FACTORS IN MATURATION OF SMALL RNAS IN THE YEAST NUCLEUS

酵母细胞核中小 RNA 成熟的因素

• 批准号: 6525509
• 负责人: MICHAEL R CULBERTSON
• 金额: $ 20.88万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6525509
• 关键词: Saccharomyces cerevisiae; gel mobility shift assay; genetic regulatory element; genetic screening; helicase; mass spectrometry; matrix assisted laser desorption ionization; molecular cloning; posttranscriptional RNA processing; protein protein interaction; protein structure function; regulatory gene; site directed mutagenesis; small nuclear RNA; western blottings; yeast two hybrid system

In eukaryotic cells, small nuclear and small nucleolar RNAs function in pre-mRNA and pre-rRNA processing.Many of these RNAs are themselves produced from primary transcripts that require modification and processing. In the yeast Saccharomyces cerevisiae, there are five small nuclear RNAs (snRNAs) involved in mRNA splicing and about 60 small nucleolar RNAs (snoRNAs) that function in ribosomal RNA processing and modification. Although a number of proteins are suspected to play a role in the maturation of small RNAs, the maturation pathways have not been well characterized. We identified a nuclear RNA helicase in yeast called Sen1p that is required for the maturation of tRNA, snRNA U5, rRNA, and numerous snoRNAs. Nine proteins, the snRNAs, and the snoRNAs have been shown to co-immunoprecipitate using antibodies against Sen1p, suggesting that the Sen1p RNA helicase assembles into one or more RNP complexes. Our recent studies show that Sen1p binds to the Sm/snRNA RNP involved in snRNA processing. Sen1p functions in conjunction with the endonuclease RNase III to form the 3' end of snRNA US. Sen1p also functions in 5' and 3' end formation of C/D box snoRNAs and may function in conjunction with other proteins including fibrillarin that bind to the C/D box. in this proposal, our specific aims will focus on the overall process of snoRNA maturation. We will screen for mutants that impair maturation, characterize snoRNA maturation pathways in vivo and in vitro, and define protein-protein and protein-RNA interactions that are important for the overall execution of steps in maturation. We will also characterize the biochemical activities of the Sen1p helicase in vitro and identify proteins that associate with Sen1p. The long-term objective of the proposal is to understand the biological implications of how small RNAs are derived from primary transcripts and how RNA helicases such as Sen1p can influence and possibly cross-regulate disparate RNA processing pathways. Further studies of RNA helicases and the processing of small RNAs in yeast are warranted and are ultimately of medical importance because numerous helicases and small RNAs have counterparts in vertebrates including humans and have been implicated in some human genetic disorders.

在真核细胞中，小核RNA和小核仁RNA在前mRNA和前rRNA的加工过程中发挥作用，其中许多RNA本身是由初级转录物产生的，需要经过修饰和加工。在酿酒酵母中，有5个小核RNA（snRNA）参与mRNA剪接，约60个小核仁RNA（snoRNA）参与核糖体RNA加工和修饰。尽管许多蛋白质被怀疑在小RNA的成熟中发挥作用，但成熟途径尚未得到很好的表征。我们在酵母中发现了一种称为Sen 1 p的核RNA解旋酶，它是tRNA、snRNA U 5、rRNA和许多snoRNA成熟所必需的。九种蛋白质，snRNA和snoRNA已经显示出使用针对Sen 1 p的抗体进行免疫共沉淀，这表明Sen 1 p RNA解旋酶组装成一个或多个RNP复合物。我们最近的研究表明，Sen 1 p与参与snRNA加工的Sm/snRNA RNP结合。Sen 1 p与核酸内切酶RNase III一起发挥作用，形成snRNA US的3'端。Sen 1 p还在C/D盒snoRNA的5'和3'末端形成中起作用，并且可以与其他蛋白质（包括结合到C/D盒的原纤蛋白）结合起作用。在这个建议中，我们的具体目标将集中在snoRNA成熟的整个过程。我们将筛选损害成熟的突变体，在体内和体外表征snoRNA成熟途径，并定义蛋白质-蛋白质和蛋白质-RNA相互作用，这对成熟步骤的整体执行很重要。我们还将在体外表征Sen 1 p解旋酶的生化活性，并鉴定与Sen 1 p相关的蛋白质。该提案的长期目标是了解小RNA如何从初级转录本中衍生的生物学意义，以及RNA解旋酶如Sen 1 p如何影响并可能交叉调节不同的RNA加工途径。RNA解旋酶和酵母中小RNA加工的进一步研究是必要的，并且最终具有医学重要性，因为许多解旋酶和小RNA在脊椎动物（包括人类）中具有对应物，并且与一些人类遗传疾病有关。