RNA Detection

Chapter 22

Real-Time Fluorescence Imaging of Single-Molecule

Endogenous Noncoding RNA in Living Cells

Hideaki Yoshimura and Takeaki Ozawa

Abstract

Visualizing RNA in living cells is increasingly important to facilitate accumulation of knowledge about the
relation between specific RNA dynamics and physiological events. Single-molecule fluorescence imaging of
target RNAs is an excellent approach to analyzing intracellular RNA motion, but it requires special
techniques for probe design and microscope setup. Herein, we present a principle and protocol of an
RNA visualization probe based on an RNA binding protein of the Pumilio homology domain (PUM-HD).
We also describe the setup and operation of a microscope, and introduce an application to visualize
telomeric repeats-containing RNA with telomeres and a telomere-related protein: hnRNPA1. This imaging
technique is applicable to visualization of different RNAs, especially including repetitive sequences, in living
cells.

KeywordsFluorescence imaging, Single molecule, Repetitive RNA, PUM-HD

1 Introduction

Generally, RNA has been considered merely as a messenger of

genetic information. However, the importance of intracellular

transportation and localization of mRNA for physiological phe-

nomena has been recognized recently [1, 2]. Furthermore, various

noncoding RNAs have been implicated in many biological events,

especially controlling chromatin states [3, 4]. Noncoding RNAs

show such unique dynamics and localization in their functions as

localization on particular loci on chromosomes and formation of

particle-like structures called speckles [5, 6]. Consequently, the

implication of dynamics and localization of RNAs in their functions

promotes them as a target of interest in the bioimaging field.

Nevertheless, live cell imaging studies of RNAs have not performed

intensively, in contrast to live cell fluorescence imaging studies of

various proteins, which have provided a great deal of information

for elucidating the mechanisms of biological phenomena. A major

reason for the delay of development on RNA imaging technique is

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_22,©Springer Science+Business Media LLC 2018

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