Looking at mRNA and proteins in cellswhole organisms

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Looking at mRNA and proteins in cells/whole
organisms -over the last 30 years or so,
numerous techniques have been developed to
visualize RNA and protein distributions in whole
organisms or tissues, and such approaches have
made an enormous contribution to understanding
gene function RNA in situ hybridization - in
situ hybridization relies on the hybridization
of a labelled single- stranded DNA or RNA probe
to a target mRNA in a tissue -early approaches
(which are still used in certain cases) used
radiolabelled probes (for example 35S or 3H) and
autoradiography (for example, by coating slide
in nuclear tracking emulsion) to detect mRNA
distribution in sectioned tissues or
organisms -use of sections is laborious, and
reconstruction with multiple sections
is required to obtain information on the 3-D
distribution of a transcript -in addition,
spatial resolution is impaired by the track of
the decaying particle emitted from
radioactive probe -in recent years,
non-radioactive detection methods have emerged
that allow visualization of mRNA in unsectioned
samples, thus allowing the immediate
visualization of 3-D transcript distribution
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Commonly used methods for labelling nucleic acid
probes non-radioactively -fluoresceinisothiocya
nate (FITC), detected by fluorescence or with
anti-FITC antibody -biotin, detected with
labelled streptavidin -digoxigenin (DIG),
detected with anti-DIG antibody -DIG-UTP is
probably the most widely used label these days
and we will be using it in the lab to look at
Drac1 transcripts in embryos -DIG is derived
from the steroid digoxin which is found
in Digitalis plants -features that make DIG a
good label -high-affinity antibodies against
DIG available -because DIG is only found in
Digitalis plants, no background problems with
antibody -DIG readily coupled to nucleotides
such as dUTP or UTP -DIG-UTP can be
incorporated into an antisense riboprobe (i.e.
RNA probe complementary to mRNA) using an in
vitro transcription reaction
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-DIG-labelled RNA probe that has hybridized to
mRNA transcripts in the sample can be detected
with alkaline phosphatase (AP)-conjugated
anti-DIG antibodies (note other labels can be
attached to anti-DIG antibodies) -two
colourless substrates for AP are then used to
visualize the AP-conjugated antibody -BCIP/X-phos
phate is oxidized by AP to a blue product by
release of a phosphate group -NBT is reduced to
diformazan -the two reaction products form a
water insoluble dark blue to brownish
precipitate
-DIG system is broadly applicable...
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High resolution fluorescent RNA in
situ hybridization -recent studies have lead to
the development of high resolution RNA in situ
techniques in which the subcellular position of
mRNA transcripts can be observed -the
DIG-labeling/AP technique is fine for looking at
overall distribution of a mRNA, but not for
subcellular localization -in the technique of
tyramide signal amplification (TSA) the probe is
still labeled with DIG or biotin/ steptavidin,
but the detection method is altered -the
anti-DIG antibodies or streptavidin are
conjugated with the enzyme horse
radish peroxidase (HRP) -when fluorescently
labeled tyramides are added, they are acted upon
by HRP to produce tyramide radicals that react
covalently with proteins at the site and
accumulate to cause signal amplification
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-mRNA labeled with Cyanine-3 tyramides -nuclear
envelope labeled with Alexafluor488- Wheat Germ
Agglutinin
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Observing mRNA localization in live
tissues -mRNA localization is an important
regulatory mechanism in gene expression -for
example, some mRNAs contain 3 UTR sequences
interacting with cytoskeletal motors that direct
them to be localized to a specific intracellular
location prior to translation this ensures that
the protein product is synthesized right at the
place where it is required -recently developed
techniques allow the visualization of mRNA
localization in live cells 3 main
approaches (i) in vivo hybridization of
fluorescent oligonucleotides (ii)
microinjection of fluorescent RNAs into living
cells (iii) detection with fluorescent
RNA-binding proteins In vivo
hybridization -the movement of poly(A) RNA from
the nucleus to the cytoplasm has been followed by
in vivo hybridization of fluorescein labelled
oligo(dT) to the poly(A) tail -studies have also
been done in which the migration of specific
mRNAs is followed in the cell using specific
oligonucleotide probes
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Microinjection of flourescent RNAs into living
cells -the first approach used to study the
localization of mRNA transcripts in living cells
involved the injection of fluorescently labelled
versions of the RNA of interest into cells -this
approach continues to be used, and has recently
proven effective for looking at mRNA movement in
whole Drosophila embryos -a full-length cDNA
corresponding to the RNA of interest is
transcribed from plasmid in an in
vitro transcription reaction to produce a 5
capped mRNA -recall that eukaryotic transcripts
are capped at the 5 end by a terminal guanine
attached in reverse orientation through
formation of 5-5 triphosphate linkage as
follows Gppp 5 5
pppApNpNp.

5-5 ---------gt
GpppApNpNp pp p -in all eukaryotes, this
terminal guanine is methylated at the 7
position -CAP increases the stability of the
mRNA and may be required for correct processing
of transcript (translation, etc.) -the CAP is
mimicked by including a CAP analog in the in
vitro transcription reaction 7mG(5)pppG
-fluorescent label is incorporated by including
labelled UTPs such as AlexaFluor488- or
AlexaFluor546-UTP or FITC-UTP
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-two recent studies in Drosophila have looked at
the localization of developmentally important
mRNAs -Wilkie and Davis showed that movement of
the mRNA of the pair-rule gene runt (run) to the
apical surface of the blastoderm embryo occurs
through dynein-mediated movement along
microtubules

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-Cha et al. recently demonstrated that
localization of bicoid, a determinant of
embryonic polarity, to the anterior cortex of the
Drosophila egg is alsomicrotubule dependent
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Detection of mRNA with fluorescent RNA-binding
proteins -this approach to viewing mRNA in live
tissues is based on our knowledge of RNA-binding
proteins -one technique exploits the interaction
between the MS2 coat protein (CP) and CP-binding
sites from the MS2 RNA (recall that this
interaction is also used in the three-hybrid
method for detecting protein-RNA
interactions) -in this system, a CP-GFP fusion
is co-expressed in the cell with the mRNA of
interest engineered with CP-binding sites in
it -in live cells, CP-GFP will bind to the mRNA
of interest, making it visible with fluorescence
optics -this approach has been used to follow
the migration of the ASH1 mRNA into the daughter
cell during yeast budding -the ASH1 protein
plays important roles in establishing the
distinct identities of yeast mother and daughter
cells -mutations in the SHE genes disrupt
localization of ASH1 -SHE1 encodes a type V
myosin
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-the MS2 based approach has also been to look at
the migration of CaMKII mRNA in
hippocampal dendrites -various movement patterns
were described -oscillatory -unidirectional
anterograde -unidirectional retrograde -neuronal
depolarization increased amount of mRNA in
anterograde pool-movement of mRNA may prepare
dendrite for mounting local translational
response to stimuli
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-another recent study looked at b-actin transport
in neuronal growth cones - b-actin has a 54
nuclotide sequence in its 3 UTR called the
zipcode -zipcode is bound by Zipcode Binding
Protein (ZBP1) -dynamics of b-actin mRNA
movement in growth cones can be followed with
GFP-labelled ZBP1 -this analysis reveals a
dynamic bidirectional movement of b-actin
mRNA/ZBP1 particles in growth cones -the
importance of the interaction between ZBP1 and
b-actin mRNA is demonstrated by looking at the
effects of blocking this interaction with an
antisense oligonucleotide that binds the zipcode
sequence -this treatment causes growth cone
retraction
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Neuron treated with control oligonucleotide
Neuron treated with antisense oligonucleotide to
the b-actin zipcode