The cardiotonic steroid digitoxin regulates alternative splicing through depletion of the splicing factors SRSF3 and TRA2B

Anderson, Erik S.; Lin, Chia-Ho; Xiao, Xinshu; Stoilov, Peter; Burge, Christopher B.; Black, Douglas L.

doi:10.1261/rna.032912.112

Cited by 58 publications

(49 citation statements)

References 43 publications

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“…We used 115 known binding sites (motifs) of RBPs from the literature (8,27,(34)(35)(36), including well-known splicing factors. We examined the enrichment of the RBPs in the exon body and in 250 bp of upstream and downstream introns separately.…”

Section: Motif Enrichment Analysismentioning

confidence: 99%

Determination of a Comprehensive Alternative Splicing Regulatory Network and Combinatorial Regulation by Key Factors during the Epithelial-to-Mesenchymal Transition

Yang

Park

Bebee

et al. 2016

Molecular and Cellular Biology

127

165

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fThe epithelial-to-mesenchymal transition (EMT) is an essential biological process during embryonic development that is also implicated in cancer metastasis. While the transcriptional regulation of EMT has been well studied, the role of alternative splicing (AS) regulation in EMT remains relatively uncharacterized. We previously showed that the epithelial cell-type-specific proteins epithelial splicing regulatory proteins 1 (ESRP1) and ESRP2 are important for the regulation of many AS events that are altered during EMT. However, the contributions of the ESRPs and other splicing regulators to the AS regulatory network in EMT require further investigation. Here, we used a robust in vitro EMT model to comprehensively characterize splicing switches during EMT in a temporal manner. These investigations revealed that the ESRPs are the major regulators of some but not all AS events during EMT. We determined that the splicing factor RBM47 is downregulated during EMT and also regulates numerous transcripts that switch splicing during EMT. We also determined that Quaking (QKI) broadly promotes mesenchymal splicing patterns. Our study highlights the broad role of posttranscriptional regulation during the EMT and the important role of combinatorial regulation by different splicing factors to fine tune gene expression programs during these physiological and developmental transitions.A lternative splicing (AS) is a process by which a single gene transcript can be differentially spliced to yield numerous splice variants that can encode different protein isoforms and thereby greatly expand the protein coding capacity of the genome. Nearly all human genes are alternatively spliced, and cell-typespecific protein isoforms have been shown to be functionally essential for cell fate and viability (1-3). This process is under complex regulation by various cis elements in pre-mRNAs and their cognate binding partners, mainly RNA-binding proteins (RBPs). Splicing-regulatory RBPs recruited to their respective binding sites can have positive or negative effects on the splicing of different exons or splice sites. Many RBPs with largely ubiquitous expression in different tissues and cells have been shown to have broad impacts on splicing and important cell functions, such as SR and hnRNP protein families (4). However, several tissue-specific RBPs, such as NOVA, PTBP2 (nPTB), MBNL, and RBFOX proteins, have recently been described as having important roles as essential regulators of tissue-or cell-type-specific splicing (5-9). In order to further define a "splicing code" that controls broad patterns of tissue-specific splicing, as well as those that occur during developmental transitions, it is essential to characterize in greater detail how these tissue-specific regulators fine-tune AS programs combinatorially with more ubiquitously expressed splicing regulators (10).The epithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells transdifferentiate into mesenchymal cells, which involves extensive changes at the cellular ...

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Section: Motif Enrichment Analysismentioning

confidence: 99%

Determination of a Comprehensive Alternative Splicing Regulatory Network and Combinatorial Regulation by Key Factors during the Epithelial-to-Mesenchymal Transition

Yang

Park

Bebee

et al. 2016

Molecular and Cellular Biology

127

165

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“…We next examined whether the arsenite-stimulated increase in Tra2␤ protein in HuR or Chk2 and p38 MAPK knockdown cells affected Tra2␤-dependent alternative splicing. To this end, we analyzed calcitonin/calcitonin gene-related peptide (CGRP), SMN1, SMN2, TAU, and RIPK2 genes, which have been reported to exhibit altered splicing patterns in a Tra2␤-dependent manner (26)(27)(28)(29). Of these targets, HCT116 cells did not express any detectable amounts of calcitonin or CGRP mRNAs before or after exposure to arsenite (data not shown).…”

Section: Fig 5 Involvement Of Chk2 and P38mentioning

confidence: 99%

“…8B and E). Tra2␤ is expected to be able to bind to SMN1 exon 7, SMN2 exon 7, RIPK2 exon 2, and TAU exon 10 and stimulate their inclusion in mRNAs (26)(27)(28)(29). Therefore, we used qPCR to measure the levels of different isoforms containing or excluding these putative Tra2␤ target exons.…”

Section: Fig 5 Involvement Of Chk2 and P38mentioning

confidence: 99%

“…Tra2␤ regulates the splice site selection of several genes, including those encoding calcitonin/calcitonin gene-related peptide (CGRP), survival motor neuron 1 and 2 (SMN1 and SMN2), mi-crotubule-associated protein tau (TAU), and receptor-interacting serine-threonine kinase 2 (RIPK2) (26)(27)(28)(29). The human TRA2␤ gene consists of 10 exons and 9 introns and generates 5 mRNA isoforms (TRA2␤1 to -5) through alternative splicing (30).…”

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confidence: 99%

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HuR Regulates Alternative Splicing of the TRA2β Gene in Human Colon Cancer Cells under Oxidative Stress

