van den Bosch L
Laboratorium voor Fysiologie, Campus Gasthuisberg, Leuven.
Verh K Acad Geneeskd Belg. 1998;60(5):441-61.
The Ca2+ transport ATPases of the sarcoplasmic or endoplasmic reticulum (SERCA) mediate the uptake of Ca2+ into intracellular stores. These Ca2+ pumps are encoded by three different genes. Alternative processing of the SERCA2 messenger generates two different protein isoforms. These two isoforms differ in their C-terminal part and this divergence is responsible for the functional difference between SERCA2a and SERCA2b. The aim of this study was to characterise the cis-active elements and the trans-acting factors required for the generation of the muscle-specific messenger during myogenic differentiation. A competition model between muscle-type splicing and non-muscle polyadenylation was excluded as inactivation of the non-muscle polyadenylation site did not induce muscle-type splicing in non-muscle or in undifferentiated muscle cells. We therefore propose that the expression of the SERCA2a isoform is due to the regulation of the muscle-specific splice process during myogenic differentiation. We characterised the spatial and sequence requirements essential for tissue-specific transcript processing. It was demonstrated that the processing signals in the transcript, i.e. both donor splice sites and the polyadenylation site located in the muscle-specific intron, have to be weak. An exception to this rule is the 3' acceptor splice site that has to be strong in order to get muscle-specific splicing. We also found an inverse relationship between intron length and splice efficiency as shortening the terminal intron resulted in muscle-specific splicing in non-muscle and in undifferentiated muscle cells. Finally, it was demonstrated that sequences around the muscle-specific acceptor region as well as in the muscle-specific exon are required to prevent muscle-specific splicing in undifferentiated muscle cells. Especially a region upstream of the 3' acceptor contains important sequence information required for the inhibition of muscle-specific splicing. It was demonstrated that the trans-acting factors regulating the alternative SERCA2 splicing are muscle specific. First, myogenin, a transcription factor that plays a key role in muscle differentiation, induced muscle-specific SERCA2 splicing in a fibroblast cell line. Second, changes in general splice and polyadenylation efficiency, as observed during B-cell maturation, did not affect SERCA2 splicing. Finally, expression and overexpression studies did not support the hypothesis that changes in the level of the alternative splice factor ASF or other arginine and serine rich proteins are involved in the regulation of muscle-specific splicing. We conclude that muscle-specific SERCA2a expression is a tightly regulated process dependent on the inhibition of the muscle-specific splice process at the 3' end of the primary transcript. During differentiation this inhibition is overcome by the downregulation of an inhibitory factor and/or the expression of a positive trans-acting factor.
肌浆网或内质网（SERCA）的Ca2+转运ATP酶介导Ca2+摄取到细胞内储存库中。这些Ca2+泵由三个不同的基因编码。SERCA2信使核糖核酸的可变加工产生两种不同的蛋白质异构体。这两种异构体在其C末端部分有所不同，这种差异导致了SERCA2a和SERCA2b之间的功能差异。本研究的目的是确定在肌源性分化过程中产生肌肉特异性信使核糖核酸所需的顺式作用元件和反式作用因子。由于非肌肉多聚腺苷酸化位点的失活并未在非肌肉或未分化的肌肉细胞中诱导肌肉型剪接，因此排除了肌肉型剪接与非肌肉多聚腺苷酸化之间的竞争模型。因此，我们提出SERCA2a异构体的表达是由于肌源性分化过程中肌肉特异性剪接过程的调控。我们确定了组织特异性转录本加工所必需的空间和序列要求。结果表明，转录本中的加工信号，即位于肌肉特异性内含子中的供体剪接位点和多聚腺苷酸化位点，必须较弱。该规则的一个例外是3'受体剪接位点，为了实现肌肉特异性剪接，该位点必须较强。我们还发现内含子长度与剪接效率之间呈反比关系，因为缩短末端内含子会导致非肌肉和未分化肌肉细胞中的肌肉特异性剪接。最后，结果表明，肌肉特异性受体区域周围以及肌肉特异性外显子中的序列对于防止未分化肌肉细胞中的肌肉特异性剪接是必需的。特别是3'受体上游的一个区域包含抑制肌肉特异性剪接所需的重要序列信息。结果表明，调节SERCA2可变剪接的反式作用因子是肌肉特异性的。首先，肌细胞生成素是一种在肌肉分化中起关键作用的转录因子，它在成纤维细胞系中诱导肌肉特异性SERCA2剪接。其次，在B细胞成熟过程中观察到的一般剪接和多聚腺苷酸化效率的变化并不影响SERCA2剪接。最后，表达和过表达研究不支持以下假设：可变剪接因子ASF或其他富含精氨酸和丝氨酸的蛋白质水平的变化参与肌肉特异性剪接的调控。我们得出结论，肌肉特异性SERCA2a表达是一个严格调控的过程，依赖于对初级转录本3'末端肌肉特异性剪接过程的抑制。在分化过程中，这种抑制通过抑制因子的下调和/或正向反式作用因子的表达而被克服。
