Laboratory for Molecular and Cellular Pharmacology, School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia.
Laboratory for Molecular and Cellular Pharmacology, School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia
Drug Metab Dispos. 2019 Mar;47(3):314-319. doi: 10.1124/dmd.118.085290. Epub 2019 Jan 3.
Sulfotransferase 4A1 (SULT4A1) is a sulfotransferase-like protein that is highly conserved between species. In human tissues, there are two transcripts, one that produces a full-length protein and one that produces an unstable truncated protein. The second transcript, which includes a pseudo-exon between exons 6 and 7 (6p), is widely expressed, whereas the first is more restricted. Differentiation of neuronal cells results in the removal of the pseudo-exon and subsequent SULT4A1 protein expression. Recent studies with SULT4A1 knockout mice showed that the protein is essential for normal development and that its absence leads to a severe neurologic phenotype. Here, the regulation of SULT4A1 6p splicing was investigated during neuronal differentiation using SH-SY5Y cells, human induced pluripotent stem cells, and mouse embryonic tissue. In all three models, pseudo-exon 6p was removed during differentiation, resulting in stable SULT4A1 protein expression. Using a minigene splicing assay, a region upstream of pseudo-exon 6p was identified that is essential for correct splicing of SULT4A1 mRNA. Within this region, there were binding motifs for four RNA processing factors (MBNL-1, MBNL-2, CELF-1, and CELF-2). Time-dependent changes in SULT4A1 protein and MBNL/CELF protein during differentiation supported their role in correctly splicing the SULT4A1 mRNA. Furthermore, ectopic expression of each factor produced efficient splicing in the minigene assay as well as correct splicing of the endogenous SULT4A1 mRNA. These results show that SULT4A1 mRNA is a target for MBNL/CELF-dependent splicing, which may be essential in producing stable, functional SULT4A1.
磺基转移酶 4A1（SULT4A1）是一种磺基转移酶样蛋白，在物种间高度保守。在人类组织中，有两种转录本，一种产生全长蛋白，另一种产生不稳定的截断蛋白。第二个转录本在 6 号外显子和 7 号外显子之间包含一个假外显子（6p），广泛表达，而第一个转录本则更为局限。神经元细胞的分化导致去除假外显子和随后的 SULT4A1 蛋白表达。最近对 SULT4A1 基因敲除小鼠的研究表明，该蛋白对正常发育至关重要，其缺失会导致严重的神经表型。在这里，使用 SH-SY5Y 细胞、人诱导多能干细胞和小鼠胚胎组织研究了 SULT4A1 6p 剪接在神经元分化过程中的调节。在所有三种模型中，假外显子 6p 在分化过程中被去除，导致稳定的 SULT4A1 蛋白表达。使用小基因剪接测定，鉴定出 6p 假外显子上游的一个区域，该区域对于正确剪接 SULT4A1 mRNA 是必需的。在该区域内，有四个 RNA 处理因子（MBNL-1、MBNL-2、CELF-1 和 CELF-2）的结合基序。在分化过程中 SULT4A1 蛋白和 MBNL/CELF 蛋白的时间依赖性变化支持它们在正确剪接 SULT4A1 mRNA 中的作用。此外，每种因子的异位表达在小基因测定中产生有效的剪接，以及内源性 SULT4A1 mRNA 的正确剪接。这些结果表明，SULT4A1 mRNA 是 MBNL/CELF 依赖性剪接的靶标，这对于产生稳定、功能性的 SULT4A1 可能是必需的。
