Biology subjects

Lin, R.-J.

Publications and source records attributed to Lin, R.-J..

3 recordsLinked to original sources

bioRxiv2024

Engineering oncogenic hotspot mutations on SF3B1 via CRISPR-directed PRECIS mutagenesis

SF3B1 is the most recurrently mutated RNA splicing factor in cancer; However, its study has been hindered by a lack of disease-relevant cell line models. Here, we compared four genome engineering platforms to establish SF3B1mutant cell lines: CRISPR-Cas9 editing, AAV HDR editing, base editing (ABEmax, ABE8e), and prime editing (PE2, PE3, PE5Max). We showed that prime editing via PE5max achieved the most efficient SF3B1 K700E editing across a wide range of cell lines. We further refined our approach by coupling prime editing with a with a fluorescent reporter that leverages a SF3B1 mutation-responsive synthetic intron to mark prime edited cells. Calling this approach prime editing coupled intron-assisted selection (PRECIS), we then introduced the K700E hotspot mutation into two chronic lymphocytic leukemia (CLL) cell lines, HG-3 and MEC-1, and demonstrated that our PRECIS-engineered cells faithfully recapitulate the altered splicing and copy number variation (CNV) events frequently found in CLL patients with SF3B1 mutation. Our results showcase PRECIS as an efficient and generalizable method for engineering genetically faithful SF3B1 mutant models, shed new light on the role of SF3B1 mutation in cancer biology, and enables generation of novel SF3B1 mutant cell lines in any cellular context.

cancer biology↗

bioRxiv2023

Pan-Cancer Biomarker miR-151a Regulates p21 Partially Through p53

Dysregulation of microRNAs (miRNAs) has been associated with a variety of cancers. We previously identified miR-151a as a potential cell fitness-regulating microRNA using a miRNA-targeted CRISPR-Cas9 genetic screen. In this study, we created mutant cell clones with loss of miR-151a expression and verified miR-151a mutations indeed decreased cell growth. In miR-151a mutant cells, there was an increase in the fraction of cells in the G1-phase of the cell cycle. This increase in G1 cells corresponded to an increase in p53 (TP53) and p21 (CDKN1A) protein levels. Both strands of miR-151a could suppress p53; miR-151a-3p was able to directly suppress p53 expression, but the miR-151a-5p suppression of p53 apparently was indirect. Re-expression of miR-151a-5p in the mutant cells significantly decreased the p53 and p21 protein levels as well as the percentage of cells in G1, while re-expression of miR-151a-3p ironically had a modest effect. These results suggest that both the 5p and 3p strands as well as additional factors are involved in the regulation of p53/p21 and the cell cycle by miR-151a. We also analyzed the TCGA database and discovered that increased miR-151a expression occurs in many tumor types; furthermore, there was an inverse correlation between miR-151a and p21 expression, and high miR-151a expression was often associated with poor overall survival. Taken together, results from this study identify a previously underappreciated role of miR-151a in cancer through regulation of the cell cycle, and they also suggest inhibiting the less abundant 5p may be more important than inhibiting the more abundant 3p of miR-151a for therapeutic considerations.

molecular biology↗

bioRxiv2018

Human Puf-A, a Novel Component of 90S Pre-ribosome, Links Ribosome Biogenesis to Cancer Progression

We describe a novel biogenesis factor of the 90S pre-ribosome, Puf-A, which is a negative transcriptional target of p53. The expression of Puf-A is not only upregulated in advanced human lung cancer and tumors of patients especially with TP53 mutation, but also is highly prognostic for stage I lung cancer. Loss of Puf-A expression prevents KrasG12D/p53-/--induced tumor progression in the lungs and induces apoptosis in TP53- mutated cancers and c-Myc/p53-/--transformed cells as well. Overexpression of Puf-A enhances proliferation of normal cells after c-Myc induction and overcomes the cell-cycle checkpoints incurred by p53 expression. Mechanistically, Puf-A interacts with double-stranded structures of the 5.8S sequence within pre-rRNA and maintains the integrity of 90S pre-ribosomes, thereby impacting early ribosome assembly and export of ribosomes from nuclei. Silencing of Puf-A disrupts the assembly of 90S pre-ribosomes and induces the translocation of its associated nucleophosmin (NPM1) from nucleoli to the nucleoplasm, resulting in impairment of ribosome synthesis. Thus, Puf-A is crucial for over-activation of ribosome biogenesis and contributes to tumor progression and cancer growth.

cancer biology↗