Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1.
Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1.
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DOI：
10.1007/s00109-014-1185-7
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发表时间：
2014-10
影响因子：
4.7
通讯作者：
Guay-Woodford, Lisa M.
中科院分区：
文献类型：
--
作者：
Boddu, Ravindra;Yang, Chaozhe;O'Connor, Amber K.;Hendrickson, Robert Curtis;Boone, Braden;Cui, Xiangqin;Garcia-Gonzalez, Miguel;Igarashi, Peter;Onuchic, Luiz F.;Germino, Gregory G.;Guay-Woodford, Lisa M.
常染色体隐性遗传性多囊肾病（ARPKD）是由人类PKHD 1基因突变引起的。该基因及其小鼠直系同源物Pkhd 1主要在肾脏和胆管结构中表达。小鼠蛋白质产物纤维囊蛋白/多导管蛋白复合物（FPC）是由跨越>500 kb基因组DNA的67个外显子转录物编码的445-kDa蛋白质。在目前的研究中，我们观察到多个选择性剪接Pkhd 1转录本，在胚胎小鼠肾脏，肝脏和胎盘样品中的大小和外显子组成不同，以及成年小鼠胰腺，大脑，心脏，肺，睾丸，肝脏和肾脏。使用逆转录PCR和RNASeq，我们确定了22个新的Pkhd 1肾脏转录物具有独特的外显子连接。观察到选择性剪接的各种机制，包括外显子跳跃、使用选择性受体/供体剪接位点以及包含新的外显子。生物信息学分析确定，外显子捕获小基因实验验证，丝氨酸/丝氨酸丰富的蛋白质，调节选择性剪接的共识结合位点。使用定点突变，我们研究了选定的剪接增强子的功能的重要性。此外，我们证明了许多新的转录本是多核糖体结合的，因此可能是翻译的。最后，我们确定了人PKHD 1 R760 H错义变体改变了剪接增强子基序，该基序在体外破坏外显子剪接，并预测会截短蛋白质。综上所述，这些数据提供了证据的复杂的转录调控Pkhd 1/PKHD 1和确定的图案，调节其剪接。我们的研究表明，Pkhd 1/PKHD 1转录的调制，部分由基因内因素，这表明异常PKHD 1剪接代表了一个不受重视的致病机制ARPKD。
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.
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影响因子：
19.6
作者：
Menezes, LFC;Cai, YQ;Onuchic, LF
通讯作者：
Onuchic, LF
影响因子：
64.5
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Pachter L
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14.9
作者：
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通讯作者：
Krainer, AR
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作者：
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通讯作者：
Swanson, Maurice S.