POLYADENYLATION OF AN ESTROGEN REGULATED MESSENGER RNA

描述（改编自申请人的摘要）：在非洲爪蟾肝脏中， 主要的血清蛋白编码mRNA在雌激素的作用下不稳定 治疗（尤其是白蛋白）。 这些mRNA可能被“标记”用于靶向 通过短（~20个残基）聚腺苷酸尾的存在与该途径连接。 首席研究员的初步结果表明， 白蛋白上的poly（A）尾是由调节其加工过程引起的。 前mRNA。 在具体目标1中，他计划使用瞬时转染来绘制 白蛋白前体mRNA中负责顺式作用序列的位置 用于调节多聚腺苷酸化。 这个序列被称为poly（A） - 限制元件或PLE。 这一目标的实验也将确定其 最小序列的诱变，并检查距离AAUAAA的影响， 它的功能。 具体目标2中的实验将使用RNA凝胶阻滞 和UV交联，以确定PLE是否直接与一个 或多个核心蛋白的3'加工、切割和 多聚腺苷酸化特异性因子（CPSF），或与特异性PLE结合 蛋白（PLE-BP）。 PLE-BP与特定序列元件的交联 将使用已知或疑似放射性标记的嵌合RNA进行证明。 结合位点。 已知的核RNA结合蛋白的抗体将在 用于确定在细胞核中观察到的PLE结合蛋白的身份， 提取物，如果确定了已知蛋白质，将在特定的 目的3研究其在调节多聚腺苷酸化中的作用。 具体结果 目的1和2将指导特定目的中的体外聚腺苷酸化实验 3使用HeLa细胞核提取物或纯化的和/或重组的蛋白质， 3'加工复合体。 调节的多聚腺苷酸化将使用 携带野生型PLE或PLE的未切割或“预切割”转录物 突变失活（DPLE）。 三个待检验的非排他性假设 在这些具体目标是：1）PLE是否改变CPSF的稳定性 结合，2）PLE是否起募集聚（A）的特定同种型的作用 聚合酶与白蛋白3'加工复合物结合，该复合物仅能够 添加短的poly（A）尾，以及3）PLE是否改变了 核层（A）结合蛋白II，该蛋白质用于刺激 将长poly（A）添加到加工的前mRNA上。

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.