【正文】 nizing 18S rRNA. (D) Western blot analysis to assess the expression of p21, cJun (39 kDa), and actin in untransfected and AS HuRexpressing RKO cells 10 h after either no treatment or exposure to 20 J/m2 UVC. pjun, phosphorylated Jun. (E) Graphs depict the rate of loss of p21 and βactin mRNAs in cells with different HuR levels after actinomycin D (2 μg/ml) addition with or without UVC irradiation. At the times indicated, total RNA was extracted and p21 and βactin mRNAs were monitored by Northern blotting。 Elevation of p21 by UVC is acpanied with increased formation of P21 3’UTRprotein plex HuR 結(jié)合 p21 mRNA（ in vivo and in vitro） (A)， (B) HuR抗體可特異結(jié)合細胞漿蛋白與 B2形成的復(fù)合物。 C。 UVC induces the formation of p213’UTRprotein plex in the cytoplasm A。 復(fù)合物形成為 P53不依賴性的，因為無論 RKO細胞是否有野生型 P53，復(fù)合物的形成無區(qū)別。 ? P21 的調(diào)控，尤其 P53調(diào)節(jié)的轉(zhuǎn)錄已被廣泛，深入研究。 Nucleic Acids Research, 33(22): 71387150, 2021 2) 3) Wang, et al。 3) EMSA (gelshift, supershift) 4) rChip, pulldown assays (using paramagic streptavidin dynabeads, biotinyllabeled transcripts) ? mRNA半衰期測定 基本思路 :終止轉(zhuǎn)錄后 ,收取不同時間點之 RNA, 定量分析 RNA降解速率 . 1）用 ActinomycinD終止轉(zhuǎn)錄 2） Tetoff/on （或類似）報告基因系統(tǒng) 3) in vitro RNA降解分析 Teton system is activated in the presence of doxycycline the DNA binding domain of the Teton regulator (rTetR) contains mutations repressor that only binds DNA in the absence of ligand is converted to a liganddependent DNA binding protein. RNApol Tetracycline controlled transactivator (tTA) TETOFF in details Manfred Gossen and Hermann Bujard The Tetoff system is repressed in the presence of the doxycycline TETVP producing vector Gene of interest expressing vector VP – RNA pol interacting part TetR tet binding part TETOFF system Tetracycline controlled transactivator (tTA) 如： EGFPinterest target chimeric… mRNA 翻譯研究特用技術(shù) 。 3）富含 U，但無 AUUUA。 2. 被 RNA結(jié)合蛋白識別，結(jié)合。 如， HuR與 AUF1均可結(jié)合于 p21 與 cyclin D1 3’UTR， 但二者有競爭，且功能相反。 TTP ? 與 HuR及 AUF1不同， TTP主要位于細胞漿，結(jié)合 II類 AREs。 1）調(diào)控翻譯效率， 如 p53， p27 mRNA, cmyc。因此也被 稱為 mRNA 穩(wěn)定因子。主要位于細胞核，但可穿梭于細胞漿 /核間，且只有細胞漿 HuR 可調(diào)控 mRNA穩(wěn)定性（及翻譯）。RNA結(jié)合蛋白與轉(zhuǎn)錄后調(diào)控 王文恭 mRNA turnover Translation level Posttranslation level Transcription level Gene Regulation DNA and chromatin levels Posttranscriptional level Maturation mRNA export Regulatory factors for mRNA decay and translation 1. RNA binding proteins 2. microRNAs RNA binding proteins (RBPs) RNA結(jié)合蛋白種類很多，估計占細胞編碼蛋白 68%者為 RNA結(jié)合蛋白 , 但迄今只有為數(shù)不多的幾種 RNA結(jié)合蛋白（如 HuR， AUF1， TTP，TIA1， CUGBP2等）被證實可特異參與 mRNA穩(wěn)定性、翻譯、或其它層面的基因調(diào)控。核內(nèi) HuR則與 mRNA成熟及 export有關(guān)。 ? 結(jié)合并穩(wěn)定 VEGF, COX2, p21, cyclin A, cyclin B1, cfos, TNFα ， IL1， MyoD， Myogenin， bcl2等 mRNA。 2）調(diào)控 mRNA export, 如 CD83， cfos, COX2。 ? 結(jié)合目標后主要使目標不穩(wěn)定。 2. RNA結(jié)合蛋白與 microRNA間相互作用。 3. 主要為 mRNA 不穩(wěn)定元件，是 mRNA 在完成使命后快速降解的結(jié)構(gòu)基礎(chǔ)。 注： 除一級結(jié)構(gòu)外， mRNA的二級結(jié)構(gòu)也與 RNA蛋白質(zhì)相互作用密切相關(guān)。 分離， Polysomal RNA, Polysomal 蛋白質(zhì)分離。 MCB, 20:760769, 2021 4) Lal, et al。 ? 先前的研究發(fā)現(xiàn)， UVC可通過 P53不依賴的方式誘導(dǎo)P21。復(fù)合物由蛋白質(zhì)與 3‘UTR間結(jié)合而成。復(fù)合物形成在 UVC 輻射半小時后明顯被誘導(dǎo)，與 P21 被誘導(dǎo)相吻合。 EMSA 后， UV交聯(lián)， SDSPAGE 分離復(fù)合物，發(fā)現(xiàn)復(fù)合物中一條大約 40Kd 的復(fù)合物（單一蛋白與大約 10個堿基的短片斷轉(zhuǎn)錄物形成），說明有一 3540 Kd RNA結(jié)合蛋白被 UVC誘導(dǎo)并與 P21 3’UTR 結(jié)合。 (C) RNase T1 Selection Assay was carried out with B2 and A1, incubated with 10 nM GST or GSTHuR (see Materials and Methods). T1, digestions with RNase T1 alone。 signals were quantitated with a PhosphorImager, normalized against 18S (not shown), and plotted on a logarithmic scale. The mRNA halflife in each treatment group is indicated in parentheses. Values represent means 177。 cells were irradiated with UVC (20 J/m2) or left untreated, and luciferase and βgalactosidase activities were examined 24 h later. (Bottom) Relative fold increase in luciferase activity after UVC exposure, seen with either pGL3FL or pGL3ΔB2 pared with that seen with the control vector pGL3. Values represent means 177。 while UVCirradiated cells also exhibit abundant nuclear GFPHuR, the treatment causes a substantial increase in the cytoplasmic GFPHuR signal, not seen in untreated cells. UVC induces cytoplasmic HuR Increased cytoplasmic HuR and p21 RNA binding after exposure to stresses. (A) Western blot analysis to monitor HuR expression in cytoplasmic and nuclear fractions after treatment with the indicated agents. Samples were collected 2 h after addition of actinomycin (Act.) D (1 μg/ml) or 4 h after exposure 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 treate