SNRNP MEDIATORS OF VERTEBRATE GENE EXPRESSION

脊椎动物的十种小核糖核蛋白 核质有助于前信使RNA的成熟， 拼接或3 '端形成机器的部件。 与此相反， 大约200个小核仁RNP（snoRNP）直接 切割和特定的核苷酸修饰，将前 核糖体RNA（rRNA）转化为成熟的18 S、5.8S和28 S rRNA。 额外 snRNP（未知定位的）指导其他的修饰， snRNAs。 它们的功能、作用机制和生物起源 snRNP和snoRNP将主要使用Hela在体外进行研究 提取物和非洲爪蟾卵母细胞。 对于U1、U2、U4/U6和U 5 snRNP 在主要的剪接体中， 引入snRNA中以分析它们的分子接触， 拼接这些snRNAs中修饰核苷酸的精确功能 将被识别，并研究其生物成因，特别是通过 确定有多少是由其他snRNA指导的。 对新 特征性的，低丰度的AT-AC剪接体，其突出的 与主要剪接体相比的差异，以及为什么它一直是 在进化过程中维持下来的东西。 AT-AC拼接体组件将 被解剖，问为什么U11/U12 snRNP作为一个复杂的， 发现是否存在AT-AC特异性外显子增强子。 分析在 体内前mRNA群体将确定AT-AC内含子去除是否 是限速的 选择性剪接（AT-AC）的必要性 相对于主要内含子去除）的转录物的表达。 果蝇的发育及其调控将被研究。 的 形成组蛋白mRNAs 3'端的机制将被解剖 通过表征允许沿着沿着 单链前体mRNA，并通过研究切割是否 因子与聚腺苷酸化所需的因子相同。 SnoRNP， 直接rRNA 2 '-O-甲基化将被纯化以表征 甲基化酶和使用snoRNA-rRNA的体外甲基化反应 将产生嵌合体。 核苷酸类似物干扰作图 （NIAM）将用于识别重要的RNA决定簇。 引导一个DNA甲基化的snoRNP之间可能的协调 将探测28 S的特定邻域。 为了阐明为什么所有 snoRNA宿主基因是5' TOP基因，5' TOP序列的核作用 可能有助于内含子编码的snoRNA释放的基因将被 寻求。 snRNP的生物发生和功能的扰动， snoRNP是某些人类疾病和衰老的决定因素。

Ten small nuclear ribonucleoproteins (snRNPs) in the vertebrate nucleoplasm contribute to the maturation of pre-messenger RNAs as components of the splicing or 3'-end formation machinery. In contrast, approximately two hundred small nucleolar RNPs (snoRNPs) direct cleavages and specific nucleotide modifications, converting the pre- ribosomal RNA (rRNA) into mature 18S, 5.8S and 28S rRNAs. Additional snRNPs (of unknown localization) direct the modification of other snRNAs. The functions, mechanisms of action and biogenesis of these snRNPs and snoRNPs will be investigated using primarily Hela in vitro extracts and the Xenopus oocyte. For the U1, U2, U4/U6 and U5 snRNPs of the major spliceosome, photoactivatable nucleotides will be introduced into the snRNAs to analyze their molecular contacts during splicing. The precise functions of modified nucleotides in these snRNAs will be identified and their biogenesis investigated, particularly by establishing how many are directed by other snRNA guides. For the newly characterized, low-abundance AT-AC spliceosome, both its salient differences compared to the major spliceosome and why it has been maintained in evolution will be probed. AT-AC spliceosome assembly will be dissected to ask why the U11/U12 snRNP acts as a complex and to discover whether AT-AC specific exonic enhancers exist. Analyses of in vivo pre-mRNA populations will establish whether AT-AC intron removal is rate-limiting. Both the necessity for alternative splicing (AT-AC versus major intron removal) of the prospero gene transcript during Drosophila development and its regulation will be investigated. The machinery that fashions the 3' ends of histone mRNAs will be dissected by characterizing the protein(s) that allow measurement along the single-stranded pre-mRNA and by investigating whether the cleavage factor is the same as that required for polyadenylation. SnoRNPs that direct rRNA 2'-O-methylation will be purified to characterize the methylase and an in vitro methylation reaction employing snoRNA-rRNA chimeras will be developed. Nucleotide analog interference mapping (NIAM) will be used to identify the important RNA determinants. Possible coordination between snoRNPs that guide methylation of a particular neighborhood of 28S will be probed. To illuminate why all snoRNA host genes are 5' TOP genes, a nuclear role for 5' TOP sequences that might contribute to the release of intron-encoded snoRNAs will be sought. Perturbations in the biogenesis and functioning of snRNPs and snoRNPs are determinants in certain human diseases and perhaps aging.