RNA Detection

Use the 63or the 100objective and use a laser power

(100 mW) of 20–30%.

13. Record movies with 150 frames and 150 ms exposure time (see
    Note 5).


14. Use the camera in stream mode to visualize movement and
    ensure the fastest acquisition.


15. After recording moving mRNAs, convert the movie into a
    kymograph using the software package MetaMorph (seeNote
    6 ). Comparable to the analysis of the movement of Rrm4
    signals (seeSubheading3.3), the velocity and the distance can
    be measured with the software package MetaMorph (seeNote
    11 ).


16. For the simultaneous detection of mRNA and protein a two-
    channel imager is used. The imager splits green and red emis-
    sion lights and detects them on separate regions of the CCD
    camera chip. Use a 100Plan-Neofluar (NA 1.3) objective
    with the respective laser lines for imaging (seeNote 12).


17. To ensure minimal photobleaching first determine the minimal
    laser power to detect the signal for each fluorophore (seeNote
    13 ). To decrease photobleaching alternating laser excitation
    (msAlex) together with DualView technology can be applied
    [31].

3.5 RNA Live
Imaging Comparing
Heterologous RBP with
and without NLS

Signal-to-noise ratio during RNA live imaging can be improved by

a fusion of the RBP to a NLS (Fig.3a).

1. PCR amplify theλN as described for PP7 coat protein in
   Subheading3.1. Fuse it in frame to GFP multimers and an
   NLS sequence. Alternatively, the plasmid “Potef-λN-3xGfp-
   NLS” can be used directly.


2. The desired plasmid is transformed using a strain (seeSubhead-
   ing3.2), which harbors 16 boxB stem-loops integrated into
   the 3^0 UTR of thecdc3mRNA (Fig.3b and c).


3. The NLS ensures the targeting of the unbound RBPs into the
   nucleus and therefore decrease the cytoplasmic background.


4. Perform microscopic analysis as described above (Subheading
   3.4,steps 8– 14 ).


5. Adjust the contrast of the recorded movies with available soft-
   ware packages (here: MetaMorph, Version 7) to visualize
   moving mRNAs (Fig.3b and c).


6. Analyze the movement of mRNAs using kymographs. Because
   of the lower cytoplasmic background using an RBP with an
   NLS, moving mRNA signals can be visualized over extended
   periods of time (Fig.3b and c, right).

RNA Live Imaging inU. maydis 329