1. Introduction
mRNA localisation is a widespread phenomenon that occurs in
organisms as diverse as yeast and humans. It contributes to many
cellular processes including the establishment of intracellular
asymmetry, directed cell motility, asymmetric cell division, and
possibly also synaptic plasticity (1).
The molecular mechanisms of mRNA localisation have been
intensively studied in Drosophila, because the formation of the
body axes of this organism depends on the correct localisation of
several maternal mRNA species to specific regions of the oocyte.
Localisation of oskar (osk) mRNA to the posterior pole of the
Jeffrey E. Gerst (ed.), RNA Detection and Visualization: Methods and Protocols, Methods in Molecular Biology, vol. 714,
DOI 10.1007/978-1-61779-005-8_17, Springer Science+Business Media, LLC 2011
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oocyte specifies the site of the pole plasm assembly, and thus
where the abdomen and germ cells develop in the embryo (24).
nanos (nos) mRNA is recruited to the pole plasm downstream of
oskar mRNA and functions as the posterior determinant to specify
the formation of the abdomen (5). Localisation of bicoid (bcd)
mRNA to the anterior pole is essential for proper patterning of
the head and the thorax (6, 7). Finally, the localisation of gurken
(grk) mRNA to the anterior-dorsal corner of the oocyte specifies
the dorso-ventral axis of the future embryo (8).
Initially, the localisation of different mRNAs was studied using
in situ hybridisation techniques. This method visualises mRNA
localisation in fixed oocytes, and therefore provides a picture of
the steady-state distribution of mRNA molecules, but suffers from
several limitations. Firstly, the hybridisation technique is not ideal
for studying the localisation of mRNA during late stages of oogenesis, since the deposition of the vitelline membrane makes the
oocyte impermeable to in situ probes. Secondly, the method does
not provide any information about the dynamics of mRNA movement, and this information is crucial for a complete understanding
of the mechanisms of mRNA transport. A great deal of effort has
therefore been directed towards developing a system that allows
visualisation of mRNA movements in living oocytes.
1.1. Methods
for Visualisation
of mRNA Localisation
In Vivo
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Fig.1. Methods for visualising mRNAs invivo. (a) Injection of fluorescently labelled mRNA. The mRNA is transcribed invitro
in the presence of a fluorescently labelled nucleotides. These labelled transcripts are injected into the living cells. (b) Injection
of molecular beacons. Beacons are small oligonucleotides with a fluorophore attached to one end and a fluorescence
quencher attached to the other. Upon hybridisation to the target mRNA, the beacon unfolds, thereby separating the quencher
from the fluorophore which can then fluorescence. (c) GFP-tagging of an RNA-binding protein. An endogenous RNA-binding
protein that is known to be specific for the mRNA of interest is tagged with GFP. (d) RNA-MS2/MS2CP-GFP tagging system.
The 19-nucleotide MS2-binding sequence is inserted into the mRNA of interest. This mRNA is co-expressed with the NLSMS2CP-GFP construct. MS2CP-GFP-NLS binds to the MS2-binding sequence and allows visualisation of the target mRNA.
Unbound MS2CP-GFP-NLS is confined to the nuclei due to the presence of the NLS signal. (e) Secondary structure of the
19-nucleotide MS2-binding RNA sequence. The structure was predicted using the mfold program (44).
