Translation of single mRNAs in live cells
In the Wu lab, we are interested in studying dynamics of nucleic acid biology in live cells using single-molecule imaging. Towards this end, we have devised an imaging system called SINAPS to study the translation dynamics of mRNAs in cells. Using this method, we can track single mRNAs for hours in live cells and uncovered previously uncharacterized bursting translation dynamics of single mRNAs. Combining experiments with mathematic modeling, we utilize this technique to identify genetic elements that regulate the bursting parameters, investigate ribosome-associated mRNA quality control, and measure mRNA looping during translation.
RNA in disease
Genetic errors or defects in mRNA synthesis are implicated in many neurological diseases. In the Wu lab, we are interested in using single-molecule imaging to study these RNAs and how they work to promote the disease state. Recently, we have studied the GGGGCC repeat expansion in the intron of C9ORF72, the main cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. We demonstrated that the spliced intron was exported to the cytoplasm in a circular form and served as the template for repeat-associated non-AUG translation that produced toxic dipeptide.
In vivo single-molecule manipulation
The Wu lab has a strong interest in developing state-of-the-art methods to manipulate single nucleic acid molecules in live cells. Recently, we established a light-activated “very fast CRISPR (vfCRISPR)”, enabling genomic manipulation at submicron and seconds scales in collaboration with Taekjip Ha’s lab. Synchronized DNA cleavage improved kinetic analysis of double-strand-breaks (DSBs) and their repair. Using imaging-guided subcellular Cas9 activation, we achieved genome manipulation at single allele resolution. In a separate study, we developed a light-deactivated Cas9 system where photocleavable guide RNA was used to rapidly and efficiently turn off Cas9 activity. To investigate mRNA metabolism, we established a Rapid Inducible Decay of RNA (RIDR) technology to degrade target mRNA within minutes. We observed rapid formation and disappearance of RNA granules, which coincided with pre-existing processing bodies (PB). We measured the time-resolved RNA distribution and decay kinetics in subcellular compartments for the first time to show that PBs offers a kinetic advantage for rapid RNA decay.