Abstract
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.
Highlights
Increased risk of nondiabetic kidney disease in African Americans has been associated with apolipoprotein L1 (APOL1) gene coding variants G1 and G2, found exclusively in people of recent African ancestry[1,2]
We analyzed the splicing pattern of APOL1 transcripts expressed by human glomerular podocytes AB8/13, primary glomerular mesangial cells (GMC), glomerular endothelial cells (GEC), and renal proximal tubular epithelial cells (RPTECs) (Fig. 1a)
Out of all the APOL family proteins, APOL1 is the only member that contains a signal peptide that allows for its secretion into blood to provide immunity against T. brucei[25,26,31,32,33,34]
Summary
Increased risk of nondiabetic kidney disease in African Americans has been associated with apolipoprotein L1 (APOL1) gene coding variants G1 and G2, found exclusively in people of recent African ancestry[1,2]. Toxicity of risk variants can be potentiated by interaction with additional factors or second hits[16] One such factor, a soluble urokinase plasminogen activator receptor (suPAR) associated with chronic kidney disease (CKD)[17], was recently shown to synergize preferentially with G1 or G2 variants for activation of αvβ[3] integrin signaling pathway, triggering podocyte injury[18]. A soluble urokinase plasminogen activator receptor (suPAR) associated with chronic kidney disease (CKD)[17], was recently shown to synergize preferentially with G1 or G2 variants for activation of αvβ[3] integrin signaling pathway, triggering podocyte injury[18] Preventing formation of this cell membrane multiprotein complex by lowering expression of extracellular suPAR or inhibiting secretion of intracellular APOL1 could block αvβ3-mediated signaling and reduce the toxicity of APOL1 kidney risk variants. The cooperative or antagonistic interactions between splicing factors affect assembly of the spliceosome, a multi-subunit ribonucleoprotein complex that together with a large number of auxiliary proteins removes introns from pre-mRNA transcripts[22,23]
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