Research
Imaging a single mRNA molecule from transcription to decay
(current project)
1. Live-cell single-molecule imaging of mRNA requires the transcript to be fused to an imaging tag. Unexpectedly, I found that the imaging tag may destabilize the corresponding mRNA. To eliminate such undesired effect, we devised an improved system, which specifically counteracted the tag-induced mRNA destabilization. Our system allows researchers to faithfully image mRNAs with minimal perturbation to the mRNAs’ stability (Li, W. Maekiniemi, A. et al. Nature Methods 2022).
2. Single-particle tracking reveals information about the type of transport (e.g., thermal or active), the medium where the particle is moving, and interactions with other particles. Tracking requires high imaging acquisition frequency, which in turn leads to photobleaching. Thus, the duration of mRNA single-particle tracking is generally limited to a few seconds, preventing us from capturing longer events, like the translation of diffusing transcripts (1-2 minutes) and the life cycle of an mRNA (tens of minutes in yeast). In a manuscript under preparation, I have established a long-term mRNA single-molecule imaging system, using which I increased the tracking time from four seconds to 20 minutes. This system enables us to image individual mRNA from transcription to decay and capture molecular events at all time scales.
References:
An improved imaging system that corrects MS2-induced RNA destabilization.
Weihan Li*†, Anna Maekiniemi*†, Hanae Sato, Christof Osman, and Robert H. Singer†. Accepted at Nature Methods, 2022. (* equal contribution, † correspondence). Link
RNA processing in the ER unfolded protein response pathway
(PhD project)
The endoplasmic reticulum (ER) is the major folding compartment for secretory proteins. The ER stress sensor Ire1 modulates ER protein folding homeostasis through two distinct pathways: non-conventional mRNA splicing and selective mRNA decay. The two pathways protect the cells from protein folding stress during development and in diseases, like diabetes and cancers. An important question in the field is to understand the molecular mechanism that differentiates the two functional outputs. I discovered that the Ire1’s protomer alignment within its homodimer structure regulates Ire1’s substrates specificity and differentiates the two functional outputs. This finding may help us design mammalian Ire1s that favor mRNA splicing over decay, and vice versa, enabling us to distinguish the biological significance of Ire1’s two functional outputs in physiological and pathological contexts.
References:
Protomer alignment modulates specificity of RNA substrate recognition by Ire1.
Weihan Li*, Kelly Crotty*, Diego Garrido Ruiz, Mark Voorhies, Carlos Rivera, Anita Sil, Dyche Mullins, Matthew Jacobson, Jirka Peschek, and Peter Walter. eLife, 2021. (* equal contribution). Link
Engineering ER-stress dependent non-conventional mRNA splicing.
Weihan Li†, Voytek Okreglak, Jirka Peschek, Philipp Kimmig, Meghan Zubradt, Jonathan S. Weissman, and Peter Walter†. eLife, 2018. († correspondence). Link