Yoshio Umezawa

Genetically-Encoded Fluorescent Probes for Imaging Endogenous mRNA in Living Cells

Localization of mRNAs plays pivotal roles in different cell types, including neurons and the cells in the developing stages. To visualize the dynamic movements of mRNAs in living cells, many methods have been emerged in the past decade. However, it has not been realized to visualize endogenous mRNAs with genetically encoded fluorescent probes. We recently developed fluorescent protein-based RNA probes for characterizing the localization and dynamics of mRNAs in single living cells. The probes consist of two RNA-binding domains of human PUMILIO1, each connected with split fragments of a fluorescent protein capable of reconstitution upon binding to a target mRNA. The probes are modified to specifically recognize a 16-base sequence of an mRNA of interest and to target into organelles by means of a short signal peptide. We have shown that ND6 mRNA is concentrated particularly on mitochondrial DNA (mtDNA) and movement of the mRNA is restricted in mitochondria. The probes provide a general means to study spatial and temporal mRNA localization and dynamics in intracellular compartments in living cells.

Imaging of endogenous RNA using genetically encoded probes.

Imaging of RNAs in single cells revealed their localized transcription and specific function. Such information cannot be obtained from bulk measurements. This unit contains a protocol of an imaging method capable of visualizing endogenous RNAs bound to genetically encoded fluorescent probes in single living cells. The protocol includes methods of design and construction of the probes, their characterization, and imaging a target RNA in living cells. The methods for RNA imaging are generally applicable to many kinds of RNAs and may allow for elucidating novel functions of localized RNAs and understanding their dynamics in living cells. Curr. Protoc. Chem. Biol. 3:27‐37