Reining in translation with optogenetics
Summary by Will Anderson: Kim, N. Y., Lee, S., Yu, J., Kim, N., Won, S. S., Park, H., & Heo, W. D. Optogenetic control of mRNA localization and translation in live cells. Nat Cell Biol 22, 341-352, doi:10.1038/s41556-020-0468-1 (2020).
Image credit: Dave Skinner, Wikimedia Commons
Much of a cell’s behavior and fate is determined by the localization and translation of its mRNA. Visualizing and experimentally manipulating these transcripts can reveal key insights about the mechanisms governing cell behavior. However, live mRNA visualization methods often offer less-than-ideal resolution, and existing translation-inhibiting approaches can be nonspecific and thus limited in their usefulness. In this paper, Kim et al.1 introduce a tool that can control localization and inhibit translation of mRNA molecules in live cells. The tool, called mRNA-light-activated reversible inactivation by assembled trap (mRNA-LARIAT), is an optogenetically controlled system that uses light to aggregate mRNA transcripts into inaccessible clusters, inhibiting their translation by blocking ribosomal access. Notably, this system is specific to chosen mRNA molecules, reversible in that the removal of light from the system will disperse the clusters, and spatially precise in that a researcher can use light to activate the system exactly where they want.
mRNA-LARIAT is based on a similar system, called “LARIAT”2. Among several iterations, the version of mRNA-LARIAT that affects endogenous, untagged transcripts consists of three components. The first component is the protein CIB1 fused to CaMKII-alpha, called “CIB1-MP”. Multiple copies of CIB1-MP will bind, creating a structure where CIB1 sticks out of a multimeric protein complex. The second component is the light-responsive protein Cry2, which upon exposure to blue light binds to CIB1 and other copies of itself. This Cry2-CIB1 binding is the basis for the clustering effect. Cry2 is fused to an anti-GFP nanobody. The third component is nuclease-dead RNA-binding Cas9 (dRCas9) fused to GFP. Cry2 binds to GFP-dRCas9 via its anti-GFP nanobody. dRCas9 itself will bind to specific mRNA transcripts when provided with guide RNAs that correspond to that transcript’s sequence. These guide RNAs can be designed by the user, granting the system its specificity. When these components are added to cells, the Cry2-anti-GFP nanobody / GFP-RCas9 complex will bind to specific transcripts. Upon exposure to blue light, the mRNA-containing complex will bind itself and CIB1-MP. This creates a cluster that is concentrated in the light-exposed area and difficult for ribosomes to access, thus inhibiting translation.
Kim et al. demonstrated the usefulness of mRNA-LARIAT by using it to investigate the role of beta-actin mRNA translation in fibroblast migration. Under normal circumstances, beta-actin protein appears at the leading edge of cell migration and is thought to play a role in fibroblast movement. The group used mRNA-LARIAT to inhibit the translation of beta-actin at the leading edge of the cell and found that cell migration was significantly inhibited. This highlights the critical role of newly synthesized beta-actin protein in fibroblast migration and demonstrates the usefulness of mRNA-LARIAT in investigating the role of specific mRNAs in the cell. The system’s specificity allowed researchers to target a beta-actin mRNA, and its spatial precision allowed them to inhibit its translation specifically at the leading edge of the fibroblast.
mRNA-LARIAT presents an exciting opportunity for researchers to investigate the effects of specific and precise mRNA translation inhibition on other transcripts in other systems.
References
1 Kim, N. Y. et al. Optogenetic control of mRNA localization and translation in live cells. Nat Cell Biol 22, 341-352, doi:10.1038/s41556-020-0468-1 (2020).
2 Lee, S. et al. Reversible protein inactivation by optogenetic trapping in cells. Nat Methods 11, 633-636, doi:10.1038/nmeth.2940 (2014).