Meharry Medical College, Center for AIDS Health Disparities Research, Department of Microbiology and Immunology, 1005 D. B. Todd Blvd, Nashville, TN, 37028, USA.
Department of Internal Medicine, 1005 D. B. Todd Blvd, Nashville, TN, 37028, USA.
Sci Rep. 2018 Jun 7;8(1):8739. doi: 10.1038/s41598-018-27104-x.
APOL1 risk alleles G1 or G2 are associated with a kidney disease phenotype exclusively in people of recent African ancestry. Here we show that exon 4 encoding a part of the APOL1 signal peptide is constitutively spliced in major APOL1 transcripts expressed in kidney glomerular and tubular cells. We demonstrate that constitutive splicing of exon 4 results from a suboptimal hnRNP A1 binding motif found in exon 4. Accordingly, a robust binding of hnRNP A1 protein to a consensus hnRNP A1 cis-acting element in exon 4 results in almost complete exclusion of exon 4 from the APOL1 minigene transcripts. Blocking the 5' splice site at the exon 4/intron boundary with a specific antisense morpholino oligonucleotide excludes exon 4 from the splicing pattern of endogenous APOL1 transcripts. These transcripts are fully functional and produce APOL1 protein isoform that is not normally detectable in podocytes. Together with our previous data showing no cytotoxicity of overexpressed APOL1 isoform lacking exon 4, we propose that morpholino-induced APOL1 isoform switch may provide a new tool to identify in vivo molecular mechanism(s) by which risk alleles promote or mediate the kidney disease phenotype.
APOL1 风险等位基因 G1 或 G2 仅与最近具有非洲血统人群的肾脏疾病表型相关。在这里，我们表明编码 APOL1 信号肽一部分的外显子 4 在肾脏肾小球和肾小管细胞中表达的主要 APOL1 转录物中是组成性剪接的。我们证明，外显子 4 的组成性剪接是由于在外显子 4 中发现了一个不合适的 hnRNP A1 结合基序。因此，hnRNP A1 蛋白与外显子 4 中的一致 hnRNP A1 顺式作用元件的强烈结合导致外显子 4 几乎完全从 APOL1 迷你基因转录物中排除。用特异性反义 morpholino 寡核苷酸阻断外显子 4/内含子边界的 5'剪接位点可将外显子 4 从内源性 APOL1 转录物的剪接模式中排除。这些转录物是完全功能性的，并产生通常在足细胞中不可检测到的 APOL1 蛋白同工型。结合我们之前的数据显示缺乏外显子 4 的过表达 APOL1 同工型没有细胞毒性，我们提出，morpholino 诱导的 APOL1 同工型转换可能为鉴定风险等位基因促进或介导肾脏疾病表型的体内分子机制提供新工具。
