The 830th MIB Seminar
(Joint Usage/Research Center for the Multi-stratified Host Defense System)
Towards quantifying and controlling endogenous translation kinetics with single-molecule precision in living cells
Tim Stasevic, Ph.D.
Colorado State University, Department of Biochemistry and Molecular Biology
Dec. 7 (Wed), 2022
Lecture Room 101, 1F, Biomedical Research Station
No.34 on the following linked map.
My lab has created technology to image mRNA translation dynamics in real time and with single-molecule precision in living cells1,2. In this talk, I’ll describe two important extensions of this technology. First, I’ll focus on our ongoing efforts to better control single-mRNA translation kinetics in living cells. For this, we have created a single-mRNA biosensor that can be tethered to individual regulatory factors to directly visualize their impact on translation dynamics3. Using this biosensor, we provide evidence that human Argonaute2 (Ago2) shuts down translation by first downregulating translation initiation on the timescale of minutes. Later, over a period of hours, Ago2-tethered mRNAs multivalently interact with other endogenous miRISC machinery, enhancing their recruitment into P-bodies. More recently, we have begun to combine this technology with optogenetic clustering tools to better control the timing and reversibility of tethering. Our dream is to create “live cell test tubes,” where we can perform controlled single-molecule biochemistry experiments in a near-natural setting.
In the second half of the talk, I will discuss progress towards our long-term goal of imaging endogenous translation dynamics. To begin to achieve this, we performed multiple rounds of CRISPR engineering to create a homozygous knock-in human cell line harboring both RNA and protein tags in the endogenous MYH9 gene. MYH9 encodes the essential non-muscle actin cytoskeletal motor protein myosin-2A (myo-2A; 230 kDa), which assembles into homotypic bipolar filaments crucial for cell morphogenesis, migration, and division. Using our CRISPR’d cells, we can track single MYH9 mRNA in one color, single myo-2A translation sites in a second color, and mature myo-2A protein in a third color. We find evidence for MYH9 transcriptional and myo-2A translational bursts, local myo-2A synthesis at cellular sites of known actomyosin activity, and tunable coupling between transcription and translation. Interestingly, while myo-2A is important for cell division, we find that translation of its mRNA is repressed as cells enter mitosis, although there is a brief yet significant upregulation during prophase. Collectively, our data provide a revealing glimpse into the full complexity of the central dogma, one that underscores the hidden variables obscuring any simple characterization of transcription-translation coupling.
Morisaki, T. et al.
Real-time quantification of single RNA translation dynamics in living cells. Science 352:1425–1429, 2016.
Morisaki, T. & Stasevich, T. J.
Quantifying Single mRNA Translation Kinetics in Living Cells. Cold Spring Harb. Perspect. Biol. 10:a032078, 2018.
Cialek, C. A. et al.
Imaging translational control by Argonaute with single-molecule resolution in live cells. Nat. Commun. 13:3345, 2022.
Division of Transcriptomics, Medical Institute of Bioregulation
TEL: 092 (642) 4534