Enhanced single RNA imaging reveals dynamic gene expression in live animals

Abstract

Imaging endogenous mRNAs in live animals is technically challenging. Here we describe an MS2-based signal amplification with the Suntag system that enables live-cell RNA imaging of high temporal resolution and with 8xMS2 stem-loops, which overcomes the obstacle of inserting a 1,300 nt 24xMS2 into the genome for the imaging of endogenous mRNAs. Using this tool, we were able to image the activation of gene expression and the dynamics of endogenous mRNAs in the epidermis of live C. elegans.

An adapted MS2-MCP system to visualize endogenous cytoplasmic mRNA with live imaging in Caenorhabditis elegans

“…For example, our method offers the flexibility to combine the MS2-MCP system with the SunTag system to visualize translation within the same mRNA. This is different from the recently reported MS2-based Signal Amplification with SunTag System (MASS) system, which uses the SunTag to amplify mRNA detection and precludes monitoring translation [ 49 ]. We note that the sensitivity of the MASS system can induce a significant number of false positives, likely resulting from signal overamplification inherent to the system itself and not dependent on the binding to an mRNA.…”

Recent advances in methods for live-cell RNA imaging

“…Besides these approaches, recent research has combined the SunTag technology 16 with the RNA hairpin methods to further increase the signal-tonoise ratio for detecting single RNA molecules. 17,18 Since its first invention, there have been multiple improvements on the MS2 tag. Because of its highly repetitive nature, the MS2 tag is prone to deletion and recombination during bacterial amplification and viral transduction.…”

“…Besides these approaches, recent research has combined the SunTag technology 16 with the RNA hairpin methods to further increase the signal-to-noise ratio for detecting single RNA molecules. 17,18…”

Clearing the slate: RNA turnover to enable cell state switching?

“…A functional test of the clearing the slate view would be to take a sharp state transition with a strong RNA turnover component, then precisely stage the turnover event with respect to a change in the functional state of the cells, such as a loss of potential for cells to return to the initial state (‘commitment’). This might involve, for example, imaging the turnover event using a fluorescent RNA-tagging approach ( Hu et al, 2023 ) in vivo and administering a treatment promoting the original cell state at varying times in the transition. At which point relative to RNA turnover does the cell state functionally change?…”