【正文】 xposure to 100 μM H2O2, MMS (100 μg/ml), 48 μM PGA2, or UVC (20 J/m2). Hybridizations using antibodies against actin and BAF57c were carried out to assess uniformity in loading and transfer among cytoplasmic and nuclear samples, respectively. (B) B2 binding activity in cytoplasmic lysates of cells treated as for panel and supershift analysis of plexes forming after exposure to such stresses. Induction of cytoplasmic HuR and its binding to p21 mRNA by UVC Summary ? UVC在不影響總 HuR水平的條件下誘導(dǎo)細(xì)胞漿 HuR水平 ? UVC誘導(dǎo) HuR與 p21 mRNA 的結(jié)合 ? HuR 結(jié)合 P21 3’UTR后使 P21mRNA 穩(wěn)定性增高，進(jìn)而引起 P21表達(dá)增高。 ? 人為降低 HuR水平可降低 HuR與 P21 3’UTR間的相互結(jié)合，降低 P21mRNA 穩(wěn)定性，降低 P21表達(dá)，提示 HuR對 UVC誘導(dǎo)的 P21 mRNA 穩(wěn)定性是必須的。 Example II AUF1 Regulates Replicative Senescence through Mediating p16 mRNA Turnover Wang, et al。 EMBO Rep., 6 : 158164, 2021. Background ? CDK inhibitor p16INK4 is induced with replicative senescence. Although transcriptional regulation of p16 has been intensively studied, regulation by posttranscriptional mechanism has not been reported. ? RNA binding protein AUF1 is expressed as a family of four protein isoforms (p37, p40, p42 and p45) arising through alternative splicing. AUF1 binds to AUrich elements (ARE) or AUUUA motifs in the 3’UTR of target mRNAs and destabilizes them (different from HuR). Question: Is AUF1 mediated mRNA turnover involved in the regulation of p16 during cellular senescence? P16 mRNA 3’UTR contains motifs for biding by RNA binding proteins 0204060801001200 2 4 6 8 10 12VCRFLSSenescence 0204060801001200 2 4 6 8 10 12VCRFLSYoung P16 3’UTR is important for the instability of EGFPp16 3’UTR (In young cells) Time in Dox (h) Time in Dox (h) AUF1 binds to p16 3’UTR. Binding of AUF1 to p16 3’UTR attenuated with cellular senescence. AUF1 levels reduced with cellular senescence. AUF1 binds to AU rich region of p16 3’UTR. A B C 0204060801001200 2 4 6 8VCRFLSP16 3’UTR confers instability to chimeric transcripts in lung carcinoma cells (H2) Time in Dox (h) Knockdown of AUF1 stabilizes EGFPp16 3’UTR chimeric transcripts in H2 cells Knockdown of AUF1 increases p16 expression and accelerates cellular senescence of WI38 cells Summary 1. mRNA turnover is important for p16 regulation during cell aging. 2. AUF1 binds to p16 3’UTR and destabilizes p16 mRNA. 3. AUF1 expression reduced with cellular senescence. Reduction of AUF1 during cellular senescence can lead to p16 upregulation and accelerate cell senescent. RNAbinding protein HuR enhances p53 translation in response to ultraviolet light irradiation. MazanMamczarz K, et al。 2021, PNAS Example Ⅲ Background ， 功能廣泛。 在 UVC輻 射下， p53被誘導(dǎo)，但機(jī)制不明。 2. UVC誘導(dǎo)細(xì)胞漿 HuR。 Questions： HuR是否調(diào)控 p53? 如何調(diào)控 ? Fig. 1. UVC induces p53 expression at protein level (A) UVC induces p53 expression (B) p53 mRNA levels is not influenced by UVC (C)p53 mRNA halflife is not altered by UVC (D)Subcellular and polysomal p53 mRNA is not altered under UVC. UVC induces p53 expression at protein level Fig. 2. (A) Western blot analysis of p53 expression in RKO wholecell lysates prepared at the times indicated after treatment with lactacystin (5 μM), UVC (15 J/m2), or a bination of both. Signals were quantitated by densitometry (graph). (B) Newly translated p53 is increased by UVC. UVC induced p53 is not associated with proteasome turnover Fig. 3. (A) REMSA (11) by using either CR or 3′ UTR p53 radiolabeled transcripts and cytoplasmic lysates prepared 4 and 6 h after treatment of RKO cells with either 15 or 30 J/m2 UVC. (B) Plasmids pGL3Luc and pGL3Lucp53(3′UTR) were used in transient transfections。 24 h after transfection, cells were either irradiated (15 J/m2) or left untreated, and the relative luciferase activity was calculated 6, 12, and 24 h later. Binding of cytoplasmic proteins to the p53 3′ UTR is linked to p53 translational upregulation. Fig. 4. (A) (REMSA) HuR binds to the p53 3’UTR (B) (pulldown assays) HuR binds to the p53 3’UTR. (C) (rCHip assays) HuR binds to the p53 3’UTR. (D)UVC induces the presence of HuR in the cytoplasm and polysome. HuR binds to the p53 3′ UTR in a UVCdependent manner Fig. 5. (A) Western blot analysis of HuR and p53 in HuRoverexpressing (S11) cells under UVC treatment. (B) Western blot analysis of HuR and p53 in HuRsilencing cells under UVC treatment. (C)Analysis of newly translated p53 in HuRsilenced cells. Modulation of HuR levels affects p53 translation and steadystate levels. Conclussion ? UVC induces p53 translation. ? HuR binds to the 3’UTR of p53. ? HuR enhances p53 translation under UVC treatment