Transport and Translational Regulation of Dendritic mRNA
During Local Protein Synthesis
ABSTRACT
The biological basis of learning and memory is enacted at the synapse through molecular
mechanisms known as Long Term Potentiation and Long Term Depression. Not surprisingly,
these forms of synaptic plasticity require protein synthesis. Increasing evidence suggests that, in
the dendrite, protein synthesis is carried out on a small, localized scale directly adjacent to
remodeling synapses. What remains unclear is how the mRNA necessary for protein translation
is delivered to these sites of local synthesis and what prevents it from being translated at the
incorrect synapse. Previous studies used RNA interference to knockdown components of a
mRNA transport complex and implicated microRNA in translational repression. Here, I propose
additional siRNA knockdown candidates, while investigating the involvement of miRNA using
Fluorescence Resonance Energy Transfer. The information from these experiments will improve
our understanding of the biochemistry and molecular biology behind complex processes like
learning and memory formation.
LITERATURE REVIEW
The basic cellular unit of the brain, the neuron, has a highly polarized structure, with a
relatively small cell body, or soma, and long dendritic and axonal processes. Given its structure,
the neuron must overcome several technical challenges when executing even basic cellular
functions like protein synthesis. For instance, neurotransmitters to be released at axonal
synapses must first be synthesized in the soma, packaged in vesicles, and then transported
rapidly down a network of microtubules to the pre-synaptic axon terminus (Sheetz, Steuer et al.
1989). In dendrites, the same microtubule network exists and thus general protein synthesis
requirements were thought to be satisfied in much the same way.
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However, an important function of dendrites is their involvement in synaptic plasticity.
Based on recent activity, a synapse modifies its efficiency to either fire more strongly, in a
process known as Long Term Potentiation (LTP), or less strongly via Long Term Depression
(LTD). These two opposing mechanisms are widely believed to be the molecular basis of
learning and memory. Perhaps unsurprisingly, both LTP and LTD require the synthesis of new
proteins to enact changes in synaptic transmission (Goelet, Castellucci et al. 1986). Yet this is
conceptually problematic, as a single dendritic shaft contains dozens or hundreds of synapses.
How then, do plasticity-related proteins (PRPs) find the correct synapse?
In a recent review (Jiang and Schuman 2002), three possible models are proposed: 1)
PRPs are synthesized in the soma and somehow tagged for a specific synapse in the dendrite. 2)
PRPs are synthesized in the soma and sent randomly down the dendritic shaft, where they are
captured and used by a specific synapse. 3) PRPs are not synthesized in the soma at all, but
rather in the dendrite, by protein synthesis machinery localized to specific synapses.
Increasingly, it seems that the third model, local protein synthesis in dendrites, is the correct one.
Local protein synthesis was first proposed when ribosomes were isolated in neuronal
dendrites over forty years ago (Bodian 1965). Even then, its potential role of modulating
synapse-specific function was recognized. It has since been demonstrated that the ribosomes
frequently translate the same mRNA transcript in polyribosome complexes, sometimes at
membranous cisterns that are molecularly analagous to rough ER (Pierce, van Leyen et al. 2000).
It is therefore possible for even integral membrane proteins like the NMDA receptors and AMPA
receptors, two receptors for the neurotransmitter glutamate and central players in LTP, to be
synthesized locally. However, while nearly every element of the protein synthesis machinery
has been duplicated at the neuronal periphery, the mRNA transcripts necessary for translation are
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still dependent on genomic DNA templates, located far away in the neuronal nucleus. Thus, the
mechanism local protein synthesis fails to overcome the problem of synapse-specific targeting.
The challenge now lies not with transporting protein, but mRNA.
Unfortunately, far less is known about the mRNA in dendrites. Current large-scale
experimental approaches to analyze the composition of mRNA in dendrites rely on subcellular
fractionation techniques and subsequent analysis with microarrays or PCR. However,
fractionation methods are highly susceptible to contamination by fragments of glial or neuronal
soma and the data from these methods are therefore suspect. Alternately, individual candidate
mRNAs can be detected through in situ hybridization. Indeed, mRNA transcripts for proteins
with known roles in LTP, such as calmodulin-dependent kinase II (CaMKII), have been
isolated by this method (Jiang and Schuman 2002). Synthetic mRNA constructs using the 3’
UTR of CaMKII mRNA have successfully targeted proteins to dendrites, suggesting the
presence of a sorting signal -an extremely useful experimental tool. However, candidate-based
in situ hybridization is tedious and also faces resolution problems with respect to expression
levels, as we cannot assume all mRNA transcripts are present in equal amounts. Unless new
experimental technologies or methods are developed, it seems unlikely that the complete
dendritic mRNA population can be identified with confidence.
More is known, however, about the mechanism of mRNA transport and its translational
regulation. Operating off the assumption that dendritic mRNA transport would utilize the
ubiquitous microtubule network, a recent study isolated “an RNA-transporting granule” that used
the same motor protein, kinesin, as vesicles and organelles in fast axonal transport (Kanai,
Dohmae et al. 2004). Because the granule was resistant to detergent treatment, the authors
concluded that the mRNA was not transported by a vesicle, but rather a large protein complex.
