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Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1

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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.

Key messages

  • Multiple mRNA transcripts are generated for Pkhd1 in renal tissues

  • Pkhd1 transcription is modulated by standard splice elements and effectors

  • Mutations in splice motifs may alter splicing to generate nonfunctional peptides

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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.

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Authors and Affiliations

  1. Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA

    Ravindra Boddu

  2. Department of Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294, USA

    Ravindra Boddu, Chaozhe Yang & Lisa M. Guay-Woodford

  3. Center for Translational Science, Children’s National Medical Center, Washington, DC, 20010, USA

    Chaozhe Yang, Amber K. O’Connor & Lisa M. Guay-Woodford

  4. Hudson Alpha Institute, Huntsville, AL, 35806, USA

    Braden Boone

  5. Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA

    Robert Curtis Hendrickson

  6. Department of Biostatistics, University of Alabama at Birmingham, Birmingham, AL, 35294, USA

    Xiangqin Cui

  7. Complexo Hospitalario de Santiago de Compostela, Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain

    Miguel Garcia-Gonzalez

  8. Department of Medicine, University of Texas Southwestern School of Medicine, Dallas, TX, 75235, USA

    Peter Igarashi

  9. Department of Medicine, University of Sao Paulo, Sao Paulo, Brazil, 01246-903

    Luiz F. Onuchic

  10. Division of Nephrology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Gregory G. Germino

  11. National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, MD, 20892, USA

    Gregory G. Germino

  12. Children’s National Medical Center, 6th Floor Main Hospital, Center 6, 111 Michigan Ave NW, Washington, DC, 20010, USA

    Lisa M. Guay-Woodford

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  1. Ravindra Boddu
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Corresponding author

Correspondence to Lisa M. Guay-Woodford.

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Supplemental Figure 1

RT-PCR data from Pkhd1 lacZ/lacZ mice suggests alternative transcript production. RT-PCR was performed on total RNA isolated from 9-month-old Pkhd1 lacZ/lacZ kidney samples [27] (animals 7 Pkhd1 lacZ/lacZ and 8 Pkhd1 lacZ/lacZ) using primer pairs designed to amplify sequence spanning exons 50–67 and exons 38–52. a Pkhd1 exon 50–67 primers generate an amplicon of 3.5 kb in both Pkhd1 lacZ/lacZ and WT mice. The integrity of the large-sized RNA was validated with Prkdc primers (9 kb product). b Pkhd1 exon 38–52 primers generated a 2.1 kb amplicon in mouse 8 Pkhd1 lacZ/lacZ and WT. Boxes indicate Pkhd1 amplicons present in presumed null animals. A second 400 bp product was amplified from Pkhd1 lacZ/lacZ 8 RNA and was determined to represent a nonspecific amplicon by sequencing. Asterisk denotes non-specific amplicons. (PDF 598 kb).

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Boddu, R., Yang, C., O’Connor, A.K. et al. Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1 . J Mol Med 92, 1045–1056 (2014). https://doi.org/10.1007/s00109-014-1185-7

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  • DOI: https://doi.org/10.1007/s00109-014-1185-7

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