Ru-Inn Lin

Transportin-SR2 mediates nuclear import of phosphorylated SR proteins

Serine/arginine-rich proteins (SR proteins) are a family of nuclear factors that play important roles in both constitutive and regulated precursor mRNA splicing. The domain rich in arginine/serine (RS) repeats (RS domain) serves as both a nuclear and subnuclear localization signal. We previously identified an importin β family protein, transportin-SR2 (TRN-SR2), that specifically interacts with phosphorylated RS domains. A TRN-SR2 mutant deficient in Ran binding colocalizes with SR proteins in nuclear speckles, suggesting a role of TRN-SR2 in nuclear targeting of SR proteins. Using in vitro import assays, we here show that nuclear import of SR protein fusions requires cytosolic factors, and that the RS domain becomes phosphorylated in the import reaction. Reconstitution of SR protein import by using recombinant transport factors clearly demonstrates that TRN-SR2 is capable of targeting phosphorylated, but not unphosphorylated, SR proteins to the nucleus. Therefore, RS domain phosphorylation is critical for TRN-SR2-mediated nuclear import. Interestingly, we found that the RNA-binding activity of SR proteins confers temperature sensitivity to their nuclear import. Finally, we show that TRN-SR2 interacts with a nucleoporin and is targeted not only to the nuclear envelope but also to nuclear speckles in vitro. Thus, TRN-SR2 may perhaps escort SR protein cargoes to nuclear subdomains.

Nuclear Pnn/DRS Protein Binds to Spliced mRNPs and Participates in mRNA Processing and Export via Interaction with RNPS1

ABSTRACT Pnn/DRS protein is associated with desmosomes and colocalizes with splicing factors in nuclear speckled domains. The potential interaction of Pnn with RNPS1, a pre-mRNA splicing factor and a component of the exon-exon junction complex, prompted us to examine whether Pnn is involved in nuclear mRNA processing. By immunoprecipitation, we found that Pnn associates preferentially with mRNAs produced by splicing in vitro. Oligonucleotide-directed RNase H digestion revealed that Pnn binds to the spliced mRNAs at a position immediately upstream of the splice junction and that 5′ splice site utilization determines the location of Pnn in alternatively spliced mRNAs. Immunoprecipitation further showed that Pnn binds to mRNAs produced from a transiently expressed reporter in vivo. Although associated with mRNPs, Pnn is a nuclear-restricted protein as revealed by the heterokaryon assay. Overexpression of an amino-terminal fragment of Pnn that directly interacts with RNPS1 leads to blockage of pre-mRNA splicing. However, although suppression of Pnn expression shows no significant effect on splicing, it leads to some extent to nuclear accumulation of bulk poly(A)+ RNA. Therefore, Pnn may participate, via its interaction with RNPS1, in mRNA metabolism in the nucleus, including mRNA splicing and export.

Differential effects of hyperphosphorylation on splicing factor SRp55

Members of the serine/arginine-rich (SR) protein family play an important role in both constitutive and regulated splicing of precursor mRNAs. Phosphorylation of the arginine/serine dipeptide-rich domain (RS domain) can modulate the activity and the subcellular localization of SR proteins. However, whether the SR protein family members are individually regulated and how this is achieved remain unclear. In this report we show that 5,6-dichloro-1 beta-D-ribofuranosyl-benzimidazole (DRB), an inhibitor of RNA polymerase II-dependent transcription, specifically induced hyperphosphorylation of SRp55 but not that of any other SR proteins tested. Hyperphosphorylation of SRp55 occurs at the RS domain and appears to require the RNA-binding activity. Upon DRB treatment, hyperphosphorylated SRp55 relocates to enlarged nuclear speckles. Intriguingly, SRp55 is specifically targeted for degradation by the proteasome upon overexpression of the SR protein kinase Clk/Sty. Although a destabilization signal is mapped within the C-terminal 43-amino acid segment of SRp55, its adjacent lysine/serine-rich RS domain is nevertheless critical for the Clk/Sty-mediated degradation. We report for the first time that SRp55 can be hyperphosphorylated under different circumstances whereby its fate is differentially influenced.

