A rapid inducible RNA decay system reveals fast mRNA decay in P-bodies.

RNA decay is vital for regulating mRNA abundance and gene expression. Existing technologies lack the spatiotemporal precision or transcript specificity to capture the stochastic and transient decay process. We devise a general strategy to inducibly recruit protein factors to modulate target RNA metabolism. Specifically, we introduce a Rapid Inducible Decay of RNA (RIDR) technology to degrade target mRNAs within minutes. The fast and synchronous induction enables direct visualization of mRNA decay dynamics in cells. Applying RIDR to endogenous ACTB mRNA reveals rapid formation and dissolution of RNA granules in pre-existing P-bodies. Time-resolved RNA distribution measurements demonstrate rapid RNA decay inside P-bodies, which is further supported by knocking down P-body constituent proteins. Light and oxidative stress modulate P-body behavior, potentially reconciling the contradictory literature about P-body function. This study reveals compartmentalized RNA decay kinetics, establishing RIDR as a pivotal tool for exploring the spatiotemporal RNA metabolism in cells.

Imaging spatiotemporal translation regulation in vivo.

Translation is regulated spatiotemporally to direct protein synthesis when and where it is needed. RNA localization and local translation have been observed in various subcellular compartments, allowing cells to rapidly and finely adjust their proteome post-transcriptionally. Local translation on membrane-bound organelles is important to efficiently synthesize proteins targeted to the organelles. Protein-RNA phase condensates restrict RNA spatially in membraneless organelles and play essential roles in translation regulation and RNA metabolism. In addition, the temporal translation kinetics not only determine the amount of protein produced, but also serve as an important checkpoint for the quality of ribosomes, mRNAs, and nascent proteins. Translation imaging provides a unique capability to study these fundamental processes in the native environment. Recent breakthroughs in imaging enabled real-time visualization of translation of single mRNAs, making it possible to determine the spatial distribution and key biochemical parameters of in vivo translation dynamics. Here we reviewed the recent advances in translation imaging methods and their applications to study spatiotemporal translation regulation in vivo.

A Rapid Inducible RNA Decay system reveals fast mRNA decay in P-bodies.

RNA decay plays a crucial role in regulating mRNA abundance and gene expression. Modulation of RNA degradation is imperative to investigate an RNA's function. However, information regarding where and how RNA decay occurs remains scarce, partially because existing technologies fail to initiate RNA decay with the spatiotemporal precision or transcript specificity required to capture this stochastic and transient process. Here, we devised a general method that employs inducible tethering of regulatory protein factors to target RNAs and modulate their metabolism. Specifically, we established a Rapid Inducible Decay of RNA (RIDR) technology to degrade target mRNA within minutes. The fast and synchronous induction enabled direct visualization of mRNA decay dynamics in cells with spatiotemporal precision previously unattainable. When applying RIDR to endogenous ACTB mRNA, we observed rapid formation and disappearance of RNA granules, which coincided with pre-existing processing bodies (P-bodies). We measured the time-resolved RNA distribution in P-bodies and cytoplasm after induction, and compared different models of P-body function. We determined that mRNAs rapidly decayed in P-bodies upon induction. Additionally, we validated the functional role of P-bodies by knocking down specific a P-body constituent protein and RNA degradation enzyme. This study determined compartmentalized RNA decay kinetics for the first time. Together, RIDR provides a valuable and generalizable tool to study the spatial and temporal RNA metabolism in cells.

One Message, Many Translations: Heterogeneity Revealed with Multicolor Imaging.

mRNA translation and its regulation shape the human proteome. Lyon et al. (2019) and Boersma et al. (2019) introduce new orthogonal peptide-tagging systems to study translation in multiple reading frames. Both groups discovered diverse translation behavior of single mRNAs derived from the same genes.