TRANSLATIONAL REGULATION DURING RED CELL DIFFERENTIATION

描述(改编自申请者摘要)：小鼠红白血病 (MEL)细胞是研究红系的成熟系统 差异化。虽然很明显，分化的诱导者必须 影响基因表达的基因，其作用机制尚不清楚。这个 调查人员最近确定，诱导剂增加了 去烯基化和降低转录因子SPI-1mRNA的稳定性 与红血球形成有关的因子。从那时起，一位将军 在诱导剂暴露后，mRNA去烯基化增加，这表明 诱导剂可能在全球范围内影响聚(A)的代谢。聚(A)-结合 蛋白质(PABP)已被提出用来介导mRNA的作用 多聚腺苷酸化。在MEL细胞中，PABP似乎存在于限制 而且几乎只在对照组和对照组的多聚体中发现 诱导剂暴露的细胞。由于诱导剂降低了细胞中的mRNA含量 并增加亚多聚体部分的mRNA量，这些 数据表明，诱导剂会导致多聚体中PABP的净损失 MRNAs。研究人员提出，基因的转录后效应 诱导剂是通过提高从mRNA中去除PABP的速度而起作用的 Poly(A)尾巴。为了验证这一假设，拟议中的实验将集中于 关于这些动因的翻译效果，并将：1)量化 分离细胞提取液中聚(A)和PABP总量的测定 如果诱导剂导致细胞内PABP/Poly(A)数量的净减少； 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mRNAs的去烯基化和稳定性，并确定这是否 影响细胞分化的诱导；以及4)确定mRNAs 需要5‘-末端的寡嘧啶束才能 诱导语的翻译效果。这些实验可能会提供有意义的 深入了解PABP的功能和该蛋白所起的作用 诱导剂暴露后MEL细胞基因表达的变化

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