MESSENGER RNA METABOLISM IN DICTYOSTELIUM AND YEAST

该提案涉及转录后转录后的两个方面 真核生物中基因表达的调控：a) 决定基因稳定性的因素和b)细胞质功能 在mRNA上存在3‘-聚(A)区。的决定因素 信使核糖核酸稳定性：最近的实验使我们得出结论 随机的核溶解过程可能会对 在确定特定信使核糖核酸的降解率方面的作用。 因此，我们建议解决具体情况的可能性 不稳定网柄苔藓的元素(序列或结构)和 酵母mRNAs是导致这些mRNAs不稳定的原因。 为了识别这种不稳定因素，我们将构建：a) 来自编码稳定和不稳定mRNAs和b)的基因的嵌合体 编码不稳定的mRNAs基因的特定区域的缺失。 这些经过修饰的基因将通过DNA导入细胞。 介导性转化及其编码基因的稳定性 在活体内测定。体外诱变程序将 随后被用来圈定不稳定元素 更准确地说。将利用体外系统来确定 不稳定元素是否是特定核酸酶的靶标。 预计对结构基础的理解 特定酵母mRNAs的不稳定性将有助于我们设计 MRNA稳定性缺陷突变体的遗传选择 监管。 Poly(A)的细胞质功能：大量间接实验 该实验和其他研究表明，Poly(A)在 翻译启动可能由细胞质介导 聚(A)结合蛋白(S)。我们建议使用无手机 翻译系统直接解决了这一假设。我们会 灭活酵母聚(A)结合蛋白，或者用特定的 多克隆抗体或通过使用温度敏感突变体， 并评价其对翻译的影响。此外，我们还将 转录具有不同离散多聚(A)尾长的mRNAs 从我们构建的修改的SP6载体和 确定其相对招聘效率 多聚体。一种特定的长度缩短的可能性 在给定的信使核糖核酸上的聚(A)尾的变化与向 较小的多聚体，或随着多聚体的转移，也将是 通过在酵母中的活体脉冲追逐实验解决。

This proposal addresses two aspects of the post-transcriptional regulation of gene expression in eukaryotic organisms: a) the determinants of mRNA stability and b) the cytoplasmic function of the 3'-poly(A) tracts present on mRNA. Determinants of mRNA Stability: Recent experiments have led us to conclude that random nucleolytic processes are likely to play a significant role in determining the rates of degradation of specific mRNAs. Therefore, we propose to address the possibility that specific elements (sequences or structures) of unstable Dictyostelium and yeast mRNAs are responsible for the instability of those mRNAs. To identify such instability elements we will construct: a) chimeras from genes encoding stable and unstable mRNAs and b) deletions of specific regions of genes encoding unstable mRNAs. These modified genes will be introduced into cells by DNA mediated transformation and the stability of the encoded mRNAs determined in vivo. In vitro mutagenesis procedures will subsequently be utilized to delineate the instability elements more precisely. An in vitro system will be utilized to determine whether instability elements are the targets of specific nucleases. It is anticipated that an understanding of the structural basis of the instability of specific yeast mRNAs will aid us in devising genetic selections for mutants defective in mRNA stability regulation. Cytoplasmic Function of Poly(A): Numerous indirect experiments from this lab and others suggest that poly(A) has a role in translational initiation which may be mediated by the cytoplasmic poly(A)-binding protein(s). We propose to utilize cell-free translation systems to address this hypothesis directly. We will inactivate the yeast poly(A)-binding protein, either with specific polyclonal antibodies or by using a temperature-sensitive mutant, and evaluate the effects on translation. Further, we will transcribe mRNAs with different discrete poly(A) tail lengths from modified SP6 vectors which we have constructed and determine their relative efficiencies of recruitment into polysomes. The possibility that a specific reduction in the length of the poly(A) tail on a given mRNA correlates with a shift to smaller polysomes, or with a shift off of polysomes, will also be addressed by an in vivo pulse-chase experiment in yeast.