Translational regulation of PKD1 by evolutionarily conserved upstream open reading frames

Area of Science:

• Genetics
• Molecular Biology
• Biochemistry

Background:

• Autosomal-dominant polycystic kidney disease (ADPKD) is a common genetic disorder caused by mutations in the PKD1 gene.
• While promoter and 3' UTR regulation of PKD1 is studied, 5' UTR regulatory elements remain less understood.
• Upstream open reading frames (uORFs) in the 5' UTR can significantly influence gene expression.

Purpose of the Study:

• To investigate the role of uORFs and associated variants in regulating PKD1 gene translation.
• To determine if uORFs in the PKD1 5' UTR affect protein output under stress conditions.
• To explore the impact of naturally occurring variants on PKD1 translation.

Main Methods:

• Bioinformatic analysis of PKD1 5' UTR sequences.
• Luciferase reporter assays to measure translational activity.
• Quantitative reverse transcription PCR (RT-qPCR) to assess mRNA levels.
• Immunoblotting to determine protein expression.
• Antisense oligonucleotide (ASO) treatment to target uORFs.

Main Results:

• PKD1 mRNA features two conserved, translation-inhibitory uORFs (uORF1 and uORF2).
• uORFs and the 5' UTR do not affect PKD1 translation under ER or oxidative stress.
• Specific single nucleotide polymorphisms (SNPs) (rs2092942382, rs1596636969, rs2092942900) alter PKD1 translation, not transcription.
• Targeting uORFs with antisense oligos reduces protein output without affecting mRNA levels.

Conclusions:

• PKD1 expression is subject to translational regulation by 5' UTR uORFs.
• Mutations within these uORFs can lead to dysregulated PKD1 protein levels.
• This provides a novel mechanism for genetic variation impacting ADPKD pathogenesis.