TRANSLATIONAL REGULATION DURING RED CELL DIFFERENTIATION

描述（改编自申请人摘要）：小鼠红白血病 (MEL)细胞是研究红细胞系的既定系统 分化 虽然很明显，分化诱导剂必须 影响基因表达，其作用机制仍不清楚。 的 研究人员最近确定，诱导剂增加了 去腺苷化，降低Spi-1 mRNA的稳定性， 与红白血病发生有关的因子。 因为，一个将军 mRNA去腺苷化的增加跟随诱导物暴露，这表明， 诱导剂可全面影响poly（A）代谢。 poly（A）结合 蛋白（PABP）已被提出来介导mRNA的影响 聚腺苷酸化 在MEL细胞中，PABP似乎以限制性的方式存在。 在对照组和对照组中， 诱导剂暴露的细胞。 由于诱导剂减少了细胞中mRNA的量， 多聚核糖体和增加mRNA的量在亚多聚核糖体组分，这些 数据表明，诱导剂导致PABP从多聚体中的净损失， mRNA。 研究人员提出，转录后的影响， 诱导剂通过增加PABP从mRNA中的去除速率来介导 聚（A）尾。 为了验证这一假设，拟议的实验将集中在 这些试剂的翻译效果，并将：1）定量 在分级细胞提取物中聚（A）和PABP的总量，以确定 如果诱导剂导致细胞中PABP/poly（A）的量净减少; 2)确定诱导剂对PABP表达的影响，并鉴定 在诱导剂暴露的细胞中调节其表达的机制; 3） 确定PABP的强制过表达对翻译的影响， MEL细胞中mRNA的失腺苷化和稳定性，并确定这是否 影响细胞分化的诱导;以及4）确定mRNA 需要5'-末端寡嘧啶段经受 诱导物的翻译作用。 这些实验可能提供重要的 深入了解PABP的功能和这种蛋白质所起的作用 MEL细胞暴露于诱导物后基因表达的变化

DESCRIPTION (Adapted from applicant's abstract): Murine erythroleukemia (MEL) cells are an established system for studying erythroid differentiation. While it is evident that inducers of differentiation must affect gene expression, their mechanism of action remains unknown. The investigators have recently determined that inducers increase the deadenylation and decrease the stability of Spi-1 mRNA, a transcription factor that is implicated in erythroleukemogenesis. Since, a general increase in mRNA deadenylation follows inducer exposure, this suggests that inducers may globally affect poly(A) metabolism. The poly(A)-binding protein (PABP) has been proposed to mediate the affects of mRNA polyadenylation. In MEL cells, the PABP appears to be present in limiting abundance and is found almost exclusively in polysomes in both control and inducer exposed cells. Since inducers decrease the amount of mRNA in polysomes and increase the amount of mRNA in subpolysomal fractions, these data suggest that inducers result in a net loss of PABP from polysomal mRNAs. The investigators propose that the post-transcriptional effect of inducers is mediated by increasing the rate of removal of the PABP from mRNA poly(A) tails. To test this hypothesis, the proposed experiments will focus on the translational effect of these agents and will: 1) Quantitate the total amount of poly(A) and PABP in fractionated cell extracts to determine if inducers cause a net decrease in the amount of PABP/poly(A) in the cells; 2) Determine the effects of inducers on expression of the PABP and identify the mechanisms that regulate its expression in inducer-exposed cells; 3) Determine the effect of forced overexpression of the PABP on translation, deadenylation and stability of mRNAs in MEL cells and determine if this affects the induction of cell differentiation; and 4) Determine if mRNAs require a 5'-terminal oligopyrimidine tract to be subject to the translational effect of inducers. These experiments may provide significant insight into the function of the PABP and the role that this protein plays in the changes in gene expression that follow inducer exposure of MEL cells