- sCMOS camera (e.g., ORCA Flash 4.0 V2; Hamamatsu Pho-
tonics K.K.). - MetaMorph software (Molecular Devices).
- 488 nm laser (e.g., Coherent Sapphire 488 LP).
- 561 nm laser (e.g., Coherent Sapphire 561 LP).
- 647 nm laser (e.g., CUBE; Coherent Inc.).
- Optical materials (Sigma Koki Co. Ltd.,seeFig. 3).
Convex lenses off¼10 and 200 for beam expanders,f¼ 50
and 250 for relay lenses. - Optical filters (Semrock Inc.).
FF662-FDi01 for split iRFP fluorescence and shorter ones.
FF580-FDi01 for split TMR and GFP fluorescence.
FF01-697/58 for iRFP fluorescence bandpass filter.
FF01-609/54 for TMR fluorescence bandpass filter.
FF03-525/50 for GFP fluorescence bandpass filter.
3 Methods
3.1 Design of PUM-
HD-Based RNA Probes
In visualization of TERRA, a PUM-HD-based RNA recognition
method and a split fluorescent protein reconstitution technique
were adopted to create the TERRA probe. Before introducing
details of the methods and protocols of sample preparation and
observation of TERRA in living cells, we herein describe the prin-
ciples of RNA recognition by PUM-HD, split fluorescent protein
fragments reconstitution technique, and the probe design.- PUM-HD is an RNA binding domain of human PUMILIO1,
an RNA binding protein that associates with 3^0 UTR of mRNAs
to control gene translation in collaboration with other proteins
such as eIF4 [16]. Hall et al. reported the crystal structure of
PUM-HD (Fig.1a)[7], and demonstrated that PUM-HD
consists of eight repeated motifs (repeat 1–8). In each motif,
three amino acids form hydrogen bonds and van der Waals
interaction specifically to a single RNA base. Actually, PUM-
HD completely recognizes an 8-base RNA sequence of
UGUAYAUA (Y is C or U) (Fig.1b and c). - Although no motifs that would recognize a cytosine base are
present in the wild-type PUM-HD, a study using screening on
a random mutagenesis method generates a motif that recog-
nizes a cytosine base selectively [17]. - Based on information from the crystal structure, PUM-HD can
be modified e.g., by site directed PCR mutagenesis to recog-
nize 8-base RNA sequences that are different from the original
one by inducing site-specific substitution of particular amino
340 Hideaki Yoshimura and Takeaki Ozawa