ALTERED RNA COMPARTMENTATION IN CARCINOGENESIS

这项提议继续研究改变的RNA 与癌发生的早期阶段相关的区室化。在 在前一个赠款期间，两个基本调查路线是 追求。首先，假定的核支架（NS）NTR参与 在RNA转运中，这里称为p46，被鉴定为N-末端 核纤层蛋白A/C.第二，出现在细胞质中的RNA序列， 致癌物处理后的RNA被发现由一个 B2序列的亚家族。这个B2亚家族对应于一个 人Alu序列的亚家族，其被活跃地转录， 参与了逆转录转座人类突变。B2转录本 显示改变的区室化是170-360 nt pol III“有义” 转录本，并且改变的区室化发生在 大鼠肝脏中轮廓清晰的病灶。我们的基本假设是 B2（β-样）转录物的区室化是一个起始事件， 使细胞永生化，并使它们倾向于 促进/渐进的改变。建议改变B2 结构/功能异常导致的分隔 由p46的表达改变而产生。两个独立的目标， 这可能是相互关联的，是要确定：1。的 改变的功能重要性 B2过渡金属的分隔。实验将 定义：A）B2转录物中的转录后改变， 与其改变的区室化有关; B）B2的调节 转录水平将使用新的策略来完成， 对细胞生长、分化和 将研究永生化/转化; C）是否 B2区室化改变与基因组不稳定性相关 和反转录转座，使用3种不同的方法。2)的作用 在早期阶段， 致癌作用。NS NTR活性在 RNA的运输已经被记录下来，我们已经确定了 推定的NT末端作为核纤层蛋白A/C（p46）的N末端。生产 p46的表达似乎是通过一种核多催化剂来完成的。 蛋白酶，其去除核定位信号（NLS）。 我们已经通过插入表位标签创建了修饰的p46构建体， 和NLS进入螺旋1和2之间的接头区域。该构建体 将检测ATP酶/激酶活性，并在细胞培养物中检测 对：A）核结构; B）NS NTR和RNA加帽; C） 细胞生长、分化和永生化/转化;以及 D）B2区室化。

This proposal continues investigations into altered RNA compartmentation associated with early stages of carcinogenesis. In the preceding grant period, two basic lines of investigation were pursued. First, the putative nuclear scaffold (NS) NTPase involved in RNA transport, here termed p46, was identified as the N-terminus of lamins A/C. Second, RNA sequences which appear in cytoplasmic RNA following carcinogen treatment were found to consist of a subfamily of B2 sequences. This B2 subfamily corresponds to a subfamily of human Alu sequences, which is actively transcribed and involved in retrotranspositional human mutations. B2 transcripts showing altered compartmentation are 170-360 nt pol III "sense" transcripts, and the altered compartmentation occurs in welldelineated foci in rat liver. Our basic hypothesis is that altered compartmentation of B2 (Alu-like) transcripts is an initiation event, which immortalizes cells and predisposes them to subsequent promotional/progressional alterations. It is proposed thataltered B2 compartmentation results from structural/functional abnormalities produced by altered expression of p46. Two independent objectives, which are likely to be interrelated, are to determine: 1. THE FUNCTIONAL IMPORTANCE OF ALTERED COMPARTMENTATION OF B2 TRANSCRIPTS. Bxperiments will define: A) Posttranscriptional alterations in B2 transcripts which relate to their altered compartmentation; B) Modulation of B2 transcript levels will be accomplished using novel strategies, and effects on cell growth, differentiation and immortalization/transformation will be investigated; C) Whether altered B2 compartmentation is associated with genomic instability and retrotransposition, using 3 distinct approaches. 2) THE ROLE OF ELEVATED NS NTPase IN EARLY STAGES OF CARCINOGENESIS. The importance of NS NTPase activity in RNA transport has been documented, and we have identified the putative NTPase as the N-terminus of lamins A/C (p46). Production of p46 appears to be accomplished by a nuclear multicatalytic proteinase,, which removes the nuclear localization signal (NLS). We have created a modified p46 construct by inserting an epitope tag and NLS into the linker region between coils l and 2. This construct will be tested for ATPase/kinase activities, and in cell culture for effects on: A) nuclear structure; B) NS NTPase & RNA capping; C) cell growth, differentiation, and immortalization/transformation; and D) B2 compartmentation.