Rrm4-containing mRNPs with the endosomal surface by a lipid-
binding FYVE domain (Fig.1a;[9, 18]). In essence, microtubule-
dependent transport of early endosomes is not only needed for
endocytic sorting to the vacuole but also for long-distance trans-
port of mRNPs and even of whole organelles such as peroxisomes
[7, 20].
To study mRNA localization fluorescent in situ hybridization is
a very powerful technique that has been perfected down to the
single molecule level. However, in order to study dynamic pro-
cesses RNA live imaging is the method of choice [21]. RNA live
imaging was first applied studying mating type switching inSaccha-
romyces cerevisiae[22]. Inserting binding sites for the MS2 phage
coat protein in the 3^0 UTR of mating type switch repressorASH1
mRNA enabled live recording of mRNA transport [22]. The strat-
egy of inserting binding sites in the 3^0 UTR was also used in other
systems to study, for example, the influence of RBP targeting on
decay or to visualize translation in vivo [23–25]. The system has to
be applied carefully because of an accumulation of intermediates of
mRNA degradation containing MS2 binding sites [23, 26, 27].
However, processively moving mRNAs are less likely to be affected
by such problems.
A related example is the coat protein of bacteriophage PP7
(PCP), an RNA phage ofPseudomonas aeruginosa,anditscognate
RNA stem-loops (PBS; [28]). Importantly, the MS2 and PP7 coat
proteins share only 15% sequence identity and their cognate RNA
stem-loops differ from each other [28]. Thus, they can be simulta-
neously used to detect two mRNAs without interference. Another
in vivo RNA labeling system is derived from the anti-terminator
protein N from phageλ. The first 22 residues of theλNpeptide
are sufficient for recognition of its boxB RNA hairpins [29]. Thus, a
small truncated version is used for RNA live imaging (Fig.1c;[11,
30 ]). All phage proteins are fused at their C-terminus to fluorescence
proteins and no alterations in RNA-binding have been described.
In this chapter we report on the use of RNA live imaging
during microtubule-dependent transport inU. maydis. The grow-
ing hyphae of this organism provide a highly organized stereotypic
microtubule cytoskeleton ideal to study transport of motile
mRNAs and their interactions with mRNP composing protein
molecules in vivo.We describe the experimental design and the
strategy of constructing such RNA tracking systems. This includes
both multimerization of the binding sites and the fluorescence
proteins (FPs) to increase sensitivity. Furthermore, it is advisable
to fine-tune the expression of the RBP-FP fusion to obtain optimal
signal-to-noise ratios. Alternatively, we present the use of a nuclear
localization signal (NLS) for the RBP-FP fusion. Thereby,
unbound RBP-FPs are targeted from the cytoplasm to the nucleus,
also improving the signal-to-noise ratio. Finally, we compare differ-
ent phage proteins as well as the quantification of the results like
velocity, amount and distances traveled.
RNA Live Imaging inU. maydis 321