Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.
RNA. 2010 Apr;16(4):839-51. doi: 10.1261/rna.2013110. Epub 2010 Mar 2.
Splicing of vertebrate introns involves recognition of three consensus elements at the 3' end. The branch point (BP) and polypyrimidine tract (PPT) are usually located within 40 nucleotides (nt) of the 3' splice site (3' ss), AG, but can be much more distant. A characteristic of the region between distant BPs (dBPs) and the 3' ss is the absence of intervening AG dinucleotides, leading to its designation as the "AG exclusion zone" (AGEZ). The human HTR4 gene, which encodes serotonin receptor 4 and has been associated with schizophrenia, bipolar disease, and gastrointestinal disorders, has four exons with extensive AGEZs. We have mapped the BPs for HTR4 exons 3, 4, 5, and g generated by in vitro splicing, and validated them by mutagenesis in exon-trapping vectors. All exons used dBPs up to 273 nt upstream of the exon. Strikingly, exons 4 and 5 used combinations of both distant and conventionally located BPs, suggesting that successful splicing of these exons can occur by distinct pathways. Our results emphasize the importance for single nucleotide polymorphism resequencing projects to take account of potential dBPs, as the extended AGEZs are vulnerable to mutations that could affect splicing itself or regulation of alternative splicing.
脊椎动物内含子的剪接涉及到在 3' 末端识别三个一致的元件。分支点（BP）和多嘧啶 tract（PPT）通常位于 3' 剪接位点（3' ss）AG 的 40 个核苷酸（nt）内，但也可能更远。远距离 BP（dBPs）和 3' ss 之间区域的一个特征是缺乏中间的 AG 二核苷酸，这导致其被指定为“AG 排除区”（AGEZ）。人类 HTR4 基因编码血清素受体 4，与精神分裂症、双相情感障碍和胃肠道疾病有关，它有四个具有广泛 AGEZ 的外显子。我们已经对体外剪接产生的 HTR4 外显子 3、4、5 和 g 的 BP 进行了作图，并通过外显子捕获载体中的诱变对其进行了验证。所有外显子都使用了距离外显子上游 273nt 的 dBPs。引人注目的是，外显子 4 和 5 同时使用了远距离和常规位置的 BP，这表明这些外显子的成功剪接可以通过不同的途径发生。我们的研究结果强调了单核苷酸多态性重测序项目需要考虑潜在的 dBPs 的重要性，因为扩展的 AGEZ 容易受到可能影响剪接本身或替代剪接调节的突变的影响。
