amaxa news #10 - Lonza AG

› Application Note
Down-regulation of a Key Component
of the RNA Localization Machinery
in Hippocampal Neurons
Manuel Zeitelhofer and Ralf Dahm, Division of Neuronal Cell Biology, Center for Brain Research,
Medical University of Vienna, Vienna, Austria
The localization of mRNAs is an efficient way to target proteins to specific regions in a cell. In mammalian neurons, RNA localization is involved in
compartmentalizing the cell during differentiation and is assumed to play an important role in synaptic plasticity. RNAs are packaged together with
RNA-binding proteins into transport-competent ribonucleoprotein particles (RNPs) and transported into dendrites. The RNA-binding protein Staufen2
(Stau2) is a key component of RNPs and implicated in dendritic RNA transport. In this study, Stau2 was successfully down-regulated by RNA interference
(RNAi). The knockdown efficiency of Stau2 expression was assessed after nucleofection ® of shRNA constructs targeting Stau2 mRNA. In contrast to
other transfection methods, the high transfection efficiencies attainable with the nucleofection ® technique allowed the determination of the extent of
Stau2 protein down-regulation by quantitative western blot analysis.
Introduction
The localization of mRNAs is an efficient
way to target gene products to specific
regions in a cell. It occurs in a wide range
of organisms and cell types 1, 2 . During
Drosophila development, for example,
localized mRNAs act as cell fate determinants
and thereby specify the body
axes 3 . In addition to its role in development,
RNA localization also occurs in
differentiated cells. In migrating cells, for
instance, mRNAs are localized to the
leading edges, thereby enabling directed
movement. This plays a role in wound
closure when fibroblasts migrate into the
lesion or when cancer cells metastasize.
Polarized cells, such as neurons, display
a functional compartmentalization of the
cytoplasm. The cell body is molecularly
distinct from the axon and the dendrites.
RNA localization is involved in compartmentalizing
neurons during differentiation
and it is assumed to also play
an important role in synaptic plasticity,
the experience-dependent remodeling
of synapses that forms the basis of
learning and memory 4 .
How is RNA localization achieved?
Localized RNAs contain cis-acting
sequence elements, often within the
3’-untranslated region (3’-UTR), which
are recognized by proteins (trans-acting
factors). The mRNAs and trans-acting
factors are packaged into ribonucleoprotein
particles (RNPs). These RNPs
are then transported to and retained
by sites of local synthesis 5 . During transport,
the RNAs must be kept translationally
silenced to prevent the ectopic
expression of the corresponding protein.
At their destinations, this translational
block can be lifted by signals inducing
translation 6 . A central hypothesis assu-
mes that translation is only activated at
synapses where synaptic plasticity has
been induced.
In this study, the nucleofection ® technique
was used to assess the efficiency of
down-regulation of Staufen2 (Stau2) in
hippocampal neurons. Stau2 is one
of the key components implicated in
dendritic RNA localization. In Drosophila,
Staufen is required for mRNA localization
in the oocyte 7 and in neuroblasts 7 .
In mammals, it is hypothesized that
Stau2 is important in the transport of
RNAs into dendrites of mature hippocampal
neurons. To assess the consequences
of a loss of Stau2 on dendritic
RNA localization as well as the morphology
of dendrites and dendritic spines,
Stau2 was down-regulated by RNAi
in primary cultures of hippocampal
neurons 8 . In order to obtain quantitative
information on the extent of down-regulation
by western blot analysis, it was
crucial that a large proportion of hippocampal
neurons was transfected by
nucleofection ® .
Material and Methods
Primary hippocampal neuron culture
Neurons were isolated from the hippocampi
of 17 day-old embryonic (E17)
rat brains and cultured as described 9 .
For nucleofection ® experiments, neurons
were immediately used after dissociation.
For the assessment of dendritic
spine phenotypes and for fluorescence
in situ hybridization (FISH) experiments,
cells were used after 15 days in vitro (DIV).
Nucleofection ® and western blot
analysis
After dissociation, neurons were cotransfected
with an expression vector
› Page 8 › amaxa news # 10
encoding citrine and shRNA constructs
(pSUPER, OligoEngine 10 ) against
Stau2, mismatch Staufen2 (misStau2),
and Septin7 (Sept7; CDC10). Cells were
plated onto 6 cm cell culture dishes
and lysed after 3 days of expression.
The knockdown efficiency was assessed
by western blot analysis.
Immunostaining
The following antibodies were used:
an affinity-purified rabbit anti-Stau1
antibody (1 µg/ml) and an affinity-purified
rabbit anti-Stau2 antibody (1 µg/ml).
Fluorophore-coupled phalloidin was used
to label F-actin. Immunostaining experiments
were performed as described 11 .
Results
Stau2 was down-regulated in hippocampal
neurons by nucleofection ® of shRNA
constructs to assess its function during
neuronal differentiation and in mature
neurons. To evaluate the success of the
down-regulation, western blot analysis
of the transfected neurons was performed.
As neurons are post-mitotic cells,
they are difficult to transfect. Conventional
transfection methods like the
calcium phosphate (CaPi) method have
very poor transfection efficiencies for
subsequent biochemical analyses and
virus-based methods are too time-consuming
and costly 12 . The nucleofection ®
technique overcomes these limitations.
Primary hippocampal neurons were
co-transfected with an expression vector
encoding citrine and shRNA constructs
(pSUPER) against Stau2, mis-Stau2,
and Septin7. First, the transfection
efficiency was evaluated. To this aim,
transfected neurons were fixed 3 days
post nucleofection ® and the percentage