HUMAN SMALL NUCLEAR RNAS WITH NOVEL NUCLEOTIDE SEQUENCES

具有新型核苷酸序列的人小核 RNA

• 批准号: 3303772
• 负责人: George L Eliceiri
• 金额: $ 16.99万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3303772
• 关键词: HeLa cells; Xenopus; antisense nucleic acid; cell free system; crosslink; evolution; human genetic material tag; laboratory mouse; liposomes; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; protein structure function; ribosomal RNA; small nuclear RNA

We have synthesized and Cloned novel cDNA probes for three new human small nucleolar RNAs of unique nucleotide sequences, that will be named R, S and T. RNAs R, S and T appear to be true small RNA species, as opposed to breakdown products of large RNAs, because their cDNAs hybridize to a small RNA, but not to large RNA, in agarose gel Northern blots. Our long-term objective is to determine the function of RNAs R, S and T. It is reasonable to suspect that these small RNAs are involved in some aspect of ribosome production, because of their nucleolar location. Sequence complementarities between RNAs R-T and pre-rRNA are compatible with possible roles in pre-rRNA processing. This proposal focuses on testing the hypothesis that these small RNAs may function in the pathway of ribosome biogenesis. Our first goal is to determine the sequence of the -42 nucleotides located at the 5' end of RNA S. Some molecules of RNAs T and S are psoralen-crosslinked in vivo to large nucleolar RNA. This large nucleolar RNA consists of two main bands when hybridized with the S DNA probe: one migrates near 28S rRNA and the other is larger. Our second goal is to find out whether a small percentage of the molecules of RNAs R-T: a) behave as if specifically associated with pre-rRNA RNP particles or pre-rRNA and b) can be specifically psoralen-crosslinked in vivo to pre-rRNA, and, if so, to map approximately the location of the intermolecular crosslink in pre-rRNA. The third goal is the identification of the probes or system necessary to specifically lower the level of available, intact molecules of RNAs R-T. One antisense oligodeoxynucleotide targets the specific degradation of the RNA T present in a nucleolar extract. The first choice is to locate the accessible regions of RNAs R-T in nucleolar extracts and whole cells, using antisense oligodeoxynucleotide-targeted degradation. Alternative approaches are discussed. Nucleotide sequences that are highly conserved through evolution tend to be functionally important. Then, in our fourth goal a phylogenetic comparison of the nucleotide sequences of RNAs R-T should identify the conserved sequences. The fifth goal is to test the effect of the induced decrease of the level of available, intact molecules of RNAs R, S and T on the pathway of ribosome formation in whole cells or cell-free systems. There are important questions about the proteins with which these small RNAs may associate, and about the 5' ends of these small RNAs, using as probes currently available antibodies. We wish to ask those questions as this project progresses, when time allows it.

我们合成并克隆了三种新的人小细胞肺癌的cDNA探针， 核仁RNA的独特的核苷酸序列，这将被命名为R，S和 T. RNA R、S和T似乎是真正的小RNA种类，而不是 大RNA的降解产物，因为它们的cDNA与小RNA杂交， 琼脂糖凝胶北方印迹法检测到RNA，但未检测到大RNA。 我们的长期 目的是确定RNA R、S和T的功能。 是 有理由怀疑这些小RNA参与了 核糖体的产生，因为它们的核仁位置。 序列 RNA R-T和pre-rRNA之间的互补性与 在前rRNA加工中的作用。 本提案侧重于测试 这些小RNA可能在以下途径中起作用的假设 核糖体生物发生 我们的第一个目标是确定 -42个核苷酸位于RNA S的5'端。 一些RNA T分子 和S在体内与大的核仁RNA发生蛋白质交联。 这个大 核仁RNA与S DNA杂交时有两条主要带 探针：一个迁移到28 S rRNA附近，另一个较大。 我们的第二个目标 是找出是否有一小部分RNA分子R-T：a） 表现为与前rRNA RNP颗粒特异性相关，或 pre-rRNA和B）可以在体内特异性地与peptide交联， 前rRNA，并且，如果是这样的话，为了近似地映射 前体rRNA中分子间交联。 第三个目标是识别 所需的探针或系统，以专门降低的水平， 可用的完整的RNA分子R-T。 一种反义 寡脱氧核苷酸靶向存在的RNA T的特异性降解 在核仁提取物中。 第一个选择是定位可访问的 核仁提取物和整个细胞中RNA R-T区域，使用反义核酸， 寡脱氧核苷酸靶向降解。 替代方法有 讨论 核苷酸序列高度保守， 进化在功能上是很重要的。 然后，在我们的第四个目标a RNA R-T核苷酸序列的系统发育比较应该 确定保守序列。 第五个目标是测试 可利用的完整RNA分子水平的诱导降低 R、S和T在全细胞或无细胞核糖体形成途径中的作用 系统. 关于这些蛋白质有一些重要的问题， 小RNA可以缔合，并且在这些小RNA的5'端附近，使用 作为探针目前可用的抗体。 我们想问这些问题 在时间允许的情况下