Akaike

Masuda

Kuwano

et al. 2014

Molecular and Cellular Biology

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bHu antigen R (HuR) regulates stress responses through stabilizing and/or facilitating the translation of target mRNAs. The human TRA2␤ gene encodes splicing factor transformer 2␤ (Tra2␤) and generates 5 mRNA isoforms (TRA2␤1 to -5) through alternative splicing. Exposure of HCT116 colon cancer cells to sodium arsenite stimulated checkpoint kinase 2 (Chk2)-and mitogen-activated protein kinase p38 (p38 MAPK )-mediated phosphorylation of HuR at positions S88 and T118. This induced an association between HuR and the 39-nucleotide (nt) proximal region of TRA2␤ exon 2, generating a TRA2␤4 mRNA that includes exon 2, which has multiple premature stop codons. HuR knockdown or Chk2/p38 MAPK double knockdown inhibited the arsenite-stimulated production of TRA2␤4 and increased Tra2␤ protein, facilitating Tra2␤-dependent inclusion of exons in target pre-mRNAs. The effects of HuR knockdown or Chk2/p38 MAPK double knockdown were also confirmed using a TRA2␤ minigene spanning exons 1 to 4, and the effects disappeared when the 39-nt region was deleted from the minigene. In endogenous HuR knockdown cells, the overexpression of a HuR mutant that could not be phosphorylated (with changes of serine to alanine at position 88 [S88A], S100A, and T118A) blocked the associated TRA2␤4 interaction and TRA2␤4 generation, while the overexpression of a phosphomimetic HuR (with mutations S88D, S100D, and T118D) restored the TRA2␤4-related activities. Our findings revealed the potential role of nuclear HuR in the regulation of alternative splicing programs under oxidative stress. The Hu/embryonic lethal abnormal vision (ELAV) protein family comprises 3 primarily neuronal proteins (HuB, HuC, and HuD) and one ubiquitously expressed protein, HuR (Hu antigen R; also known as HuA). Hu family proteins contain 3 RNA recognition motifs (RRMs) that mediate the specific interaction of Hu proteins with RNA (1). RRMs specifically bind to short, singlestranded stretches of uridines separated by adenosines or, less commonly, other bases (1, 2), which are also known as RNA recognition elements (RREs) or AU-rich elements (AREs), in the 3= untranslated region (UTR) of target mRNAs. HuR plays a crucial role in the regulation of gene expression in cells exposed to mitogenic, differentiation, immune, and stress-inducing agents (1, 3) through stabilizing and/or facilitating the translation of ARE-containing mRNAs for various proteins, including tumor suppressors (p53 and von Hippel-Lindau tumor suppressor), cyclins (A, B1, and D1), proto-oncogene products (c-Fos and c-Myc), growth factors (vascular endothelial growth factor), cytokines (transforming growth factor ␤ and tumor necrosis factor alpha), cyclindependent kinase (Cdk) inhibitors (p21 and p27), antiapoptotic factors (prothymosin ␣ [ProT␣], B-cell CLL/lymphoma 2 [Bcl-2], and myeloid cell leukemia sequence 1 [Mcl-1]), and signaling molecules, such as mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) (4-15). Recently, several key aspects of HuR signaling have emerged; for example, the set of RNAs (...

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“…Small molecule inhibitors that inhibit Tra2β have already been identified [76]. Tra2β is also targeted by protein kinases and phosphatases, so might be manipulated by signaling pathways within cancer cells.…”

Section: Clinical and Scientific Significance Of Tra2β Protein In Brementioning

confidence: 99%

Transformer2 proteins protect breast cancer cells from accumulating replication stress by ensuring productive splicing of checkpoint kinase 1

Best

James

Hysenaj

et al. 2015

Front. Chem. Sci. Eng.

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Increased expression levels of the RNA splicing regulator Transformer2β (abbreviated Tra2β) have been reported in several types of cancer. Recent work has revealed an intimate cross-regulation between Tra2β and the highly similar Tra2α protein in human breast cancer cells, though these two proteins are encoded by separate genes created by a gene duplication that occurred over 500 million years ago. This cross-regulation involves splicing control of a special class of exons, called poison exons. Down-regulation of Tra2β reduces splicing inclusion of a poison exon in the mRNA encoding Tra2α, thereby up-regulating Tra2α protein expression. This buffers any splicing changes that might be caused by individual depletion of Tra2β alone. Discovery of this cross-regulation pathway, and its by-pass by joint depletion of both human Tra2 proteins, revealed Tra2 proteins are essential for breast cancer cell viability, and led to the identification of important targets for splicing control. These exons include a critical exon within the checkpoint kinase 1 (CHK1) gene that plays a crucial function in the protection of cancer cells from replication stress. Breast cancer cells depleted for Tra2 proteins have reduced CHK1 protein levels and accumulate DNA damage. These data suggest Tra2 proteins and/or their splicing targets as possible cancer drug targets.

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The cardiotonic steroid digitoxin regulates alternative splicing through depletion of the splicing factors SRSF3 and TRA2B

Cited by 58 publications

References 43 publications

Determination of a Comprehensive Alternative Splicing Regulatory Network and Combinatorial Regulation by Key Factors during the Epithelial-to-Mesenchymal Transition

Determination of a Comprehensive Alternative Splicing Regulatory Network and Combinatorial Regulation by Key Factors during the Epithelial-to-Mesenchymal Transition

HuR Regulates Alternative Splicing of the TRA2β Gene in Human Colon Cancer Cells under Oxidative Stress

Transformer2 proteins protect breast cancer cells from accumulating replication stress by ensuring productive splicing of checkpoint kinase 1

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