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Project 1
Mechanism and Modeling of Human Disease-Associated Intronic Variants that Perturb RNA Splicing
Mechanism and Modeling of Human Disease-Associated Intronic Variants that Perturb RNA Splicing
It is estimated that 10-30% of disease-associated genetic variants affect splicing. Splicing variants may generate deleteriously altered gene product and are potential therapeutic targets. However, experimental diagnosis for splicing variants is time-consuming and reliable computational prediction tools have not been established, especially for the near-exon intronic splice region. The major challenge lies in the redundant and ill-defined branch sites and other splicing motifs therein. Here, we carried out unbiased massively parallel splicing assays on 5,307 disease-associated variants overlapped with branch sites and collected 5,884 variants across the 5’ splice region. From the 11% variants significantly altering 3’ss splicing, we identified two distinct splicing altering mechanisms, novel splice site competition and branch site dysfunction, whereas the splicing altering mechanism of the 5’ intronic variants is mainly the splice site destruction. Statistical learning combined with these molecular features allows precise prediction for altered splicing from an intronic end variant.
This statistical model provides identity and ranking of biological features that determine splicing, which serves as transferable knowledge for future studies, and out-performs the benchmarking predictive tool.
Mechanism and modeling of human disease-associated near-exon intronic variants that perturb RNA splicing (Chiang et al., Nature Structural & Molecular Biology, 2022)
Moreover, we demonstrated that intronic splicing variants may associate with disease risks in human population. Our study elucidates the mechanism of splicing response of intronic variants, which classify disease-associated splicing variants for the promise of precision medicine.
Project 2
Study translational control of diseases related SNPs in 3’ UTR
Study translational control of diseases related SNPs in 3’ UTR
Genome-wide association study has focused on the change of amino acid in the coding region and/or the transcriptional regulation of gene expression. The translational control has been overlooked partly due to the absence of the protein expression profile.
Massively Parallel Polyribosome Profiling Reveals Translation Defects of Human Disease-Relevant UTR Mutations (Li et al., eLife 2025)
However, post-transcriptional regulation determines the level, the location and the timing of protein expression. Therefore, I propose to study how SNPs affect gene expression translationally with a massive parallel assay. From the publicly available database, such as dbSNP, Exome Aggregation Consortium (ExAC) data and The Cancer Genome Atlas (TCGA), we can identify SNPs and mutations overlapping with the microRNA and the RNA-binding protein binding sites in the 3’ UTR.
Multiplexed Assays of Human Disease-relevant Mutations Reveal UTR Dinucleotide Composition as a Major Determinant of RNA Stability (Su et al., eLife 2025)
We will then clone 3’ UTR of the minor allele and their corresponding major allele into a translation reporter, such as a luciferase reporter, and test how these minor alleles affect the protein expression.
More specifically, 3’UTRs with SNPs/mutations are cloned after luciferase coding region and within sequencing adaptors. These sequences are then in vitro transcribed, transfected into cell lines (HEK293, HeLa and SH-SY5Y neuronal cell lines). The transcripts level before and after transfection is compared to calculate the transcript stability. The luciferase reading normalized to the transcript level at harvest is compared for translation efficiency. Further analysis will be carried out to dissect the effect of SNPs/mutations that interrupt microRNA binding and RNA-protein interaction. I expect to learn how SNPs/mutations at 3’ UTR regulate RNA stability and translation efficiency. This study will greatly help us to understand the protein expression regulation in physiological and pathological background.
Our lab is supported by:
Our lab is supported by:
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