Identification of a nuclear localization signal in the polo box domain of Plk1

Polo-like kinase 1 plays an essential role in mitosis and cytokinesis. Expression and nuclear localization of Plk1 during the S phase are necessary for its functions. Although it was reported that a bipartite nuclear localization signal located at the N-terminal kinase domain is required for nuclear import of Plk1, Plk1 carrying mutations in the polo box I of the polo box domain exhibited increased cytoplasmic accumulation. We further showed that the polo box domain was able to confer nuclear import of beta-galactosidase in vivo and GST-EGFP in vitro. The import carriers transportin and importin alpha were found to interact with the polo box domain directly in a Ran-GTP sensitive manner. These results indicate the presence of a nuclear localization signal in the polo box domain. A 38 amino acid sequence with the function of nuclear localization signal was identified to interact with transportin. Our findings demonstrated that a transportin-dependent nuclear localization signal is present in the polo box domain of Plk1, possibly required for efficient nuclear import. Showing little similarity to the M9 sequence, the 38 amino acid sequence identified here likely represents a novel nuclear localization signal.

Interaction between human papillomavirus type 5 E2 and polo-like kinase 1.

The E2 protein of the papillomavirus plays an essential role in the viral life cycle. Through a yeast two‐hybrid screening, human polo‐like kinase 1 was found to interact with human papillomavirus type 5 E2. Further characterization identified that the domains responsible for the interaction are the transactivation domain of HPV‐5 E2 and the sequence between the kinase and the polo box domains of Plk1. In vivo, Plk1 and HPV‐5 E2 are colocalized at the nuclear speckles. In the skin epithelium not infected with epidermodysplasia verruciformis associated HPVs, Plk1 is expressed in the stratum basale, indicating that the Plk1–HPV‐5 E2 interaction likely occurs in the keratinocytes at the basal layer of the epithelium upon infection of HPV‐5. Both HPV‐5 E2 and Plk1 also interact with the E2 binding domain of Brd4. The E2 binding domain of Brd4 is phosphorylated by Plk1 in vitro, and this phosphorylation event is blocked by the presence of HPV‐5 E2. Hence, these findings suggest the possibility that the cellular function of Brd4 is de‐regulated by forming a complex with HPV‐5 E2 in the infected epithelial cells. J. Med. Virol. 81:536–544, 2009.

Abstract 74: E2F6-mediated ceRNA and epigenetic silencing of miR193a lead to cancer stemness and anticancer immunity in ovarian cancer

