| Full text | |
| Author(s): Show less - |
Boddu, Ravindra
[1, 2]
;
Yang, Chaozhe
[2, 3]
;
O'Connor, Amber K.
[3]
;
Hendrickson, Robert Curtis
[4]
;
Boone, Braden
[5]
;
Cui, Xiangqin
[6]
;
Garcia-Gonzalez, Miguel
[7]
;
Igarashi, Peter
[8]
;
Onuchic, Luiz F.
[9]
;
Germino, Gregory G.
[10, 11]
;
Guay-Woodford, Lisa M.
[2, 3, 12]
Total Authors: 11
|
| Affiliation: Show less - | [1] Univ Alabama Birmingham, Dept Cell Biol, Birmingham, AL 35294 - USA
[2] Univ Alabama Birmingham, Dept Genet, Birmingham, AL 35294 - USA
[3] Childrens Natl Med Ctr, Ctr Translat Sci, Washington, DC 20010 - USA
[4] Univ Alabama Birmingham, Dept Microbiol, Birmingham, AL 35294 - USA
[5] Hudson Alpha Inst, Huntsville, AL 35806 - USA
[6] Univ Alabama Birmingham, Dept Biostat, Birmingham, AL 35294 - USA
[7] Complexo Hosp Santiago de Compostela, Inst Invest Sanitaria IDIS, Santiago De Compostela - Spain
[8] Univ Texas Southwestern Sch Med, Dept Med, Dallas, TX 75235 - USA
[9] Univ Sao Paulo, Dept Med, BR-01246903 Sao Paulo - Brazil
[10] Johns Hopkins Univ, Sch Med, Div Nephrol, Baltimore, MD 21205 - USA
[11] NIDDK, NIH, Bethesda, MD 20892 - USA
[12] Childrens Natl Med Ctr, Washington, DC 20010 - USA
Total Affiliations: 12
|
| Document type: | Journal article |
| Source: | JOURNAL OF MOLECULAR MEDICINE-JMM; v. 92, n. 10, p. 1045-1056, OCT 2014. |
| Web of Science Citations: | 17 |
| 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. (AU) | |