Homologous recombination (HR) is critical for error-free repair of DNA double-strand breaks. Chromatin loading of RAD51, a key protein that mediates the recombination, is a crucial step in the execution of the HR repair. Here, we present evidence that SUMOylation of RAD51 is crucial for the RAD51 recruitment to chromatin and HR repair. We found that topoisomerase 1-binding arginine/serine-rich protein (TOPORS) induces the SUMOylation of RAD51 at lysine residues 57 and 70 in response to DNA damaging agents. The SUMOylation was facilitated by an ATM-induced phosphorylation of TOPORS at threonine 515 upon DNA damage. Knockdown of TOPORS or expression of SUMOylation-deficient RAD51 mutants caused reduction in supporting normal RAD51 functions during the HR repair, suggesting the physiological importance of the modification. We found that the SUMOylation-deficient RAD51 reduces the association with its crucial binding partner BRCA2, explaining its deficiency in supporting the HR repair. These findings altogether demonstrate a crucial role for TOPORS-mediated RAD51 SUMOylation in promoting HR repair and genomic maintenance.

Background: SF3B4, a critical component of U2 pre-mRNA spliceosomal complex, has been recently indicated as a potential oncogene in hepatocellular carcinoma (HCC). However, limited information exists on how SF3B4 expression is regulated in HCC. Materials and Methods: To determine the regulatory factor for SF3B4 expression, small interfering RNA (siRNA), real-time polymerase chain reaction (qRT-PCR) and western blotting assay were performed. The in vivo expression profiles of SRSF3 and SF3B4 were analyzed using public datasets and clinical samples. Results: Among 10 liver-specific splicing factors, only SRSF3 knockdown resulted in a significant increase in SF3B4 mRNA and consequently protein levels in SNU-368 HCC cells, probably via the retardation of SF3B4 mRNA decay rates. Using green fluorescent protein-SF3B4 fusion construct, the coding region of SF3B4 was found to be involved in SRSF3-mediated regulation of SF3B4 expression. Publicly available data from paired normal and tumor tissues in HCC and results from patients with HCC suggest that SRSF3 and SF3B4 possess an inverse relationship. Conclusion: SRSF3 is a key molecule for determining SF3B4 levels in HCC cells.The splicing factor 3B subunit 4 (SF3B4), also known as the spliceosome-associated protein (SAP) 49, is a core protein of the SF3B complex in the U2 small nuclear ribonucleoprotein (snRNP). This splicing factor also plays an important role in tethering U2 snRNP to the pre-mRNA region located at the branched point of the prespliceosome complex (1). Happloinsufficiency or mutations of SF3B4 gene have been implicated as a major cause of acrofacial dystosis such as Nager syndrome and Rodriguez syndrome, suggesting that an accurate mRNA processing is critical for craniofacial development (2-5). Recently, Ueno et al. clearly demonstrated that SF3B4 functions as a bridging factor to facilitate the assembly of polysomes on the endoplasmic reticulum, which confers an enhanced translation of secretory proteins including collagen A1s. This showed that the impairment in the biosynthesis of collagen is a molecular base leading to the poor craniofacial formation caused by SF3B4 mutation (6).A large-scale systematic genomic analysis identified frequent alterations in the splicing regulatory components in diverse types of cancers (7). Mutations in pre-mRNA regulatory sequences including exonic enhancers, exon silencers or those in splice site in the oncogene or tumor suppressor gene might affect the rate and pattern of splicing in key cancer-associated genes, which in turn might contribute to the modulation of cancer phenotypes. In addition to the mutations in the pre-mRNA cis-elements, mutations in genes encoding the trans-acting splicing factors, which include SF3B1, U2AF1, SRSF2, and ZRSR2, have been recurrently found predominantly in hematological malignancies (8, 9). In solid tumors, the occurrence of mutations in genes encoding splicing factors is very low but the alteration of expression levels of several regulatory splicing factors such as SRSF1, SRSF2, SRSF...

The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.

DNA double-strand breaks (DSBs) are one of the most lethal types of DNA damage due to the fact that unrepaired or mis-repaired DSBs lead to genomic instability or chromosomal aberrations, thereby causing cell death or tumorigenesis. The classical non-homologous end-joining pathway (c-NHEJ) is the major repair mechanism for rejoining DSBs, and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a critical factor in this pathway; however, regulation of DNA-PKcs expression remains unknown. In this study, we demonstrate that miR-145 directly suppresses DNA-PKcs by binding to the 3′-UTR and inhibiting translation, thereby causing an accumulation of DNA damage, impairing c-NHEJ, and rendering cells hypersensitive to ionizing radiation (IR). Of note, miR-145-mediated suppression of DNA damage repair and enhanced IR sensitivity were both reversed by either inhibiting miR-145 or overexpressing DNA-PKcs. In addition, we show that the levels of Akt1 phosphorylation in cancer cells are correlated with miR-145 suppression and DNA-PKcs upregulation. Furthermore, the overexpression of miR-145 in Akt1-suppressed cells inhibited c-NHEJ by downregulating DNA-PKcs. These results reveal a novel miRNA-mediated regulation of DNA repair and identify miR-145 as an important regulator of c-NHEJ.