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

Abstract

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 (...

Diverse roles of RNA‐binding proteins in cancer traits and their implications in gastrointestinal cancers

“…Some RBPs can also regulate multiple posttranscriptional processes. For example, NF90 can regulate both mRNA translation and turnover, whereas HuR and TIA1 can affect mRNA translation, turnover, and pre‐mRNA splicing (Akaike et al, ; Del Gatto‐Konczak et al, ; Hamada et al, ; Masuda et al, ; Piecyk et al, ; Shim, Lim, Yates, & Karin, ; Y. H. Xu, Busald, & Grabowski, ; Y. H. Xu & Grabowski, ; Y. H. Xu, Leonova, & Grabowski, ).…”

Cytoplasmic translocation of HuR contributes to angiotensin II induced cardiac fibrosis

“…We recently have revealed that RNA binding protein could promote fibrogenic response through stabilizing TGFb in cardiac fibroblasts [10]. In addition, HuR was found to be activated under multiple stress conditions, such as oxidative stress [11], UV light exposure [12], inflammation [13] and hypoxia [14]. Taken together, it is reasonable to hypothesize that angiotensin II might activate HuR and then promotes fibrosis via TGFb signal pathway.…”

Roles of silent information regulator 1–serine/arginine-rich splicing factor 10–lipin 1 axis in the pathogenesis of alcohol fatty liver disease

“…25 HuR functions in nucleus and mediates stress responses by stabilizing and/or promoting the expression of target mRNAs, while SFRS10 includes AU-rich elements in exon 2, in which exists a HuR-binding motif. 16 HuR may participate in splicing regulation of SFRS10 pre-mRNA after exposure to oxidative stress ( Figure 1). Akaike et al have demonstrated that both Chk2-and mitogen-activated protein kinase p38 (p38MAPK)-dependent phosphorylation of HuR induce SFRS10 exon 2 retention and interact with the 39-nucleotide proximal domain of exon 2, reducing the expression of SFRS10 in HCT116 cells exposed to sodium arsenite.…”

“…Akaike et al have demonstrated that both Chk2-and mitogen-activated protein kinase p38 (p38MAPK)-dependent phosphorylation of HuR induce SFRS10 exon 2 retention and interact with the 39-nucleotide proximal domain of exon 2, reducing the expression of SFRS10 in HCT116 cells exposed to sodium arsenite. 16 Chk2-and p38MAPK-mediated phosphorylation of nuclear HuR at residue serine 88 and/or tyrosine 118 urges the association between HuR and exon 2a of SFRS10 pre-mRNA in colon cancer cells exposure to oxidants, and promotes the formation of TRA2b 4 transcript that does not translate. 16 Oxidative stress may promote the retention of SFRS10 exon 2 to reduce SFRS10 expression via phosphorylating HuR.…”

“…16 Chk2-and p38MAPK-mediated phosphorylation of nuclear HuR at residue serine 88 and/or tyrosine 118 urges the association between HuR and exon 2a of SFRS10 pre-mRNA in colon cancer cells exposure to oxidants, and promotes the formation of TRA2b 4 transcript that does not translate. 16 Oxidative stress may promote the retention of SFRS10 exon 2 to reduce SFRS10 expression via phosphorylating HuR. Nevertheless, SFRS10 has been confirmed to be induced in cultured astrocytes during hypoxia followed by reoxygenation, 31 middle carotid artery occlusion, silicosis, arteriosclerosis, nerve injury, 32 and breast cancer.…”