Zhou Hua-Lin, Baraniak Andrew P, Lou Hua
Department of Genetics, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106, USA.
Mol Cell Biol. 2007 Feb;27(3):830-41. doi: 10.1128/MCB.01015-06. Epub 2006 Nov 13.
Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3' splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF(65) binding to the 3' splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.
尽管已知多种调控元件和蛋白质因子可调节降钙素/降钙素基因相关肽（CGRP）前体mRNA的非神经元选择性加工途径，但控制神经元特异性途径的机制仍不清楚。在此，我们报告鉴定出Fox-1和Fox-2蛋白是介导神经元特异性剪接模式的新型调控因子。Fox-1和Fox-2蛋白的作用是抑制外显子4的包含，这种效应取决于降钙素特异性外显子4的3'剪接位点周围的两个UGCAUG元件。在类神经元细胞中，UGCAUG元件子集的突变会促进包含外显子4的非神经元模式。在HeLa细胞中，Fox-1或Fox-2蛋白的过表达会减少外显子4的包含。Fox-1和Fox-2蛋白与UGCAUG元件特异性相互作用，并通过阻止U2AF(65)与外显子4上游的3'剪接位点结合来调节剪接。我们进一步研究了UGCAUG沉默元件与先前鉴定的内含子和外显子剪接调控元件之间的相互关系，发现外显子4受正负调控的复杂平衡调节。这些结果确定了Fox-1和Fox-2蛋白在降钙素/CGRP前体mRNA外显子4包含中的关键作用，并建立了一个控制外显子4命运的调控网络。
