Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1

Abstract

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.

The Future of Polycystic Kidney Disease Research—As Seen By the 12 Kaplan Awardees

“…Efforts to identify QTL in human ARPKD cohorts are confounded by several factors, most notably: (1) ARPKD is a relatively rare disorder; (2) the PKHD1 gene is transcriptionally complex 34 ; and (3) most patients are compound heterozygotes for different PKHD1 mutations. 35 Genetic studies in experimental models would be a reasonable alternative, but of the eight currently available mouse Pkhd1 models, none expresses a renal lesion that phenocopies the human disease.…”

Aberrant transcriptional regulation could explain phenotypic variability in autosomal recessive polycystic kidney disease

“…In this month's issue of J Mol Med, Boddu and colleagues [10] studied alternative splicing of PKHD1. The human gene spans 469 kb, has 86 exons, 15 of which exhibit alternative splicing boundaries.…”

“…Their analysis of PKHD1 missense variants suggests that dysregulated PKHD1 splicing represents an unappreciated pathogenic mechanism in ARPKD. Boddu et al [10] 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. They then used real-time PCR and RNA sequencing to identify 22 novel Pkhd1 kidney transcripts with unique exon junctions.…”

“…They extended their findings with bioinformatics analyses and exontrapping experiments to validate consensus-binding sites for serine/arginine-rich proteins that modulate alternative splicing. Boddu et al [10] then used site-directed mutagenesis to study the functional importance of selective splice-site enhancers. They then demonstrated that many of the novel transcripts were polysome-bound.…”

“…The contribution by Boddu et al [10] underscores the fact why clinicians need to be aware of alternate splicing. More than 60 % of all human disease-causing mutations may affect splicing rather than directly affecting coding sequences.…”