POLYADENYLATION OF AN ESTROGEN REGULATED MESSENGER RNA

描述(改编自申请人的摘要)：在非洲爪哇肝脏， 雌激素引起的主要血清蛋白编码基因的不稳定 治疗(最明显的是白蛋白)。这些mRNAs可能被“标记”为靶向 通过存在短的(~20个残基)聚(A)尾巴到这条途径。 首席调查员的初步结果表明，短期内 白蛋白上的Poly(A)尾巴是其加工过程调节的结果 前信使核糖核酸。在特定的目标1中，他计划使用瞬时转基因来映射 顺式作用序列在白蛋白前mRNA中的位置 用于调节多聚腺苷酸化。这个序列被称为聚(A) -限制元素或PLE。这一目标的实验也将定义其 通过突变获得最小序列并检查距离AAUAAA的距离效应 它的功能。在特定目标2中的实验将使用RNA凝胶延迟 和UV交联，以确定PLE是否直接与 或更多的核心蛋白的3‘加工，裂解和 多聚腺苷酸化特异性因子(CPSF)或与PLE特异性结合 蛋白质(PLE-BP)。PLE-BP与特定序列元件的交联 将使用已知或可疑的放射性标记的嵌合RNA进行演示 结合部位。已知的核RNA结合蛋白的抗体将是 用于确定在核中观察到的PLE结合蛋白的身份 提取，如果鉴定出一种已知的蛋白质，将对其进行特定的测试 目的3研究其在调节多聚腺苷基化中的作用。来自特定项目的结果 AIMS 1和AIMS 2将指导特定靶点的体外多聚腺苷作用实验 3使用HeLa核提取物或纯化和/或重组蛋白 3‘加工复合体。受调控的多腺苷酸化将用 携带野生型PLE或PLE的未切割或预切割的转录本 突变失活(DPLE)。有待检验的三个非排他性假设 这些具体目标是：1)PLE是否改变了CPSF的稳定性 结合，2)PLE是否起到招募聚(A)的特定亚型的作用 聚合酶到白蛋白3‘处理复合体，该复合体仅能够 添加短的Poly(A)尾巴，以及3)PLE是否改变了 核层(A)结合蛋白II，用于刺激 在处理后的前信使核糖核酸中加入长聚(A)。

DESCRIPTION (Adapted from the applicant's abstract): In Xenopus liver, major serum protein-coding mRNAs are destabilized in response to estrogen treatment (most notably albumin). These mRNAs may be "marked" for targeting to this pathway by the presence of a short (~20 residue) poly(A) tail. Preliminary results from the principal investigator indicate that the short poly(A) tail on albumin results from regulation of processing of its pre-mRNA. In Specific Aim 1, he plans to use transient transfections to map the location within albumin pre-mRNA of the cis-acting sequence responsible for regulating polyadenylation. This sequence is termed the poly(A) -limiting element or PLE. Experiments in this aim will also define its minimal sequence by mutagenesis and examine distance effects from AAUAAA for its function. Experiments in Specific Aim 2 will use RNA gel retardation and UV crosslinking to determine whether the PLE interacts directly with one or more of the components of the core protein of 3' processing, Cleavage and Polyadenylation Specificity Factor (CPSF), or with a specific PLE-binding protein (PLE-BP). Crosslinking of PLE-BP to specific sequences elements will be demonstrated using chimeric RNAs radiolabeled at known or suspected binding sites. Antibodies to known nuclear RNA-binding proteins will be used to determine the identity of PLE-binding proteins observed in nuclear extract, and if a known protein is identified, it will be tested in Specific Aim 3 for its role in regulating polyadenylation. Results from Specific Aims 1 and 2 will guide in vitro polyadenylation experiments in Specific Aim 3 using HeLa nuclear extract or purified and/or recombinant proteins of the 3' processing complex. Regulated polyadenylation will be reproduced using either uncleaved or "precleaved" transcripts bearing wild type PLE or PLE inactivated by mutation (DPLE). Three non-exclusive hypotheses to be tested in these Specific Aim are; 1) does the PLE alter the stability of CPSF binding, 2) does the PLE act to recruit a specific isoform of poly(A) polymerase to the albumin 3' processing complex that is capable of only adding a short poly(A) tail, and 3) does the PLE alter the binding of nuclear ply(A)-binding protein II, the protein that serves to stimulate addition of long poly (A) onto the processed pre-mRNA.