Van den Bosch L, Eggermont J, De Smedt H, Mertens L, Wuytack F, Casteels R
Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Belgium.
Biochem J. 1994 Sep 1;302 ( Pt 2)(Pt 2):559-66. doi: 10.1042/bj3020559.
Tissue-specific alternative processing of sarco/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) transcripts generates functionally different Ca2+ pump isoforms in muscle compared with non-muscle tissues. In non-muscle cells, the SERCA2 pre-mRNA can be polyadenylated at a site located between the donor and acceptor splice site of an intron which is only removed in muscle tissues. To define the cis-active elements involved in differential processing, we constructed a minigene (pCM beta SERCA2) containing the 3' end of the SERCA2 gene. When stably transfected into a myogenic cell line, minigene transcripts were differentially processed depending on the differentiation state of the cells. This proves that the essential elements required for regulated processing are present in the construct. Furthermore, co-transfection of the pCM beta SERCA2 minigene and a myogenin expression vector in a fibroblast cell line induced muscle-specific splicing of transcripts from pCM beta SERCA2. This shows that trans-acting factor(s) responsible for muscle-specific processing can be induced by one of the important regulatory genes of muscle differentiation. Inactivation of the non-muscle poly(A) site did not induce splicing in non-muscle cells. This excludes a simple competition model between splicing and polyadenylation, but it is consistent with splicing being very inefficient in non-muscle cells. Moreover, splicing could be induced in non-muscle cells by optimizing the muscle-specific donor splice site and/or by shortening the intron length. We therefore propose that expression of the muscle-specific SERCA2a isoform is the result of activation of an otherwise inefficient splicing process.
肌浆网/内质网Ca(2+)-ATP酶2（SERCA2）转录本的组织特异性可变加工在肌肉组织中产生了与非肌肉组织功能不同的Ca2+泵亚型。在非肌肉细胞中，SERCA2前体mRNA可在一个位于内含子供体和受体剪接位点之间的位点进行多聚腺苷酸化，而该内含子仅在肌肉组织中被去除。为了确定参与差异加工的顺式作用元件，我们构建了一个包含SERCA2基因3'端的小基因（pCMβSERCA2）。当稳定转染到成肌细胞系中时，小基因转录本会根据细胞的分化状态进行差异加工。这证明了构建体中存在调控加工所需的关键元件。此外，在成纤维细胞系中共同转染pCMβSERCA2小基因和肌细胞生成素表达载体可诱导pCMβSERCA2转录本的肌肉特异性剪接。这表明负责肌肉特异性加工的反式作用因子可由肌肉分化的重要调控基因之一诱导产生。非肌肉多聚腺苷酸化位点的失活并未在非肌肉细胞中诱导剪接。这排除了剪接和多聚腺苷酸化之间的简单竞争模型，但与非肌肉细胞中剪接效率非常低的情况一致。此外，通过优化肌肉特异性供体剪接位点和/或缩短内含子长度，可在非肌肉细胞中诱导剪接。因此，我们提出肌肉特异性SERCA2a亚型的表达是原本低效的剪接过程被激活的结果。
