NUCLEAR ROLE OF YEAST POLY(A)-BINDING PROTEIN

描述（改编自申请人的摘要）：拟议的研究旨在 了解poly（A）结合蛋白的多种功能， 与大多数真核生物mRNA的poly（A）尾相关。有充分 有证据表明poly（A）尾及其结合蛋白的存在 对基因的表达有着深远的影响。这种RNA/蛋白质复合物 刺激翻译的起始并促进mRNA的稳定， 细胞质在细胞核中，mRNA 3 '端需要poly（A）结合蛋白 新生mRNA的形成和输出到细胞质。这项提议， 使用酵母酿酒酵母作为模型系统，解决 poly（A）-binding protein（Pab 1 p）的核功能。因素被 确定，与Pab 1 p一致，调节多聚腺苷酸化的程度。 如果编码这些因子中最具特征的基因PBP 1， 破坏后，poly（A）尾的长度显著减少， 在无细胞提取物中合成，并抑制 HIS 4报告基因中的早期多聚腺苷酸化位点。PBP 1的破坏也 抑制PAB 1缺失的致死性而不直接影响mRNA 稳定性或翻译，这与 先前分离的PAB 1抑制剂。双杂交分析表明，Pbp 1 p 可能是参与RNA加工的几个调节因子之一 设备.探讨Pbp 1及其相关因素在调节人外周血淋巴细胞凋亡中的作用。 Pab 1 p在3 '端处理中的作用PI建议：1）分析 Pbp 1 p和其他Pbp的生化活性，包括评估 pbp 1 p调节poly（A）核酸酶或poly（A）活性的能力 聚合酶; 2）鉴定其表达依赖于相对表达量的mRNA。 使用寡核苷酸探针阵列的聚腺苷酸化程度;和3）分析 通过测定PAB 1和PBP 1的相互作用， 与编码切割和/或多聚腺苷酸化因子的其它基因， 表征生长缺陷的高拷贝抑制剂， 这些基因的过度表达。

DESCRIPTION (Adapted from applicant's abstract): The proposed research seeks to understand the diverse functions of the poly(A)-binding protein that is associated with the poly(A) tail of most eukaryotic mRNAs. There is ample evidence indicating that the presence of the poly(A) tail and its bound protein has profound consequences on the expression of genes. This RNA/protein complex stimulates initiation of translation and promotes mRNA stabilization in the cytoplasm. In the nucleus, poly(A)-binding protein is required for mRNA 3'-end formation and export of the nascent mRNA from to the cytoplasm. This proposal, using the yeast Saccharomyces cerevisiae as a model system, addresses the nuclear functions of poly(A)-binding protein (Pab1p). Factors have been identified that, in concert with Pab1p, regulate the extent of polyadenylation. If the gene encoding the most well-characterized of these factors, PBP1, is disrupted, there is a substantial reduction in the lengths of poly(A) tails synthesized in cell-free extracts and inhibits the in vivo utilization of an early polyadenylation site in a HIS4 reporter. Disruption of PBP1 also suppresses the lethality of a PAB1 deletion without directly effecting mRNA stability or translation, which stands in contrast to the phenotypes of previously isolated pab1 suppressors. Two-hybrid analyses suggest that Pbp1p may be one of several regulatory factors recruited to the RNA processing apparatus. To study the role of Pbp1 and related factors in the regulation of Pab1p's function in 3'-end processing the PI proposes to: 1) analyze the biochemical activities of Pbp1p and the other Pbps, including an assessment of Pbp1p's ability to regulate the activity of poly(A) nuclease or poly(A) polymerase; 2) identify mRNAs whose expression is dependent on the relative extent of polyadenylation using oligonucleotide probe arrays; and 3) analyze the genetic relationships of PAB1 and PBP1 by determining their interactions with other genes encoding cleavage and/or polyadenylation factors by characterizing high copy suppressors of the growth defects resulting from overexpression of these genes.