Carlo T, Sterner D A, Berget S M
Verna and Marrs McClean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.
RNA. 1996 Apr;2(4):342-53.
The average length of a vertebrate axon is approximately 130 nt. Decreasing the size of an internal axon to less than 51 nt induces axon skipping, implying a minimal size for exons. A few constitutively included internal exons, however, are extremely small. To investigate if such micro-exons require special mechanisms for their inclusion, we studied the sequences necessary for inclusion of a 6-nt axon from chicken cardiac troponin T (cTNT). In vivo, the cTNT micro-exon was not included in mRNA unless accompanied by a 134-nt sequence located next to the micro-exon in the downstream intron. Increasing the length of the micro-exon alleviated the requirement for the intron element, indicating that the lack of inclusion of the micro-exon in the absence of a facilitating sequence was due to its small size, rather than suboptimal splice sites. The intron element contained six copies of a G-rich 7-nt sequence. Multimers of the repeat supported exon inclusion, indicating that the repeat sequence is an important part of the intron element. The entire intron element activated inclusion of a heterologous 7-nt exon, suggesting that the intron element is a general enhancer for the splicing of micro-exons. In vitro, the intron element and the repeated sequence facilitated splicing of a heterologous exon. Because of the ability of the cTNT intron element to facilitate the splicing of heterologous exons, we have termed the element an intron splicing enhancer (ISE). Interestingly, the ISE demonstrated position independence in that it facilitated inclusion of the heterologous micro-exon when placed either upstream or downstream of the micro-exon. In vitro, the ISE or copies of the ISE G-rich repeat stimulated splicing of an adjacent intron. The ISE thus becomes one of only a few characterized ISEs containing a G-rich repeat and the first to work both upstream and downstream of a target axon.
脊椎动物轴突的平均长度约为130个核苷酸。将内部轴突的大小减小到小于51个核苷酸会导致轴突跳跃，这意味着外显子有一个最小尺寸。然而，一些组成型包含的内部外显子非常小。为了研究这些微外显子的包含是否需要特殊机制，我们研究了鸡心肌肌钙蛋白T（cTNT）中一个6个核苷酸的轴突包含所需的序列。在体内，cTNT微外显子不包含在mRNA中，除非在下游内含子中紧邻微外显子的位置有一个134个核苷酸的序列。增加微外显子的长度减轻了对内含子元件的需求，表明在没有促进序列的情况下微外显子不被包含是由于其尺寸小，而不是剪接位点不理想。内含子元件包含六个富含G的7个核苷酸序列的拷贝。该重复序列的多聚体支持外显子包含，表明重复序列是内含子元件的重要组成部分。整个内含子元件激活了一个异源7个核苷酸外显子的包含，表明内含子元件是微外显子剪接的一般增强子。在体外，内含子元件和重复序列促进了异源外显子的剪接。由于cTNT内含子元件能够促进异源外显子的剪接，我们将该元件称为内含子剪接增强子（ISE）。有趣的是，ISE表现出位置独立性，即当置于微外显子的上游或下游时，它都能促进异源微外显子的包含。在体外，ISE或ISE富含G的重复序列拷贝刺激相邻内含子的剪接。因此，ISE成为少数几个含有富含G重复序列且第一个在靶轴突上游和下游均起作用的已表征ISE之一。
