RNA Detection

3.4 RNA Live
Imaging of Endosomal
mRNA Transport
in U. maydis

Shuttling mRNAs in hyphae ofU. maydiscan be also detected

using theλN system.

1. Fuse a modifiedλN* peptide to GFP multimers (either double
   or triple GFP can be used; Fig.2c and d;seeNote 7). The
   whole construct can be expressed either with a constitutively
   active promoter or an inducible promoter (Potef,or Pcrg, respec-
   tively; [33]).


2. For the generation of an mRNA construct, PCR amplify an
   array of boxB RNA stem-loops (typically 16 repeats) and insert
   it into the 3^0 UTR of the target mRNA (e.g.,cdc3;seeNote 8).


3. To analyze possible colocalization and comigration of the
   mRNA of interest (cdc3B^16 ) and cognate RBP (Rrm4 fused to
   mCherry) use high-speed dual-color microscopy (Fig.2e).


4. To this end cotransform theλN*-GFP plasmid and the gener-
   ated cdc3B^16 expression plasmid (λN system) into a strain
   expressing a functional RBP-FP fusion.


5. Comparably, colocalization experiments with an mRNA and
   the encoded protein can be performed.


6. For this purpose a strain can be used in which the GOI is fused
   to mCherry to visualize the protein localization and boxB
   stem-loops are integrated into the 3^0 UTR of the same con-
   struct. Here, the plasmid “Potef-mCherry-cdc3-16boxB-3^0
   UTR” can be used directly.


7. Coexpress theλN* peptide fused to a triple GFP and the fusion
   protein of Cdc3-mCherry to visualize and analyze the coloca-
   lization of both mRNA and encoded protein (Fig.2f).


8. After transformation ofU. maydis(seeSubheading3.2), inocu-
   late yeast-like cells in 3 mL of CM-glc in a glass tube and grow
   culture on rotation wheel for 24 h at 28C.


9. Inoculate a main-culture in a 250 mL baffled flask with 30 mL
   CM-ara (1% w/v f.c.) medium (dilute the 24 h culture to
   ~1:1000) to induce the Pcrgpromoter or CM-glc (1% w/v f.c.)
   medium (dilute the 24 h culture to ~1:2000) if the expression is
   controlled by Potefpromoter. Incubate cells 15–20 h shaking
   with 200 rpm at 28C(seeNote 9).


10. For induction of hyphal growth adjust OD 600 to 0.5 in 20 mL
    and shift the cells to NM-ara or NM-glc media by centrifuga-
    tion at 3500gfor 5 min (seeNote 10). Wash once in the
    same media and resuspend cells in 20 mL. Continue shaking at
    200 rpm at 28C for 7–8 h (seedilute the 24 h culture to ~).


11. To prepare microscopy followsteps 7and 8 in Subheading3.3.


12. To visualize moving mRNA choose appropriate illumination
    for the excitation of chosen fluorophores (GFP or mCherry).

328 Sabrina Zander et al.