Autosomal recessive polycystic kidney disease (ARPKD) results from mutations in the human PKHD1 gene. Both this gene, and its mouse ortholog, Pkhd1, are primarily expressed in renal and biliary ductal structures. The mouse protein product, fibrocystin/polyductin complex (FPC), is a 445-kDa protein encoded by a 67-exon transcript that spans >500 kb of genomic DNA. In the current study, we observed multiple alternatively spliced Pkhd1 transcripts that varied in size and exon composition in embryonic mouse kidney, liver, and placenta samples, as well as among adult mouse pancreas, brain, heart, lung, testes, liver, and kidney. Using reverse transcription PCR and RNASeq, we identified 22 novel Pkhd1 kidney transcripts with unique exon junctions. Various mechanisms of alternative splicing were observed, including exon skipping, use of alternate acceptor/donor splice sites, and inclusion of novel exons. Bioinformatic analyses identified, and exon-trapping minigene experiments validated, consensus binding sites for serine/arginine-rich proteins that modulate alternative splicing. Using site-directed mutagenesis, we examined the functional importance of selected splice enhancers. In addition, we demonstrated that many of the novel transcripts were polysome bound, thus likely translated. Finally, we determined that the human PKHD1 R760H missense variant alters a splice enhancer motif that disrupts exon splicing in vitro and is predicted to truncate the protein. Taken together, these data provide evidence of the complex transcriptional regulation of Pkhd1/PKHD1 and identified motifs that regulate its splicing. Our studies indicate that Pkhd1/PKHD1 transcription is modulated, in part by intragenic factors, suggesting that aberrant PKHD1 splicing represents an unappreciated pathogenic mechanism in ARPKD.
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Acknowledgments The authors acknowledge Drs. Mary Ann Accavitti, Feng Qian, Brad Yoder, and members of the Guay-Woodford laboratory for their helpful advice. This work was supported by the NIDDK and the NIDDK intramural program (R01DK51259 and ZIA DK75042) to G.G.G., the UAB Hepato-Renal Fibrocystic Disease Core Center (P30 DK074038) to LGW, from the Foundation for Research Support of the State of São Paulo (2004/02622-0 to L.O.) and by the NIH-funded UAB Center for Clinical and Translational Science (UL1 RR025777). The authors have no conflicts to disclose.
- Alternative splicing
- Autosomal recessive polycystic kidney disease
- Exon splice enhancers