Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1

Ravindra Boddu, Chaozhe Yang, Amber K. O'Connor, Robert C urtis Hendrickson, Braden Boone, Xiangqin Cui, Miguel Garcia-Gonzalez, Peter Igarashi, Luiz F. Onuchic, Gregory G. Germino, Lisa M. Guay-Woodford

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)1045-1056
Number of pages12
JournalJournal of molecular medicine (Berlin, Germany)
Volume92
Issue number10
DOIs
StatePublished - Oct 1 2014

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Exons
Kidney
Autosomal Recessive Polycystic Kidney
RNA Splice Sites
Alternative Splicing
Mutation
Proteins
Polyribosomes
Liver
Site-Directed Mutagenesis
Computational Biology
Serine
Placenta
Genes
Reverse Transcription
Arginine
Testis
Pancreas
Binding Sites
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Boddu, R., Yang, C., O'Connor, A. K., Hendrickson, R. C. U., Boone, B., Cui, X., ... Guay-Woodford, L. M. (2014). Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1. Journal of molecular medicine (Berlin, Germany), 92(10), 1045-1056. https://doi.org/10.1007/s00109-014-1185-7

Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1. / Boddu, Ravindra; Yang, Chaozhe; O'Connor, Amber K.; Hendrickson, Robert C urtis; Boone, Braden; Cui, Xiangqin; Garcia-Gonzalez, Miguel; Igarashi, Peter; Onuchic, Luiz F.; Germino, Gregory G.; Guay-Woodford, Lisa M.

In: Journal of molecular medicine (Berlin, Germany), Vol. 92, No. 10, 01.10.2014, p. 1045-1056.

Research output: Contribution to journalArticle

Boddu, R, Yang, C, O'Connor, AK, Hendrickson, RCU, Boone, B, Cui, X, Garcia-Gonzalez, M, Igarashi, P, Onuchic, LF, Germino, GG & Guay-Woodford, LM 2014, 'Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1', Journal of molecular medicine (Berlin, Germany), vol. 92, no. 10, pp. 1045-1056. https://doi.org/10.1007/s00109-014-1185-7
Boddu, Ravindra ; Yang, Chaozhe ; O'Connor, Amber K. ; Hendrickson, Robert C urtis ; Boone, Braden ; Cui, Xiangqin ; Garcia-Gonzalez, Miguel ; Igarashi, Peter ; Onuchic, Luiz F. ; Germino, Gregory G. ; Guay-Woodford, Lisa M. / Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1. In: Journal of molecular medicine (Berlin, Germany). 2014 ; Vol. 92, No. 10. pp. 1045-1056.
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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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