Ovarian cancer is one of the most lethal cancers in the female reproductive system. Previous study suggested that long term treatment of estrogen such as hormonal replacement therapy (HRT) may increase the risk of ovarian cancer, however the role of estrogen in ovarian carcinogenesis is still controversial. To decipher this complicated process, we generated a mathematical model and found that estrogen-mediated up-regulation of E2F6 could upregulate the ovarian cancer stem/initiating marker, c-kit by two means one through epigenetic silencing of their co-targeted miR193a by binding of E2F6 which subsequently recruit EZH2 to miR-193a promoter; and second, by competing endogenous (ceRNA) mechanism. To confirm this model, treatment of E2 or environmental hormone, BPA resulted in upregulation of both E2F6 and c-kit but down-regulation of miR-193a in immortalized ovarian surface epithelial cells. Further bisulfite pyrosequencing, ChIP-qPCR and epigenetic treatment found that miR193a was epigenetically silenced by DNA methylation and H3K27me3 in CP70 but not HeyC2 ovarian cancer cells. Overexpression of miR193a inhibited tumor growth in vitro and in vivo. Depletion of EZH2 or E2F6 in CP70 restored miR-193a expression and decreased the number of “ovo” spheroid by reversing the repressive chromatin status of miR-193a promoter. To further explore the biological significance of this E2F6 ceRNA network, integrative RNA-Seq and computational analysis found that PBX1, a miR-193a target and transcriptional activator of the immunosuppressive cytokine IL-10, was down-regulated in E2F6 and EZH2 knockdown CP70 cells. Overexpression of E2F6 39UTR containing miR-193a MRE but not MRE mutant increased the expression of PBX1 and IL10 in ovarian cancer cells. Importantly, co-culture of conditional media from E2F6 39UTR overexpressing CP70 cells inhibited the differentiation of THP-1 monocytes into dendritic cell and the T-cell activating function of this THP-1 derived DC. This phenomenon can be rescued by incubation of anti-IL-10 antibody or pretreatment of CP70 cells with EZH2 inhibitor. Finally, clinical studies demonstrated that patients with higher promoter methylation of miR193a were associated with poor survival. Serum IL10 level was found to be higher in high staged ovarian cancer patients and patients with higher E2F6 mRNA level. Additional analysis from TCGA ovarian cancer expression microarray dataset demonstrated that ovarian cancer patients with low expression of EZH2, showed a positive correlation between E2F6, c-KIT and PBX1 resembling the ceRNA phenomenon between these mRNAs. Taken together, our results showed that estrogen-mediated E2F6 ceRNA network can regulate cancer stemness and anti-tumor immunity of DC through epigenetic silencing of miR-193a. Anti-estrogen therapy together with the EZH2 inhibitor may be a novel strategy against this deadly cancer. Citation Format: Frank Hsueh-Che Cheng, Hon-Yi Lin, Yin-Chen Chen, Tzy-Wei Hwang, Rui-Lan Huang, Chia-Bin Chang, Ru-Inn Lin, Ching-Wen Lin, Gary C.W. Chen, Jora M. J. Lin, Yu-Ming Chuang, Jian-Liang Chou, Chin Li, Alfred S.L. Cheng, Hung-Cheng Lai, Shu-Fen Wu, Je-Chiang Tsai, Michael W.Y. Chan. E2F6-mediated ceRNA and epigenetic silencing of miR193a lead to cancer stemness and anticancer immunity in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 74.

Abstract 1501: Aberrant JAK/STAT signaling orchestrates global promoter methylation and promotes TGF-β mediated EMT through epigenetic silencing of miR-193a in gastric cancer

Gastric cancer is the fourth leading cause of cancer-related death worldwide. Previous studies demonstrated that activation of the JAK/STAT3 signaling pathway is frequently observed in H. pylori infected gastric cancer. However, the role of aberrant JAK/STAT signaling in the global epigenetic changes remains unclear. In this regard, we compared the global methylomic changes in AGS gastric cancer cells showing constitutive activation of STA3 vs its STAT3 knock-down subclone by MBDcap-Seq followed by PrEMeR-CG analysis. Together with RNA-Seq, we identified 97 targets showing concomitant hypomethylation and over-expression while 76 targets showing concomitant hypermethylation and down-regulation after STAT3 knock down. Genes showing hypomethylaton/over-expression were subjected to transcription factor binding site analysis by MEME CentriMo. Interestingly, the transcriptional repressors binding site for ETS1 (p = 2.90E-06) and EHF (p = 3.50E-06) were overrepresented in those identifed STAT3 targets suggesting the cooperative binding with STAT3 in the epigenetic silencing of the targets. Further gene ontology analysis by DAVID showed that genes involved in cell cycle and apoptosis were significantly enriched in the hypomethylated/over-expressed targets while genes involved in protein degradation and ubiquitination were found among the hypermethylation/down-regulated targets. To experimentally confirm our result, we analyzed the functional role of one of the hypomethylated targets, miR-193a in gastric cancer. Concomitant with MBDcap-Seq, bisulphite pyrosequencing confirmed that promoter region of miR-193a was hypomethylated in AGS cells depleted with STAT3 but hypermethylated in MKN28 gastric cancer cells overexpressed with constitutive activated STAT3. Cell lines studies also found that promoter region of miR-193a was hypermethylated in gastric cancer cells which did not express miR-193a. Over-expression of miR-193a in AGS cells inhibited cell proliferation (p Citation Format: Jora M.j. Lin, Jiang-Liang Chou, David E. Frankhouser, Yu-Ming Chuang, Alex Liang-Yu Chang, Li-Han Zeng, Szu-Shan Chen, Ru-Inn Lin, Cheng-Shyong Wu, Kuo-Liang Wei, Enders K.W. Ng, Pearlly S. Yan, Alfred S.L. Cheng, Chin Li, Michael W. Y. Chan. Aberrant JAK/STAT signaling orchestrates global promoter methylation and promotes TGF-β mediated EMT through epigenetic silencing of miR-193a in gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1501.