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Fig.2. Visualisation of osk mRNA with the RNA-MS2/MS2CP-GFP system. (a, d, g) Egg chambers expressing both oskMS2
and MS2CP-GFP. (a) oskMS2 is localised to the middle of the stage 8 oocyte. (d, g) oskMS2 mRNA localises to the posterior of the oocyte from stage 9 onwards. The pattern of localisation of oskMS2 mRNA is indistinguishable from that of
the endogenous osk mRNA at all stages of oogenesis (b, e, h). (c, f, i) When MS2CP-GFP is expressed on its own, the GFP
signal remains confined to the nuclei of the nurse and follicle cells. Scale bars are 50mm. (j) Overlay of six frames from
a high magnification time-lapse movie of an oskMS2/MS2CP-GFP expressing oocyte to show the movements of osk
mRNA particles. Examples of individual tracks are highlighted by coloured rectangles, and are shown in separate boxes
with higher magnification (l, m). (k) Co-visualisation of oskMS2/MS2CP-GFP particles and Tral-RFP in a Drosophila
oocyte. The red and green channels were imaged sequentially. (l) Example of the fast-moving particle track highlighted
by the green rectangle in (j). (m) Example of a stationary particle highlighted by the orange rectangle in (j).
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2. Materials
2.1. Preparation
of Drosophila Oocytes
for Live Cell Imaging
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3. Methods
The major steps in the development of mRNA-MS2/MS2-FP
labelling system for the visualisation of mRNA localisation in
Drosophila oocytes are outlined in Fig.3.
3.1. C
loning
3.1.1. Generation
of mRNA-MS2 Construct
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3.1.3. Creation
of Transgenic Flies
Expressing mRNA-MS2
and MS2CP-FP Constructs
3.2. Preparation
of Samples for Live
Imaging
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The data from FRAP and FLIP experiments is analysed by measuring the changes in fluorescence intensities of bleached and
unbleached regions of the oocyte over time. There are different
software packages that allow such analyses, such as Metamorph.
The resulting data can be used to estimate the dynamics of mRNAMS2 in the analysed region of the cell. With such experiments, it
is important to correct for the photobleaching due to image acquisition. This can be done by measuring the changes in fluorescence
intensity in a cell that is in the same field of view, but was not subjected to bleaching (for example a follicle cell nucleus or a neighbouring oocyte).
For the analysis of high-resolution movies, it is often desirable to obtain quantitative information about the parameters of
mRNA particle movement, and this requires the tracking of many
individual fluorescent particles over time. It is important to track
a statistically sufficient number of particle trajectories to be able
to draw significant conclusions from these data. In addition, it is
important to analyse a sufficient number of oocytes per genotype
in order to account for the variation between oocytes. The exact
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number required for the analysis will depend on the quality of the
data and on the type of conclusions one wishes to draw.
There are a number of different software packages available
for the automatic tracking of fluorescent particles. These programs are worth investigating to see whether they are capable of
detecting all the particles of interest in a particular setting, and
ignore other fluorescent or autofluorescent objects (e.g. yolk and
vesicles). All those we have tested on oskar mRNA failed to track
all of the particles because of the high particle density and the difficulty in distinguishing between fast moving particles that pass
close to each other.
In many cases, the fluorescence of RNA-MS2/MS2CP-GFP
particles is too low, while the background fluorescence of the
ooplasm too high to use these types of software. In such cases,
one has to use manual tracking algorithms. One software package
that can allow this is MetaMorph Premier Offline (using Track
points application; [Molecular Devices]). This software allows
manual tracking of all the particles of interest and then logs the
coordinates of each data point into a separate file. These data can
then be used for the determination of parameters, such as particle
speed, direction of movement, and length of movement. Analysis
of these data can be performed in MatLab or Microsoft Excel.
When performing manual tracking, it is important to track all of
the detectable particles in the cell, in order to avoid introducing
of biases into the data.
4. Notes
1. In cases where no movement of RNA particles is observed,
one should check whether the oocyte is still alive by acquiring
images every 10s to see whether there are any cytoplasmic
movements. In young oocytes, there are noticeable shortrange movements of limited amounts of cytoplasm called
cytoplasmic seething. In older oocytes, the entire ooplasm
moves in circular fashion around the oocyte a phenomenon
called cytoplasmic streaming. The presence of these movements indicates that the oocyte is viable.
Acknowledgements
K.B. was supported by the Darwin Trust of Edinburgh. D. St J. was
supported by a Wellcome Trust Principal Research Fellowship.
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