ABSTRACT
Transcribed-ultraconserved regions (T-UCRs), which contain conserved sequences with 100% identity across human, rat and mouse species, are a novel category of functional RNAs. The human transformer 2ß gene (TRA2B) encodes a UCR that spans exon 2 (276 bp) and its neighboring introns. Among five spliced RNA variants (TRA2ß1-5) transcribed from the TRA2B gene, only TRA2ß4 contains the conserved exon 2. TRA2ß4 is overexpressed in colon cancer cells and accelerates cell growth by blocking the transcription of CDKN1A. However, the mechanisms underlying the overexpression of TRA2ß4 in colon cancer cells are unknown. Using biotinylated RNA pull-down assays followed by liquid chromatography-mass spectrometric analysis, we identified nucleolin as a TRA2ß4-binding protein. Knockdown of nucleolin reduced the nuclear retention of TRA2ß4 and accelerated its degradation in the cytoplasm, whereas nucleolin overexpression increased TRA2ß4 levels and its mitogenic activity. Nucleolin directly bound to TRA2ß4 exon 2 via the glycine/arginine-rich (GAR) domain. Overexpression of GAR-deficient nucleolin failed to increase TRA2ß4 expression and growth of colon cancer cells. RNA fluorescence in situ hybridization showed that TRA2ß4 co-localized with nucleolin in nuclei but not with the mutant lacking GAR. Our results suggest that specific interactions between nucleolin and UCR-containing TRA2ß4 may be associated with abnormal growth of colon cancer cells.
ABSTRACT
Hu antigen R (HuR) regulates stress responses through stabilizing and/or facilitating the translation of target mRNAs. The human TRA2ß gene encodes splicing factor transformer 2ß (Tra2ß) and generates 5 mRNA isoforms (TRA2ß1 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 TRA2ß exon 2, generating a TRA2ß4 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 TRA2ß4 and increased Tra2ß protein, facilitating Tra2ß-dependent inclusion of exons in target pre-mRNAs. The effects of HuR knockdown or Chk2/p38(MAPK) double knockdown were also confirmed using a TRA2ß 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 TRA2ß4 interaction and TRA2ß4 generation, while the overexpression of a phosphomimetic HuR (with mutations S88D, S100D, and T118D) restored the TRA2ß4-related activities. Our findings revealed the potential role of nuclear HuR in the regulation of alternative splicing programs under oxidative stress.
Subject(s)
Alternative Splicing/genetics , Colonic Neoplasms/genetics , ELAV Proteins/genetics , Nerve Tissue Proteins/genetics , Oxidative Stress/genetics , RNA-Binding Proteins/genetics , Alternative Splicing/drug effects , Arsenites/pharmacology , Cell Line, Tumor , Checkpoint Kinase 2/genetics , Checkpoint Kinase 2/metabolism , Colonic Neoplasms/metabolism , ELAV Proteins/metabolism , Exons/drug effects , Exons/genetics , HCT116 Cells , Humans , Nerve Tissue Proteins/metabolism , Oxidative Stress/drug effects , Phosphorylation/drug effects , Phosphorylation/genetics , Protein Interaction Maps/drug effects , Protein Interaction Maps/genetics , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , Serine-Arginine Splicing Factors , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
BACKGROUND: Transformer (Tra) 2ß is a member of the serine/arginine-rich (SR)-like protein family that regulates alternative splicing of numerous genes in a concentration-dependent manner. Several types of cancer cells up-regulate Tra2ß expression, while the regulatory mechanism of Tra2ß expression remains to be elucidated. In this study, we examined the transcriptional regulation and possible functions of Tra2ß in human colon cancer cells. METHODS: We cloned 959 bp-upstream of the human TRA2ß 5'-flank into luciferase constructs. Chromatin immunoprecipitation (ChIP) was employed to identify crucial cis element(s) and trans activator(s) of the TRA2ß promoter. Tra2ß expression in the human colon and colon cancer tissues was examined by immunohistochemistry. RESULTS: In response to sodium arsenite, colon cancer cells (HCT116) increased levels of TRA2ß1 mRNA encoding a functional, full-length Tra2ß with a peak around 6 h without changing its mRNA stability. Transient expression assays using a reporter gene driven by serially truncated TRA2ß promoters and Chip assay demonstrated that an Ets1-binding site present at -64 to -55 bp was crucial for basal transcription, while three heat shock elements (HSEs) located at -145 to -99 bp mediated the oxidant-induced transactivation of TRA2ß. Tra2ß knockdown caused apoptosis of HCT116 cells. Tra2ß were preferentially expressed in proliferative compartment of normal human colonic glands and adenocarcinomas, where Ets1 and heat shock factor 1 were also highly expressed. CONCLUSIONS: Our results suggest that oxidative stress-responsive Tra2ß may play an important role in colon cancer growth.
Subject(s)
Adenocarcinoma/metabolism , Colonic Neoplasms/metabolism , DNA-Binding Proteins/physiology , Nerve Tissue Proteins/genetics , Proto-Oncogene Protein c-ets-1/physiology , RNA-Binding Proteins/genetics , Transcription Factors/physiology , Adenocarcinoma/genetics , Apoptosis , Arsenites/pharmacology , Base Sequence , Colonic Neoplasms/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Heat Shock Transcription Factors , Heat-Shock Response/genetics , Humans , Molecular Sequence Data , Neoplasm Proteins/physiology , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/metabolism , Oxidative Stress/genetics , Promoter Regions, Genetic , Proto-Oncogene Protein c-ets-1/metabolism , RNA, Messenger/genetics , RNA, Neoplasm/genetics , RNA-Binding Proteins/biosynthesis , RNA-Binding Proteins/metabolism , Serine-Arginine Splicing Factors , Sodium Compounds/pharmacology , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
Alternative splicing (AS) not only regulates the gene expression program in response to surrounding environment, but also produces protein isoforms with unique properties under stressful conditions. However, acute psychological stress-initiated AS events have not been documented in human studies. After assessments of changes in salivary cortisol levels and anxiety among 28 fourth-grade medical students 7 weeks prior to, 1 day before, immediately after, and 1 week after an examination for promotion, we selected 5 male students, who showed a typical stress response, and screened AS events in their circulating leukocytes using the GeneChip human exon 1.0 ST array. AS events of 27 genes with splicing indices >1.0 could be detected between immediately after and either 7 weeks before, 1 day before, or 1 week after the examination. The examination stress preferentially caused skipping rather than inclusion: 21 out of the 27 pre-mRNAs underwent skipping of exons, and skipping in 3'UTR was observed in 8 genes. Among the candidate genes, real-time reverse transcription PCR validated the stress-initiated skipping of exon 63 of SMG-1 that encodes a phosphatidylinositol 3-kinase-related protein kinase crucial for activations of p53-dependent pathways and nonsense-mediated mRNA decay. Our results indicate a significant impact of brief naturalistic stressors on AS-mediated regulation of gene expression in peripheral leukocytes, and suggest the SMG-1 splice variant as a potential biomarker for acute psychological stress.
