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Distinct Evolutionary Origins of Intron Retention Splicing Events in NHX1 Antiporter Transcripts Relate to Sequence Specific Distinctions in Oryza Species.

ABSTRACT: The genome of Asian cultivated rice (Oryza sativa L.) shows the presence of six organelle-specific and one plasma membrane (OsNHX1-7) NHX-type cation proton antiporters. Of these, vacuolar-localized OsNHX1 is extensively characterized. The genus Oryza consists of 27 species and 11 genome-types, with cultivated rice, diploid O. sativa, having an AA-type genome. Oryza NHX1 orthologous regions (gene organization, 5' upstream cis elements, amino acid residues/motifs) from closely related Oryza AA genomes cluster distinctly from NHX1 regions from more ancestral Oryza BB, FF and KKLL genomes. These sequence-specific distinctions also extend to two separate intron retention (IR) events involving Oryza NHX1 transcripts that occur at the 5' and 3' ends of the NHX1 transcripts. We demonstrate that the IR event involving the 5' UTR is present only in more recently evolved Oryza AA genomes while the IR event governing retention of the 13th intron of Oryza NHX1 (terminal intron) is more ancient in origin, also occurring in halophytic wild rice, Oryza coarctata (KKLL). We also report presence of a retro-copy of the OcNHX1 cDNA in the genome of O. coarctata (rOcNHX1). Preferential species and tissue specific up- or down-regulation of the correctly spliced NHX1 transcript/5' UTR/13th intron-retaining splice variants under salinity was observed. The implications of IR on NHX1 mRNA stability and ORF diversity in Oryza spp. is discussed.

SUBMITTER: Sellamuthu G

PROVIDER: S-EPMC7078337 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Distinct Evolutionary Origins of Intron Retention Splicing Events in NHX1 Antiporter Transcripts Relate to Sequence Specific Distinctions in Oryza Species.

Sellamuthu Gothandapani G Jegadeeson Vidya V Sajeevan Radha Sivarajan RS Rajakani Raja R Parthasarathy Pavithra P Raju Kalaimani K Shabala Lana L Chen Zhong-Hua ZH Zhou Meixue M Sowdhamini Ramanathan R Shabala Sergey S Venkataraman Gayatri G

Frontiers in plant science 20200311

The genome of Asian cultivated rice (Oryza sativa L.) shows the presence of six organelle-specific and one plasma membrane (OsNHX1-7) NHX-type cation proton antiporters. Of these, vacuolar-localized OsNHX1 is extensively characterized. The genus Oryza consists of 27 species and 11 genome-types, with cultivated rice, diploid O. sativa, having an AA-type genome. Oryza NHX1 orthologous regions (gene organization, 5' upstream cis elements, amino acid residue ...[more]

PMID: 32218795

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Project description:BackgroundMutations in minor spliceosome components such as U12 snRNA (cerebellar ataxia) and U4atac snRNA (microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1)) result in tissue-specific symptoms. Given that the minor spliceosome is ubiquitously expressed, we hypothesized that these restricted phenotypes might be caused by the tissue-specific regulation of the minor spliceosome targets, i.e. minor intron-containing genes (MIGs). The current model of inefficient splicing is thought to apply to the regulation of the ~ 500 MIGs identified in the U12DB. However this database was created more than 10 years ago. Therefore, we first wanted to revisit the classification of minor introns in light of the most recent reference genome. We then sought to address specificity of MIG expression, minor intron retention, and alternative splicing (AS) across mouse and human tissues.ResultsWe employed position-weight matrices to obtain a comprehensive updated list of minor introns, consisting of 722 mouse and 770 human minor introns. These can be found in the Minor Intron DataBase (MIDB). Besides identification of 99% of the minor introns found in the U12DB, we also discovered ~ 150 new MIGs. We then analyzed the RNAseq data from eleven different mouse tissues, which revealed tissue-specific MIG expression and minor intron retention. Additionally, many minor introns were efficiently spliced compared to their flanking major introns. Finally, we identified several novel AS events across minor introns in both mouse and human, which were also tissue-dependent. Bioinformatics analysis revealed that several of the AS events could result in the production of novel tissue-specific proteins. Moreover, like the major introns, we found that these AS events were more prevalent in long minor introns, while retention was favoured in shorter introns.ConclusionHere we show that minor intron splicing and AS across minor introns is a highly organised process that might be regulated in coordination with the major spliceosome in a tissue-specific manner. We have provided a framework to further study the impact of the minor spliceosome and the regulation of MIG expression. These findings may shed light on the mechanism underlying tissue-specific phenotypes in diseases associated with minor spliceosome inactivation. MIDB can be accessed at https://midb.pnb.uconn.edu .

