REGULATION OF ALTERNATIVE PROCESSING OF CALCITONIN/CGRP

该项目旨在了解替代mrna前加工用于生成编码不同蛋白质的组织特异性rna的机制。所研究的基因是编码肽激素降钙素（CT）和降钙素基因相关肽（CGRP）的基因。这两种肽由单个前mrna产生，包括另一个3'端外显子，外显子4，产生CT或排除外显子4产生CGRP。降钙素及其含外显子4的mRNA主要在甲状腺C细胞中产生；CGRP和不包括外显子4的mRNA主要在神经元细胞中产生。恶性甲状腺C细胞和小肺细胞癌同时表达rna和肽，可能是由于加工调节的缺失。我们已经确定了一个有趣的内含子增强子，它通过改变可选3'端外显子的聚腺苷化切割效率来调节这种加工选择。该增强子位于受调节外显子的下游，是唯一已知的调节聚腺苷酸化的远端组织特异性增强子。在之前的工作中，我们已经证明增强子具有类似伪外显子的CORE，并结合剪接因子，包括U1 snRNPs， SR蛋白（ASF/SF2和SRp20）和PTB。此外，我们已经证明，增强子含有额外的序列，对包含和排除外显子4都很重要，这些外显子4结合未知因子，可能对神经元细胞和恶性细胞的外显子跳跃很重要。本提案包含进一步表征正常和恶性细胞中的增强子的实验。实验针对增强子在大多数细胞中促进聚腺苷化和外显子包含的机制，或在神经元和恶性细胞中促进外显子排除的机制。此外，该提案还包含了针对伪外显子内含子元素在更广泛的剪接事件中可能发挥作用的实验。具体目标包括：1)确定参与增强子功能的新因子，2)确定这些因子在组织特异性外显子4的包含和排除中的作用；3)确定增强子在一般剪接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.