Related Experiment Video
Updated: Feb 5, 2026
Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells
Published on: April 26, 2017
Human-Specific Abnormal Alternative Splicing of Wild-Type PKD1 Induces Premature Termination of Polycystin-1
Wendy A Lea1,2, Stephen C Parnell1,3, Darren P Wallace1,2,4
1The Jared Grantham Kidney Institute and Departments of.
Insights
Human PKD1 gene mutations cause autosomal dominant polycystic kidney disease. Unusual DNA structures in the human PKD1 gene lead to abnormal splicing, reduced full-length polycystin-1 (PC1) protein, and potentially drive cyst formation.
Area of Science:
- Genetics
- Molecular Biology
- Human Disease Genetics
Background:
- Autosomal dominant polycystic kidney disease (ADPKD) is primarily caused by heterozygous mutations in the PKD1 gene, encoding polycystin-1 (PC1).
- Human PKD1 possesses unique, long polypyrimidine tracts in introns 21 and 22, unlike other mammals.
- These tracts can form stable triplex DNA, potentially stalling DNA polymerase and increasing mutation rates, but their impact on transcription and splicing was unknown.
Purpose of the Study:
- To investigate the impact of polypyrimidine tracts in human PKD1 introns on transcription and splicing efficiency.
- To characterize the protein products resulting from alternative splicing events in human PKD1.
Main Methods:
- RT-PCR and Western blotting were employed to analyze splicing events across exons 20-24 in human and mouse PKD1.
- Nanopore sequencing was utilized to confirm the presence of various splice forms in human PKD1.
Main Results:
- Human PKD1 produces a smaller protein, Trunc_PC1, not observed in mice, due to abnormal differential splicing across introns 21 and 22.
- A significant percentage (28.8%-61.5%) of human PKD1 transcripts exhibit premature termination.
- The presence of polypyrimidine tracts reduces the levels of full-length PKD1 mRNA from normal alleles.
Conclusions:
- Polypyrimidine tracts in human PKD1 introns impair the production of full-length mRNA and PC1 protein.
- Reduced levels of full-length PC1 in heterozygous individuals may fall below a critical threshold, contributing to cyst development in ADPKD.
Background:
The major form of autosomal dominant polycystic kidney disease is caused by heterozygous mutations in PKD1, the gene that encodes polycystin-1 (PC1). Unlike PKD1 genes in the mouse and most other mammals, human PKD1 is unusual in that it contains two long polypyrimidine tracts in introns 21 and 22 (2.5 kbp and 602 bp, respectively; 97% cytosine and thymine). Although these polypyrimidine tracts have been shown to form thermodynamically stable segments of triplex DNA that can cause DNA polymerase stalling and enhance the local mutation rate, the efficiency of transcription and splicing across these cytosine- and thymine-rich introns has been unexplored.
Methods:
We used RT-PCR and Western blotting (using an mAb to the N terminus) to probe splicing events over exons 20-24 in the mouse and human PKD1 genes as well as Nanopore sequencing to confirm the presence of multiple splice forms.
Results:
Analysis of PC1 indicates that humans, but not mice, have a smaller than expected protein product, which we call Trunc_PC1. The findings show that Trunc_PC1 is the protein product of abnormal differential splicing across introns 21 and 22 and that 28.8%-61.5% of PKD1 transcripts terminate early.
Conclusions:
The presence of polypyrimidine tracts decreases levels of full-length PKD1 mRNA from normal alleles. In heterozygous individuals, low levels of full-length PC1 may reduce polycystin signaling below a critical "cystogenic" threshold.
Related Concept Videos
02:18Alternative RNA Splicing
There are five types of alternative RNA splicing that vary in the ways the pre-mRNA segments are removed or retained in the mature mRNA. The first...
02:18Alternative RNA Splicing
01:32RNA Splicing
01:44Termination of Translation
01:44Termination of Translation
02:23Abnormal Proliferation