Serine/arginine-rich splicing factor 3 (SRSF3) likely has wide-ranging roles in gene expression and facilitation of tumor cell growth. SRSF3 knockdown induced G1 arrest and apoptosis in colon cancer cells (HCT116) in association with altered expression of 833 genes. Pathway analysis revealed 'G1/S Checkpoint Regulation' as the most highly enriched category in the affected genes. SRSF3 knockdown did not induce p53 or stimulate phosphorylation of p53 or histone H2A.X in wild-type HCT116 cells. Furthermore, the knockdown induced G1 arrest in p53-null HCT116 cells, suggesting that p53-dependent DNA damage responses did not mediate the G1 arrest. Real-time reverse transcription-polymerase chain reaction and western blotting confirmed that SRSF3 knockdown reduced mRNA and protein levels of cyclins (D1, D3 and E1), E2F1 and E2F7. The decreased expression of cyclin D and E2F1 likely impaired the G1-to-S-phase progression. Consequently, retinoblastoma protein remained hypophosphorylated in SRSF3 knockdown cells. The knockdown also induced apoptosis in association with reduction of BCL2 protein levels. We also found that SRSF3 knockdown facilitated skipping of 81 5'-nucleotides (27 amino acids) from exon 8 of homeodomain-interacting protein kinase-2 (HIPK2) and produced a HIPK2 Δe8 isoform. Full-length HIPK2 (HIPK2 FL) is constantly degraded through association with an E3 ubiquitin ligase (Siah-1), whereas HIPK2 Δe8, lacking the 27 amino acids, lost Siah-1-binding ability and became resistant to proteasome digestion. Interestingly, selective knockdown of HIPK2 FL induced apoptosis in various colon cancer cells expressing wild-type or mutated p53. Thus, these findings disclose an important role of SRSF3 in the regulation of the G1-to-S-phase progression and alternative splicing of HIPK2 in tumor growth.

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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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MicroRNAs (miRNAs) play key roles in regulation of cellular processes in response to changes in environment. In this study, we examined alterations in miRNA profiles in peripheral blood from 25 male medical students two months and two days before the National Examination for Medical Practitioners. Blood obtained one month after the examination were used as baseline controls. Levels of seven miRNAs (miR-16, -20b, -26b, -29a, -126, -144 and -144*) were significantly elevated during the pre-examination period in association with significant down-regulation of their target mRNAs (WNT4, CCM2, MAK, and FGFR1 mRNAs) two days before the examination. State anxiety assessed two months before the examination was positively and negatively correlated with miR-16 and its target WNT4 mRNA levels, respectively. Fold changes in miR-16 levels from two days before to one month after the examination were inversely correlated with those in WNT4 mRNA levels over the same time points. We also confirmed the interaction between miR-16 and WNT4 3′UTR in HEK293T cells overexpressing FLAG-tagged WNT4 3′UTR and miR-16. Thus, a distinct group of miRNAs in periheral blood may participate in the integrated response to chronic academic stress in healthy young men.