the difficulty of labeling target RNAs in living cells. RNA cannot be
fused with fluorescent or tag proteins through genetic engineering.
A possible approach to label target RNAs in living cells is to use
an RNA binding protein that specifically addresses the target RNAs.
In the simplest design, fusing the RNA binding protein to a fluo-
rescent protein such as enhanced green fluorescent protein (EGFP)
produces a fluorescent probe to label the target RNA. To generalize
this approach, the RNA binding protein must have the following
features: selective recognition of substantial length of RNA
sequence to assure the specificity to the target RNA, capability of
tailor-made design for variety of RNA sequences, and sufficient
affinity to the target RNA sequence for labeling in living cells.
Pumilio homology domain (PUM-HD) of human PUMILIO 1 is
a promising candidate for RNA binding proteins to be used in RNA
probes. The PUM-HD consists of eight repeated motifs, each of
which binds specifically to an RNA base through formation of
hydrogen bonds and van der Waals interaction [7]. Then an 8-
base sequence of RNA is captured completely by a PUM-HD. A
noteworthy feature of PUM-HD is its design flexibility [8].
Reported crystal structures of PUM-HD allow the production of
tailor-made design of PUM-HD mutants to recognize particular
RNA sequences. We previously succeeded in labeling endogenous
β-actin mRNA in living cells using a PUM-HD based probe [9–11]
consisting of two subunits including different PUM-HD mutants
targeting particular sites in the 3^0 UTR ofβactin mRNA and split
fluorescent protein fragments. Upon these two subunits attached
on aβactin mRNA at a time, fluorescent protein reconstitution is
induced: the probe fluoresces. Consequently,βactin mRNA can be
visualized selectively in living cells using fluorescence microscopy.
Using a PUM-HD based probe, we recently visualized single-
molecule motion of telomeric repeat-containing RNA (TERRA),
which is a noncoding RNA transcribed from telomeres, and which
therefore contains a telomeric repeat sequence region [12]. In
addition to the biological importance of TERRA and telomeres,
RNAs including repetitive sequences have arisen as targets of inter-
est in many biological fields. For instance, genes containing CAG
repeats are known to introduce diseases [13]. The origin of the
diseases is suspected not only as the translated proteins but also
RNAs. In addition, several repetitive sequences provide markers for
RNA splicing or translation [14, 15]. About 20% of the total
genome is now regarded as regions of repetitive sequences. There-
fore, RNAs including repetitive sequences are anticipated as impor-
tant targets to elucidate functions of the RNAs and mechanisms of
physiological events and diseases.
This chapter introduces the principles and methods of simulta-
neous visualization of TERRA, telomeres, and a telomere related
protein hnRNPA1 in living cells [12]. We designed a fluorescent
probe that targets the telomeric repeat region to label using a338 Hideaki Yoshimura and Takeaki Ozawa