Abstract 3314: DNA methylome analysis identifies epigenetic silencing of FHIT as a determining factor for radiosensitivity in oral cancer and its implication in treatment and outcome prediction

Radioresistance is still an emerging problem for radiotherapy of oral cancer. Aberrant epigenetic alterations play an important role in cancer development, yet the role of such alterations in radioresistance of oral cancer is not fully explored. Using Illumina 27K methylation BeadChip microarray, we identified promoter hypermethylation of FHIT (fragile histidine triad) in radioresistant OML1-R cells, established from hypo-fractionated irradiation (5-Gy by 10 fractions) of parental OML1 radiosensitive oral cancer cells. Further analysis confirmed that transcriptional repression of FHIT was due to promoter hypermethylation and H3K27me3 as demonstrated by MBDcap-PCR, bisulfite pyrosequencing and ChIP-PCR. These phenomenon were partially attributed to overexpression of EZH2 and DNMT3a, 3b in OML1-R cells. In consistent with these observations, treatment of 5-azaDC, EZH2 inhibitor (GSK343) or depletion of EZH2 by lentiviral knockdown restored FHIT expression in OML1-R cells. Interestingly, knockdown of EZH2 also reversed histone modifications (increased of H3K4me3 and decreased of H3K27me3) and reduced promoter methylation of FHIT thus suggesting that H3K27me3 linked to DNA methylation in this loci. We also analyzed the expression of FHIT in primary human oral keratinocyte (HOK) and four other oral cancer cell lines (OCSL, SCC25, SAS, and SCC4). FHIT expression demonstrated a tight inverse relationship with its promoter methylation. Ectopic expression of FHIT restored radiosensitivity (single fraction, 10-Gy) in OML1-R cells and oral cancer cells (SAS, SCC25) showing epigenetic silencing of FHIT. These phenomenon may be due to restoration of Chk2 phosphorylation, induction of apoptosis and G2/M check point. Reciprocal experiments also showed that depletion of FHIT in OSCL cells, which highly express FHIT, slightly enhanced radioresistance. Clinically, bisulfite pyrosequencing and iummnohistochemistry revealed that promoter hypermethylation of FHIT inversely correlated with its expression. Patients with higher FHIT methylation (methylation>10%, n = 22) are associated with lower locoregional control (P Citation Format: Hon-Yi Lin, Shih-Kai Hung, Moon-Sing Lee, Wen-Yen Chiou, Tze-Ta Huang, Chih-En Tseng, Liang-Yu Shih, Ru-Inn Lin, Jora Lin, Yi-Hui Lai, Chia-Bin Chang, Feng-Chun Hsu, Liang-Cheng Chen, Shiang-Jiun Tsai, Yu-Chieh Su, Szu-Chi Li, Hung-Chih Lai, Wen-Lin Hsu, Dai-Wei Liu, Chien-Kuo Tai, Shu-Fen Wu, Michael W. Chan. DNA methylome analysis identifies epigenetic silencing of FHIT as a determining factor for radiosensitivity in oral cancer and its implication in treatment and outcome prediction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3314. doi:10.1158/1538-7445.AM2015-3314