Project description:Background: Splicing factors are essential for nascent pre-mRNA processing and critical in cancer progression, suggesting that proteins with splicing functions represent potential molecular targets for cancer therapy. Here, we investigate the role of splicing factors in glioblastoma multiforme (GBM) progression and the possibility of targeting them for the treatment of the disease. Methods: The TCGA and CGGA public databases were used to screen for differentially expressed mRNA splicing factors. Immunohistochemistry and qRT-PCR were used to analyze the expression of non-POU domain-containing octamer-binding protein (NONO), a Drosophila behavior human splicing (DBHS) protein. Knockdown/overexpression of NONO with siRNA and lentiviral expression constructs was used to examine cell growth, apoptosis, and invasion in GBM cells. RNA sequencing was used to identify potential downstream molecular targets of NONO. RIP-PCR and RNA pulldown were used to determine the interaction between NONO and pre-mRNA. JC-1 staining and the seahorse assay were performed to assess redox homeostasis. Results: Expression of NONO was increased in GBM samples and associated with poor survival in patients (P = 0.04). Knockdown of NONO suppressed GBM growth, and overexpression of NONO promoted GBM tumorigenesis in vitro and in vivo. RNA sequencing-based transcriptomic profiling confirmed that knockdown of NONO in U251 and P3 cells resulted in global intron retention of pre-mRNA and led to abnormal splicing of specific pre-mRNAs for GPX1 and CCN1. NONO bound to a consensus motif in the intron of GPX1 pre-mRNA in association with another DBHS protein family member, PSPC1. Knockdown of NONO impaired tumor growth, invasion, and redox homeostasis through aberrant splicing of GPX1. Finally, Auranofin, a small molecule inhibitor of NONO, suppressed GBM tumor growth in an orthotopic xenograft model in mice. Conclusions: We demonstrated that intron retention was a critical alternative RNA splicing event to occur in GBM progression, and that NONO was a key regulator of mRNA splicing in GBM. Targeting NONO represents a novel, potential therapeutic strategy for GBM treatment.

Project description:BackgroundAdvanced prostate cancer (PC) is characterized by insensitivity to androgen deprivation therapy and chemotherapy, resulting in poor outcome for most patients. Thus, advanced PC urgently needs novel therapeutic strategies. Mounting evidence points to splicing dysregulation as a hallmark of advanced PC. Moreover, pharmacologic inhibition of the splicing process is emerging as a promising option for this disease.MethodBy using a representative androgen-insensitive PC cell line (22Rv1), we have investigated the genome-wide transcriptomic effects underlying the cytotoxic effects exerted by three splicing-targeting drugs: Pladienolide B, indisulam and THZ531. Bioinformatic analyses were performed to uncover the gene structural features underlying sensitivity to transcriptional and splicing regulation by these treatments. Biological pathways altered by these treatments were annotated by gene ontology analyses and validated by functional experiments in cell models.ResultsAlthough eliciting similar cytotoxic effects on advanced PC cells, Pladienolide B, indisulam and THZ531 modulate specific transcriptional and splicing signatures. Drug sensitivity is associated with distinct gene structural features, expression levels and cis-acting sequence elements in the regulated exons and introns. Importantly, we identified PC-relevant genes (i.e. EZH2, MDM4) whose drug-induced splicing alteration exerts an impact on cell survival. Moreover, computational analyses uncovered a widespread impact of splicing-targeting drugs on intron retention, with enrichment in genes implicated in pre-mRNA 3'-end processing (i.e. CSTF3, PCF11). Coherently, advanced PC cells displayed high sensitivity to a specific inhibitor of the cleavage and polyadenylation complex, which enhances the effects of chemotherapeutic drugs that are already in use for this cancer.ConclusionsOur study uncovers intron retention as an actionable vulnerability for advanced PC, which may be exploited to improve therapeutic management of this currently incurable disease.

Dataset Information

Distinct Evolutionary Origins of Intron Retention Splicing Events in NHX1 Antiporter Transcripts Relate to Sequence Specific Distinctions in Oryza Species.

Publications

Distinct Evolutionary Origins of Intron Retention Splicing Events in <i>NHX1</i> Antiporter Transcripts Relate to Sequence Specific Distinctions in <i>Oryza</i> Species.

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