The role of mRNA decay in p53-induced gene expression

The p53 tumor suppressor is a DNA damage responsive sequence-specific transcriptional activator. The sustained activation of the p53 response is incompatible with cell growth and viability. To circumvent this issue, a variety of negative feedback loops exist to limit the duration of p53 activation. Despite our understanding of p53-regulation, very little is known about the effect of transient p53 activation on the long term expression of p53 target genes. Here we used a temperature sensitive variant of p53 and oligonucleotide microarrays to monitor gene expression during and following reversible p53 activation. The expression of most p53-induced transcripts was rapidly reversible, consistent with active mRNA decay. Representative 3’UTRs derived from short-lived transcripts (i.e. DDB2 and GDF15) conferred instability on a heterologous mRNA while 3’UTRs derived from more stable transcripts (i.e. CRYAB and TP53I3) did not. The 3’UTRs derived from unstable p53-induced mRNAs were significantly longer than those derived from stable mRNAs. These 3’UTRs had high uridine and low cytosine content, leading to a higher density of U-, AU- and GU-rich sequences. Remarkably, short-lived p53 targets were induced faster reaching maximum transcript levels earlier than the stable p53-targets. Taken together, the p53 transcriptional response has evolved with primarily short-lived target mRNAs and that post-transcription processes play a prominent role in the p53 response. HT29-tsp53 cells expressing a temperature sensitive variant of p53 were used to study the reversal of the p53 response. Gene expression was assessed using Affymetrix Gene 1.0 ST oligonucleotide microarrays. Controls were maintained at the restrictive temperature (time 0) and treatments were collected following 16 hours at the permissie temperature (16 hours) and following a subsequent 6 hour recovery period (16 hours at 32 + 6 hour at 37).

p53肿瘤抑制因子（p53 tumor suppressor）是一种响应DNA损伤的序列特异性转录激活因子。p53应答通路的持续激活与细胞生长及存活能力无法兼容。为规避这一问题，机体存在多种负反馈环路以限制p53激活的持续时长。尽管我们对p53的调控机制已有一定认识，但对于瞬时p53激活对p53靶基因长期表达的影响，目前仍知之甚少。本研究利用温度敏感型p53变体与寡核苷酸微阵列（oligonucleotide microarrays），在可逆性p53激活过程中及激活结束后监测基因表达情况。多数p53诱导转录本的表达可快速恢复，这与活跃的mRNA降解过程相符。源自短命转录本（如DDB2与GDF15）的代表性3'非翻译区（3' untranslated region，3'UTR）可使异源mRNA产生不稳定特性，而源自稳定转录本（如CRYAB与TP53I3）的3'非翻译区则无此效果。源自不稳定p53诱导mRNA的3'非翻译区，长度显著长于源自稳定mRNA的3'非翻译区。此类3'非翻译区的尿苷（uridine）含量较高，胞嘧啶（cytosine）含量较低，因此富含U、AU及GU的序列密度更高。值得注意的是，短命型p53靶基因的诱导速度更快，其转录本水平达到峰值的时间早于稳定型p53靶基因。综上，p53转录应答在进化过程中以短命型靶mRNA为主要特征，且转录后调控过程在p53应答中发挥重要作用。本研究使用表达温度敏感型p53变体的HT29-tsp53细胞，探究p53应答的逆转过程。基因表达水平通过Affymetrix Gene 1.0 ST寡核苷酸微阵列进行检测。对照组细胞维持于限制性温度下（记为时间0点）；处理组样本分别于许可温度下培养16小时后（记为16小时组），以及后续经过6小时恢复培养（32℃培养16小时后转移至37℃培养6小时）后收集。