RNA Detection

3.6 RNA Live
Imaging Comparing
Different Heterologous
RBPs

1. In contrast to the already described boxB loops, the PP7 stem-
   loop consists of 25 nucleotides (nt) and the MS2 stem-loop has
   a size of 19 (nt). For binding of the MS2 coat protein both of
   the single stranded-adenosine (A) residues at the top of the
   stem and the bulged-A are essential (Fig.4a). For the PP7
   stem-loop (PBS) a similar bulged-A is needed for tightest
   binding of the RPB (Fig.4a).


2. The RBP deriving from the bacteriophageλbinds to the RNA
   stem-loops as a monomer (Fig.1c;[36]). Twenty-two amino
   acids of the RBP are sufficient for binding. Furthermore, three
   point mutations in theλN-peptide increase the binding to the
   cognate boxB stem-loops (λN*; [36]).


3. The coat proteins of MS2 and PP7 bind as dimers to their
   RNA-binding sites (Fig.1c;[28, 37]). In contrast to the
   PCP, the MCP is already expressed as a dimer which is then
   fused to different numbers of fluorescent proteins.


4. PCR amplify the PP7 or MS2 stem-loops (typically 12 or 24
   repeats, respectively) and insert it into the 3^0 UTR of the target
   (e.g.,cdc3)as described in Subheading3.1 (seeNotes 1and 14 ).


5. Generate a fusion of the cognate RBP fused to GFP multimers
   as described in Subheading3.1.

Fig. 3mRNA visualization with and without a NLS sequence. (a) Schematic representation showing the
difference between RNA live imaging using a RBP-FP with or without a nuclear localization sequence (NLS). (b)
Background adjusted fluorescence image of a strain expressingλN coupled to GFP andcdc3mRNA with boxB
stem-loops (left, scale bar 10μm). mRNA signal is marked byarrowheads, note the strong cytoplasmic
background. Kymograph corresponding to the indicated area (right) visualizes movement of the corresponding
signal (inverted images). (c) Background-adjusted fluorescence image of a strain expressingλN coupled to
GFP with an NLS andcdc3mRNA with 16 boxB stem-loops (left, scale bar 10μm).Arrowheadmarks motile
mRNA. Kymograph on the right visualizes movement of the corresponding signal (inverted images)

330 Sabrina Zander et al.