The nucleic acid specificity and endogenous substrate of Rei1

Rei1的核酸特异性和内源底物

• 批准号: 7274504
• 负责人: Kenneth M Parnell
• 金额: $ 5.13万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7274504
• 关键词: Affect; Affinity; Amino Acid Sequence; Binding; Biogenesis; Biological Assay; Cell physiology; Characteristics; Class; Defect; Double-Stranded RNA; Elements; Growth; Hydroxyl Radical; In Vitro; Lead; Length; Mus; Mutate; Nucleic Acids; Pathway interactions; Phenotype; Play; Protein Biosynthesis; Proteins; RNA; RNA Binding; RNA Sequences; RNA-Binding Proteins; Ribosomal RNA; Ribosomes; Role; Role playing therapy; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Series; Specificity; Structure; Testing; Xenopus; Zinc Fingers; cancer type; cell growth; ds RNA-Binding Proteins; human TYRP1 protein; in vivo; mutant; nucleocytoplasmic transport; research study

DESCRIPTION (provided by applicant): RNA binding proteins play key roles in important cellular pathways. A new class of zinc finger proteins that bind double-stranded RNA has recently been discovered. My broad aim is to characterize zinc finger proteins that bind double-stranded RNA to determine what cellular roles they play. I will first investigate the Saccharomyces cerevisiae protein Reilp, a zinc finger protein that I have shown binds to double-stranded RNA. Reilp has been shown to be involved in the maturation of the large ribosomal subunit, perhaps by regulating the nucleocytoplasmic shuttling of other ribosome maturation factors. It is my hypothesis that Rei1 p binds to double-stranded or highly structured RNA regions on the 60S subunit, and that this interaction is crucial for its cellular function. Therefore, my specific aims are 1) To identify the endogenous RNA substrate of the S. cerevisiae protein Rei1 p; and 2) To determine the regions of Rei1 p that contribute to RNA binding in vitro and ribosome biogenesis in vivo. To identify the endogenous substrate of Reilp, I will utilize hydroxyl radical probing to determine ReMp's large subunit RNA "footprint". Having established the region of RNA to which Reilp binds, I will more precisely determine, through in vitro binding experiments, the specific RNA sequence/structure that is bound by Reilp. Finally, I will confirm the Rei1p-RNA interaction in vivo by mutating key residues of the ribosomal RNA and demonstrating an accompanying loss in Reilp-ribosome association. To determine the regions of Reilp that contribute to RNA binding and ribosome biogenesis, I will create a series of truncation, deletion and point mutants, all of which will be probed for in vitro RNA binding as well as in vivo function. This will allow me to determine the regions of Reilp that affect its RNA binding, as well as to determine the extent to which RNA binding by Reilp correlates with in vivo function. In addition, these studies will allow me to identify regions that, while not important for RNA binding, are important for Rei1 p's cellular function-such as regions involved in interactions with other cellular factors. Reilp appears to play an important regulatory role in creating ribosomes, and thus in protein synthesis. It is increasingly clear that control and coordination of protein synthesis is a critical issue in cell growth control, and defects in the biogenesis of ribosomes lead to certain types of cancer.

描述（由申请人提供）：RNA结合蛋白在重要的细胞途径中发挥关键作用。最近发现了一类新的结合双链RNA的锌指蛋白。我的主要目标是描述结合双链RNA的锌指蛋白，以确定它们在细胞中扮演的角色。我将首先研究酿酒酵母蛋白Reilp，一种我已经证明与双链RNA结合的锌指蛋白。Reilp已被证明参与核糖体大亚基的成熟，可能是通过调节其他核糖体成熟因子的核质穿梭。我的假设是，Rei 1 p与60 S亚基上的双链或高度结构化的RNA区域结合，这种相互作用对其细胞功能至关重要。因此，本研究的具体目标是：1）鉴定S.酿酒蛋白Reilp;和2）确定Reilp的有助于体外RNA结合和体内核糖体生物合成的区域。 为了确定Reilp的内源性底物，我将利用羟基自由基探针来确定ReMp的大亚基RNA“足迹”。在确定了Reilp结合的RNA区域之后，我将通过体外结合实验更精确地确定Reilp结合的特定RNA序列/结构。最后，我将确认Reilp-RNA相互作用在体内的核糖体RNA的关键残基突变，并证明在Reilp-核糖体协会伴随的损失。 为了确定有助于RNA结合和核糖体生物合成的Reilp区域，我将创建一系列截短、缺失和点突变体，所有这些突变体都将被探测用于体外RNA结合以及体内功能。这将使我能够确定影响其RNA结合的Reilp区域，以及确定Reilp的RNA结合与体内功能相关的程度。此外，这些研究将使我能够确定区域，而不是重要的RNA结合，是重要的Rei 1 p的细胞功能，如区域参与与其他细胞因子的相互作用。 Reilp似乎在核糖体的产生中起着重要的调节作用，因此在蛋白质合成中也起着重要的调节作用。越来越清楚的是，蛋白质合成的控制和协调是细胞生长控制中的关键问题，核糖体生物合成的缺陷导致某些类型的癌症。