- For the generation of kymographs other software packages
than MetaMorph like the Zeiss software (Zen; Zeiss) or Fiji
(ImageJ,https://imagej.nih.gov/ij/[40, 41]) are available. - The gene encoding theλN-peptide carries three point muta-
tions [36]. These mutations increase the binding to the cog-
nate boxB stem-loops. The peptide is expressed ectopically in
theipslocus ofU. maydis. The expression of the RBP is driven
by the arabinose inducible promoter Pcrg1or the constitutively
active promoter Potef. Three GFP molecules are fused to the C-
terminus of theλN-peptide without a visible effect on RNA
binding. - Sixteen boxB stem-loops are integrated in the 3^0 UTR of the
cdc3mRNA. The stem-loops are integrated 100 bp down-
stream of the stop codon of Cdc3, followed by 600 bp
30 UTR sequence. In between each stem-loop a linker sequence
of 8 bp is used. The whole construct is integrated at the
endogenous locus by homologous recombination. - The switch to arabinose as single carbon source for the induc-
tion of the Pcrgresults in a slower growth rate. To obtain
comparable culture densities to the strains expressing the
RBP with the Potefpromoter the ratio of inoculation has to
be adjusted. - In comparison to strains grown in glucose containing media, a
delay in filament induction can be observed in strains growing
in media containing arabinose. This is due to arabinose as
single carbon source. - Measured velocities for moving mRNAs should be comparable
to the velocity of moving RBPs if they localize in identical
complexes. - Using camera chip binning 2 can improve signal-to-noise ratio
thus facilitating detection. - The 488 nm laser also bleaches mCherry. Therefore, laser
power has to be adjusted carefully. - Make sure that an increased number of integrated stem-loops
and therefore extended 3^0 UTR do not decrease mRNA
stability.
Acknowledgments
We thank lab members for critical comments on the manuscript as
well as Dr. Jeffrey A. Chao for plasmids and helpful advice on RNA
live imaging. Our research was in part financed by grants from the
German Science Foundation through DFG-FOR2333, DFG-
Fe448/8-1; DFG-Fe448/9-1, DFG-EXC1024 CEPLAS and
DFG-CRC1208.RNA Live Imaging inU. maydis 333