and in mouse and human brain. Using RT-PCR from cell lines HeLa, HEK293 and COS-7 throughout the open reading frame of ataxin-2 together with PCR-sequencing, we found novel splice variants lacking exon 12 and exon 24. These findings were corroborated in murine and human brain. The splice variants were also found in human skin fibroblasts from SCA2 patients and controls, indicating that the polyglutamine expansion does not abolish the splicing.ConclusionsGiven that Ataxin-2 interacts with crucial splice modulators such as TDP-43 and modulates the risk of Amyotrophic Lateral Sclerosis, its own splice isoforms may become relevant in brain tissue to monitor the RNA processing during disease progression and neuroprotective therapy.
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Project description:Nitrogen (N), a critical macronutrient for plant growth and development, is a major limiting factor in most agricultural systems. Microarray analyses have been conducted to investigate genome-wide gene expression in response to changes in N concentrations. Although RNA-Seq analysis can provide a more precise determination of transcript levels, it has not previously been employed to investigate the expression of N-starvation-induced genes.We constructed cDNA libraries from leaf sheaths and roots of rice plants grown under N-deficient or -sufficient conditions for 12 h. Sequencing the libraries resulted in identification of 33,782 annotated genes. A comparison of abundances revealed 1,650 transcripts that were differentially expressed (fold-change???2) due to an N-deficiency. Among them, 1,158 were differentially expressed in the leaf sheaths (548 up-regulated and 610 down-regulated) and 492 in the roots (276 up, 216 down). Among the 36 deficiency-induced genes first identified via RNA-Seq analyses, 34 were subsequently confirmed by qRT-PCR. Our RNA-Seq data identified 8,509 multi-exonic genes with 19,628 alternative splicing events. However, we saw no significant difference in alternative splicing between N-sufficient and -deficient conditions. We found 2,986 novel transcripts, of which 192 were regulated under the N-deficiency.We identified 1,650 genes that were differentially expressed after 12 h of N-starvation. Responses by those genes to a limited supply of N were confirmed by RT-PCR and GUS assays. Our results provide valuable information about N-starvation-responsive genes and will be useful when investigating the signal transduction pathway of N-utilization.
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Project description:There is increasing evidence to suggest that splicing decisions are largely made when the nascent RNA is still associated with chromatin. Here we demonstrate that activity of histone deacetylases (HDACs) influences splice site selection. Using splicing-sensitive microarrays, we identified ∼700 genes whose splicing was altered after HDAC inhibition. We provided evidence that HDAC inhibition induced histone H4 acetylation and increased RNA Polymerase II (Pol II) processivity along an alternatively spliced element. In addition, HDAC inhibition reduced co-transcriptional association of the splicing regulator SRp40 with the target fibronectin exon. We further showed that the depletion of HDAC1 had similar effect on fibronectin alternative splicing as global HDAC inhibition. Importantly, this effect was reversed upon expression of mouse HDAC1 but not a catalytically inactive mutant. These results provide a molecular insight into a complex modulation of splicing by HDACs and chromatin modifications.
| S-EPMC3032741 | biostudies-literature