Exon recognition during constitutive pre-mRNA splicing

描述（由申请人提供）：RNA分子在许多水平上指导细胞过程的基因表达。它们通过与rna结合蛋白（rbp）的特定分子相互作用来形成多蛋白核糖核蛋白复合物（RNPs）。每个RBP都表现出与一组特定RNA序列结合的偏好，但这组RNA序列可以很多。我们将建立控制哺乳动物细胞中特定rbp与其特定RNA序列靶点之间发生的分子相互作用的生物物理参数。这种特异性通常是连续的，最紧密的结合体伴随着越来越不紧密的结合序列。后者可能会在细胞中竞争rbp，事实上，它们较弱的结合可能对它们的功能很重要，例如，短暂的结合是更稳定的多蛋白复合物组装的先决条件。通过使用高通量DNA测序技术（Illumina），我们将测量所有8个碱基的65,536个可能的RNA序列的生物物理结合常数；长度为8的RNA比典型的RNA靶标要大一些。这些结合参数将由平衡解离常数（Kd）组成，用于测量整体紧密性，以及重要的打开速率和关闭速率动力学常数，分别用于测量可及性和稳定性。重组rbp将在培养的哺乳动物细胞中表达，然后在瞬时转染后纯化并在珠上共价固定，只需一步。这些小珠子将暴露在包含所有可能的8-mers的RNA分子库中。在不同时间和平衡状态下分离头结合分子，并通过深度测序确定每个RNA序列结合的分子数。在使用一个模型RBP （pre- mRNA剪接因子Fox-2）建立方法后，该方法将扩展到其他相关剪接因子。随着这种额外rbp数据的积累，我们将能够模拟细胞中发生的rna -蛋白质相互作用，并更好地理解使这些rbp如此复杂的组合因素。此外，这些基本信息将使我们能够更好地预测遗传疾病和癌症中常见的RNA代谢紊乱如何影响细胞行为。

DESCRIPTION (provided by applicant): RNA molecules guide cellular processes at many levels of gene expression. They do so through specific molecular interactions with RNA-binding proteins (RBPs) to form multi-protein ribonucleoprotein complexes (RNPs). Each RBP exhibits a preference for binding to a particular set of RNA sequences, but this set could be many. We will establish the biophysical parameters governing the molecular interactions that take place between specific RBPs and their specific RNA sequence targets in mammalian cells. This specificity is typically a continuous function, with the tightest binders accompanied by less and less tightly bound sequences. These latter are likely to compete for RBPs in the cell and indeed their weaker binding may be important to their function, as for example a transient binding that is a prerequisite in the assembly of a more stable multi-protein complex. Through the use of high throughput DNA sequencing technology (Illumina) we will measure biophysical binding constants for all 65,536 possible RNA sequences of 8 bases; a length of 8 is somewhat larger than a typical RNA target. These binding parameters will be comprised of the equilibrium dissociation constant (Kd) to measure overall tightness, and the important kinetic constants of the on-rate and the off-rate, a measure of accessibility and stability, respectively. Recombinant RBPs will be expressed in cultured mammalian cells and then purified and covalently immobilized on beads in a single step following transient transfection. The beads will be exposed to a library of RNA molecules comprising all possible 8-mers. The bead-bound molecules will be isolated after various times as well as at equilibrium and the number of molecules of each RNA sequence bound will be determined by deep sequencing. After establishment of the methodology using one model RBP (the pre- mRNA splicing factor Fox-2), the method will be extended to additional related splicing factors. As this sort of data is accumulated for additional RBPs, we will be able to model RNA-protein interactions that occur in the cell and better understand the combinatorial factors that make these RNPs so complex. In addition, such fundamental information will allow us to better predict how disturbances in RNA metabolism, commonly seen in genetic diseases and in cancer, can affect cell behavior.