RNA Detection

Chapter 17

Live Imaging of Nuclear RNPs in Mammalian Complex

Tissue with ECHO-liveFISH

Dan Ohtan Wang

Abstract

Multiplex RNA detection with fluorescence microscopy offers high spatial and temporal resolution required
for addressing complex behaviors of RNA in living cells. Using chemically engineered linear oligonucleotide
probes that emit fluorescence upon hybridization to target RNA, we have devised an imaging method
suitable for studies of the dynamic regulation of nuclear RNPs, an important and yet poorly understood
cellular pathway of gene expression. This new method labels specific sequences of RNA components in
RNPs and thus avoids overexpression of fluorescent marker proteins that may result in entangled experi-
mental results. Using this method, we observe in living brain tissue spatially constrained nuclear RNA foci
under dynamic regulation in response to cellular transcriptional activity with individual cell heterogeneity.

Key wordsExciton, Fluorescence microscopy, RNA labeling, Thiazole orange, In vivo

electroporation

1 Introduction

In the nucleus, local concentrations of specific RNA and proteins

(RNPs: ribonucleoprotein complex) have long been observed at

subnuclear organelles such as nucleoli, nuclear speckles, para-

speckles, Cajal bodies, histone locus bodies, and promyelocytic

leukemia bodies. These functionally specialized regions are promi-

nent under light and electron microscopy and have shown close

relation to nuclear structures and organization [1–4]. Concen-

trated nuclear RNPs have also been observed at pathological ribo-

nuclear foci that contain abnormally lengthened repeat sequences

in degenerative human muscular and nervous systems [5, 6]. Fur-

thermore, nuclear-retained noncoding RNA species have emerged

to be critical regulators of chromatin structure, transcription activ-

ity, RNA processing and modification, gene expression dynamics,

and nuclear compartmentalization [7–10]. These discoveries

impose new demands for a suitable imaging method that labels

specific RNA species and effectively monitors nuclear RNPs with

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_17,©Springer Science+Business Media LLC 2018

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