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Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1
Ist Teil von
Journal of molecular medicine (Berlin, Germany), 2014-10, Vol.92 (10), p.1045-1056
Ort / Verlag
Berlin/Heidelberg: Springer Berlin Heidelberg
Erscheinungsjahr
2014
Quelle
SpringerLink
Beschreibungen/Notizen
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