CONTROL MECHANISMS IN HUMAN TUMOR CELLS - SMALL RNAS

我们正在研究紫外线辐射的一些明显的新影响，这是一种主要的 环境致癌物和诱变剂，对动物细胞。我们的主要目标是 以确定三个意外事件背后的详细分子机制 在这个实验室里对紫外光的影响进行了观察 小核糖核酸U1、U2、U3、U4和U5(U)的合成 信使RNA前体的加工需要短链RNA)：a)有 SnRNA转录对紫外光的直接、高敏感性，尽管 已知的SnRNA前体非常短(相反，转录 只有由长的初级转录本产生的RNA物种对紫外线敏感 光)；b)紫外线照射后立即，U1、U2、U3的一小部分 U5 RNA的合成不受(即使是高剂量)紫外线的影响(在 相比之下，对于合成对紫外线敏感的RNA物种 辐射，即合成是完全敏感的)；以及c)存在延迟 紫外线诱导的SnRNA转录抑制，这需要紫外光 流量远低于可比的 立即抑制水平，这似乎是一种不同于 早期的抑制，这在任何其他RNA中都没有观察到 物种。我们的工作将主要集中在哺乳动物的表达上 U1 RNA的基因，因为对它们的了解更多。我们提出了一些 假设，并建议它们可以通过相关的 紫外线照射后U1 RNA基因的表达和结构。这个 表达研究(U1 RNA合成的测定)包括：a)哺乳动物 DNA转染法转化U1基因的细胞；b)转录 分离的细胞核；c)将人U1基因显微注射到非洲爪哇 卵母细胞核；d)U1基因在全细胞提取液中的转录；以及e) U1基因在哺乳动物细胞中的瞬时表达这个 U1 RNA基因及其侧翼区的结构研究包括 DNA和染色质的分析。RNA转录的抑制，但 不是RNA的加工，已知是紫外线照射的早期效应。 相反，我们的结果表明成熟，而不是 UV照射后不久，KB中U6SnRNA的转录受到抑制 细胞。该项目的次要目标是测试两个提议的模型 这种抑制的机制。这些研究应该提供重要的 关于以下机制的信息：a)表达独一无二的 (RNA聚合酶II)U单链RNA转录单位；和b)一些明显 紫外线辐射对动物细胞的新影响。

We are studying some apparently novel effects of UV radiation, a major environmental carcinogen and mutagen, on animal cells. Our main goals are to determine the detailed molecular mechanisms underlying three unexpected observations made in this laboratory about the effect of UV light on the synthesis of small nuclear RNA (snRNA) species U1, U2, U3, U4 and U5 (U snRNAs are required for processing messenger RNA precursors): a) there is an immediate, high sensitivity of snRNA transcription to UV light, although the known snRNA precursors are very short (instead, the transcription of only RNA species generated from long primary transcripts is sensitive to UV light); b) immediately after UV irradiation, a small fraction of U1, U2, U3 and U5 RNA synthesis is unaffected by (even high doses of) UV light (in contrast, in the case of RNA species whose synthesis is sensitive to UV radiation, that synthesis is fully sensitive); and c) there is a delayed UV-induced inhibition of snRNA transcription, that requires UV light fluences that are considerably lower than those needed for a comparable level of immediate inhibition, that appears to be a reaction distinct from the early inhibition, and that has not been observed with any other RNA species. Our work will focus primarily on the expression of the mammalian genes for U1 RNA, since more is known about them. We propose some hypotheses and suggest that they can be tested by correlation of the expression and structure of U1 RNA genes after UV irradiation. The expression studies (assays of U1 RNA synthesis) include: a) mammalian cells transformed with U1 genes by DNA transfection; b) transcription in isolated nuclei; c) microinjection of human U1 genes into Xenopus laevis oocyte nuclei; d) transcription of U1 genes in whole cell extracts; and e) transient expression of U1 genes transfected into mammalian cells. The structural studies of U1 RNA genes and their flanking regions involve analysis of DNA and chromatin. The suppression of RNA transcription, but not of RNa processing, is known to be an early effect of UV irradiation. In contrast, our results suggest that the maturation, but not the transcription, of U6 snRNA is inhibited shortly after UV irradiation in KB cells. A secondary goal of this project is to test two proposed models of the mechanism of this inhibition. These studies should provide important information about the mechanisms of: a) expression of the unique group of (RNA polymerase II) U snRNA transcription units; and b) some apparently novel effects of UV radiation on animal cells.