Cooper T A, Cardone M H, Ordahl C P
Department of Anatomy, University of California, San Francisco 94143.
Nucleic Acids Res. 1988 Sep 12;16(17):8443-65. doi: 10.1093/nar/16.17.8443.
The cardiac troponin T (cTNT) pre-mRNA splices 17 exons contiguously but alternatively splices (includes or excludes) the fifth exon. Because both alternative splice products are processed from the same pre-mRNA species, the cTNT pre-mRNA must contain cis-acting sequences which specify exon 5 as an alternative exon. A cTNT minigene (SM-1) transfected into cultured cells produces mRNAs both including and excluding exon 5. The junctions of exons 4-5-6 and 4-6 in the cTNT minigene mRNAs are identical to those of endogenous cTNT mRNAs and no other exons are alternatively spliced. Thus, the SM-1 pre-mRNA is correctly alternatively spliced in transfected cells. To circumscribe the pre-mRNA regions which are required for the alternative nature of exon 5, we have constructed a systematic series of deletion mutants of SM-1. Transfection of this series demonstrates that a 1200 nt pre-mRNA region containing exons 4, 5, and 6 is sufficient to direct alternative splicing of exon 5. Within this region are two relatively large inverted repeats which potentially sequester the alternative exon via intramolecular base-pairing. Such sequestration of an alternative exon is consistent with models which propose pre-mRNA conformation as being determinative for alternative splicing of some pre-mRNAs. However, deletion mutants which remove the majority of each of the inverted repeats retain the ability to alternatively splice exon 5 demonstrating that neither is required for cTNT alternative splice site selection. Taken together, deletion analysis has limited cis elements required for alternative splicing to three small regions of the pre-mRNA containing exons 4, 5, and 6. In addition, the cTNT minigene pre-mRNA expresses both alternative splice products in a wide variety of cultured non-muscle cells as well as in cultured striated muscle cells, although expression of the cTNT pre-mRNA is normally restricted to striated muscle. This indicates that cis elements involved in defining the cTNT exon 5 as an alternative exon do not require muscle-specific factors in trans to function.
心肌肌钙蛋白T（cTNT）前体mRNA连续剪接17个外显子，但对第五个外显子进行可变剪接（包括或排除）。由于两种可变剪接产物均由同一前体mRNA产生，因此cTNT前体mRNA必定含有将外显子5指定为可变外显子的顺式作用序列。转染到培养细胞中的cTNT小基因（SM-1）产生包含和不包含外显子5的mRNA。cTNT小基因mRNA中外显子4-5-6和4-6的连接与内源性cTNT mRNA的连接相同，且没有其他外显子进行可变剪接。因此，SM-1前体mRNA在转染细胞中被正确地可变剪接。为了确定外显子5可变性质所需的前体mRNA区域，我们构建了一系列系统的SM-1缺失突变体。对该系列的转染表明，包含外显子4、5和6的1200 nt前体mRNA区域足以指导外显子5的可变剪接。在该区域内有两个相对较大的反向重复序列，它们可能通过分子内碱基配对隔离可变外显子。这种可变外显子的隔离与提出前体mRNA构象决定某些前体mRNA可变剪接的模型一致。然而，去除每个反向重复序列大部分的缺失突变体仍保留可变剪接外显子5的能力，这表明两者都不是cTNT可变剪接位点选择所必需的。综合起来，缺失分析已将可变剪接所需的顺式元件限制在前体mRNA包含外显子4、5和6的三个小区域。此外，cTNT小基因前体mRNA在多种培养的非肌肉细胞以及培养的横纹肌细胞中均表达两种可变剪接产物，尽管cTNT前体mRNA的表达通常限于横纹肌。这表明将cTNT外显子5定义为可变外显子所涉及的顺式元件在反式作用中不需要肌肉特异性因子来发挥作用。
