REGULATION OF ALTERNATIVE PROCESSING OF CALCITONIN/CGRP

这个项目的目的是了解替代的前mRNA处理被用来产生组织特异性RNA编码不同蛋白质的机制。正在研究的基因是编码多肽荷尔蒙降钙素(CT)和降钙素基因相关肽(CGRP)的基因。这两个多肽由单一的Pre-mRNA产生，以包括替代的3‘末端外显子，外显子4，以产生CT或排除外显子4，以产生CGRP。降钙素和含外显子4的mRNA主要在甲状腺C细胞中产生，CGRP和不含外显子4的mRNA主要在神经细胞中产生。恶性甲状腺C细胞和小肺细胞癌同时表达RNA和多肽，可能是由于失去了加工调节。我们已经确定了一个有趣的内含子增强子，它通过改变替代的3‘端外显子的聚腺苷酸化切割效率来调节这种加工选择。该增强子位于受调控外显子的下游，是已知的唯一调节多聚腺苷酸化的远端组织特异性增强子。在以前的工作中，我们已经证明该增强子有一个类似于假外显子的核心，并结合剪接因子，包括U1 SnRNPs、SR蛋白(ASF/SF2和SRp20)和PTB。此外，我们还发现，增强子包含对包含和排除外显子4都很重要的额外序列，这些序列结合了可能对神经元和恶性肿瘤细胞中的外显子跳过重要的未知因子。这项提议包含了进一步确定增强子在正常和恶性细胞中的特征的实验。实验针对的是增强子促进大多数细胞中的多聚腺苷化和外显子包涵体，或神经细胞和恶性肿瘤细胞中的外显子排斥的机制。此外，该提案还包含了针对伪外显子内含子元件在更广泛的剪接事件中可能扮演的角色的实验。具体目标包括：1)确定与增强子功能有关的新因子；2)确定这些因子在组织特异性包含和排除外显子4中的作用；以及3)确定增强子在一般和调节剪接中的作用。这些实验的总体目标是了解类似外显子的增强子如何参与邻近外显子的聚腺苷酸化和剪接。

This project is aimed at understanding the mechanism whereby alternative pre-mRNA processing is used to generate tissue-specific RNAs coding for different proteins. The gene under study is that coding for the peptide hormones calcitonin (CT) and calcitonin-gene-related peptide (CGRP). The two peptides are produced from a single pre-mRNA to include an alternative 3' terminal exon, exon 4, to produce CT or exclude exon 4 to produce CGRP. Calcitonin and mRNA including exon 4 are produced mainly in thyroid C cells; CGRP and mRNA excluding exon 4 are produced mainly in neuronal cells. Malignant thyroid C cells and small lung cell carcinomas express both RNAs and peptides, presumably due to a loss of processing regulation. We have identified an intriguing intron enhancer that regulates this processing choice by altering polyadenylation cleavage efficiency of the alternative 3'-terminal exon. This enhancer is located downstream of the regulated exon and is the only known distal tissue-specific enhancer known to regulate polyadenylation. In previous work we have shown that the enhancer has a CORE resembling a pseudo exon and binds splicing factors, including U1 snRNPs, SR proteins (ASF/SF2 and SRp20), and PTB. In addition, we have shown that the enhancer contains additional sequences important for both inclusion and exclusion of exon 4 that bind unknown factors that may be important for exon skipping in neuronal and malignant cells. This proposal contains experiments to further characterize the enhancer in both normal and malignant cells. Experiments are directed at the mechanism whereby the enhancer facilitates polyadenylation and exon inclusion in most cells, or exon exclusion in neuronal and malignant cells. In addition, the proposal contains experiment directed at the role pseudo exon intron elements might play in more generalized splicing events. Specific aims include: 1) Identify new factors involved in enhancer function, 2) Determine the role of these factors in tissue-specific inclusion and exclusion of exon 4; and 3) Determine the role of the enhancer in general and regulated splicing. The overall goal of these experiments is to understand how an enhancer resembling an exon participates in both polyadenylation and splicing of neighboring exons.