Symmetry-breaking malachite green as a near-infrared light-activated fluorogenic photosensitizer for RNA proximity labeling.

Cellular RNA is asymmetrically distributed in cells and the regulation of RNA localization is crucial for proper cellular functions. However, limited chemical tools are available to capture dynamic RNA localization in complex biological systems with high spatiotemporal resolution. Here, we developed a new method for RNA proximity labeling activated by near-infrared (NIR) light, which holds the potential for deep penetration. Our method, termed FAP-seq, utilizes a genetically encoded fluorogen activating protein (FAP) that selectively binds to a set of substrates known as malachite green (MG). FAP binding restricts the rotation of MG and rapidly activates its fluorescence in a wash-free manner. By introducing a monoiodo modification to MG, we created a photosensitizer (MG-HI) with the highest singlet oxygen generation ability among various MG derivatives, enabling both protein and RNA proximity labeling in live cells. New insights are provided in the transcriptome analysis with FAP-seq, while a deeper understanding of the symmetry-breaking structural arrangement of FAP-MG-HI was obtained through molecular dynamics simulations. Overall, our wash-free and NIR light-inducible RNA proximity labeling method (FAP-seq) offers a powerful and versatile approach for investigating complex mechanisms underlying RNA-related biological processes.

Differential subcellular localization of ASPM RNA and protein.

RNAs encoding some centrosomal components are trafficked to the organelle during mitosis. Some RNAs, including ASPM , localize to the centrosome co-translationally. However, the relative position of these RNAs and their protein after trafficking to centrosomes remained unclear. We find that mislocalization of ASPM RNA from the centrosome does not affect the localization of ASPM protein. Further, ASPM RNA and ASPM protein reside in two physically close yet distinct subcellular spaces, with ASPM RNA on the astral side of the centrosome and ASPM protein on the spindle side. This suggests subtly distinct locations of ASPM RNA translation and ASPM protein function.