Cooper T A, Ordahl C P
Department of Anatomy, University of California, San Francisco 94143.
Nucleic Acids Res. 1989 Oct 11;17(19):7905-21. doi: 10.1093/nar/17.19.7905.
The cardiac troponin T (cTNT) pre-mRNA contains a single alternative exon (exon 5) which is either included or excluded from the processed mRNA. Using transient transfection of cTNT minigenes, we have previously localized pre-mRNA cis elements required for exon 5 alternative splicing to three small regions of the pre-mRNA which include exons 4, 5, and 6. In the present study, nucleotide substitutions were introduced into the region containing exon 5 to begin to define specific nucleotides required for exon 5 alternative splicing. A mutation within the 5' splice site flanking the cTNT alternative exon that increases its homology to the consensus sequence improves splicing efficiency and leads to increased levels of mRNAs that include the alternative exon. Surprisingly, substitution of as few as four nucleotides within the alternative exon disrupts cTNT pre-mRNA alternative splicing and prevents recognition of exon 5 as a bona fide exon. These results establish that the cTNT alternative exon contains information in cis that is required for its recognition by the splicing machinery.
心肌肌钙蛋白T（cTNT）前体mRNA包含一个单一的可变外显子（外显子5），该外显子在加工后的mRNA中可以被包含或排除。通过cTNT小基因的瞬时转染，我们之前已经将外显子5可变剪接所需的前体mRNA顺式元件定位到前体mRNA的三个小区域，其中包括外显子4、5和6。在本研究中，将核苷酸替换引入包含外显子5的区域，以开始确定外显子5可变剪接所需的特定核苷酸。cTNT可变外显子侧翼5'剪接位点内的一个突变增加了其与共有序列的同源性，提高了剪接效率，并导致包含可变外显子的mRNA水平增加。令人惊讶的是，可变外显子内仅四个核苷酸的替换就会破坏cTNT前体mRNA的可变剪接，并阻止外显子5被识别为真正的外显子。这些结果表明，cTNT可变外显子包含顺式信息，这是剪接机制识别它所必需的。