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This novel transport mechanism is sensitive to RNase treatment, which both confirms the
inclusion of RNA in the granule and suggests an integral role for mRNA in maintaining granule
structure. But the precise mechanism of mRNA transport remains unclear as the molecular size
of the granule is too small to include, for instance, a single CaMKII mRNA.
An extremely thorough analysis of the RNA-transporting granule by gel electrophoresis
and mass spectrometry identified 42 proteins. Some proteins had known RNA-binding
properties like staufen, which responsible for mRNA localization during the development of
Drosophila melanogaster, and the RNA helicases, DDX3/DDX1. Others have heretofore no
demonstrated function. It was suggested that proteins with no direct role in RNA-binding could
also be involved in silencing mRNA contained in the granule. Indeed, mRNA transcripts
targeted to dendritic synapses would be unique in that extra precautions are necessary to avoid
premature translation of mRNA before arriving at the appropriate synapse. Interestingly, there is
some evidence that dendritic mRNA may be translationally silenced in part by microRNA
(miRNA): small endogenous hairpin-loops of single stranded RNA. In a separate study, RISC
proteins, which are involved with miRNA processing, were detected in the dendrite and one
RISC protein known as Armitage was degraded during memory formation (Ashraf and Kunes
2006). These provocative findings suggest an elegant mechanism: mRNA transcripts, destined
for translation in the dendrite, could be kept dormant in a miRNA-dependent manner during and
potentially after delivery to the appropriate synapse. Provided sufficient stimulation, these
silenced mRNA transcripts could be activated by disbanding the RISC complex. Protein
synthesis could then begin immediately, without any sort of communication with the soma.
Clearly, many features of the proposed mechanisms regulating dendritic mRNA transport
and translation are purely speculative. It should also be noted that not all relevant proteins are
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synthesized locally; many proteins undoubtedly are synthesized conventionally in the cell body
and later transported into the dendrite. But while aspects of local dendritic protein synthesis
remain largely theoretical, it is clearly a significant modulator of synaptic plasticity. Several key
experiments could illuminate many gaps in our understanding. First: more information is needed
about the identity and function of proteins that bind mRNA in the transport granule. Additional
knockdown data could illuminate which proteins are involved in granule-formation and
movement and which, if any, are involved in translational repression of transported mRNA.
Second: the RISC complex was initially detected in Drosophila neurons and was not identified
as part of the initial 42 granule proteins purified from mouse hippocampus cells. Mouse cells
expressing transgenic RISC proteins with fluorescent tags could simultaneously assay for the
presence of RISC proteins at the mammalian synapse and for RISC complex degradation upon
synapse stimulation. Together, these experiments could elucidate how an mRNA transcript
moves from the neuronal nucleus to a specific dendrite and initiates translation at the appropriate
time. If successful, these experiments would ground our understanding of dendritic protein
synthesis and its underlying mechanisms in fact, not speculation.
EXPERIMENTAL PROPOSAL
Aim I: Knockdown of putative mRNA-transport proteins using complementary siRNA sequences.
In the original study, Hirokawa and colleagues identified 42 proteins in the RNAtransporting granule and organized them by putative function (Kanai, Dohmae et al. 2004). Of
particular interest are the 6 RNA transport proteins and 3 RNA helicases, of which only Pur ,
staufen, and DDX3 were knocked down. Because of their known interactions with RNA, a
systematic knockdown of FMR1, FXR1, FXR2, Pur , DDX1 and DDX5 could yield
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informative results. The complementary siRNA constructs should be transfected into cultured
hippocampal neurons from E16 mouse embryos to maintain original experimental conditions.
The appropriate negative controls should also be included; that is, cells transfected with noncomplementary siRNA constructs. To assay the effect of the knockdowns, in situ hybridization
probes against the CaMKII 3’ UTR and antibody against CaMKII protein must be generated.
The in situ probes will measure distance traveled of CaMKII mRNA, while the antibodies will
assay for increased/decreased amounts of CaMKII in the dendrite.
Expected Results, Interpretations:
If the protein knockdowns disrupted normal mRNA transport, the CaMKII 3’ UTR
should appear to have traveled a shorter distance compared to control when measured from the
base of the dendrite, orthogonal to the cell body. It’s also possible that some of the proteins
involved do not interact with kinesin, but with dynein, the motor protein responsible for
retrograde motion. While there is no direct evidence of granule-interaction with dynein,
previous studies of vesicles have seen close association between kinesin and dynein in a
molecular “tug-of-war.” A knockdown of a granule-dynein interaction could drive the granule a
larger distance down the dendrite compared to control. Alternately, it’s possible that all
CaMKII 3’ UTR appears to stay in the cell body, since the disruption of a key RNA-binding
protein could cause the entire granule to disassemble, leading to a build up of CaMKII mRNA
in the cytoplasm near the soma. Lastly, there could be no observed effect on the distance
traveled, in which case the protein was either non-essential or has some other function, such as
translational regulation.
While dendritic mRNA is regulated by miRNA, more than one form of translational
regulation is possible. If a knockdown protein was involved in repressing translation of mRNA
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cargo, I would expect an increase of CaMKII protein at synapses near the base of the dendritic
shaft, which would be detected by immunocytochemistry. Any apparent increase in CaMKII in
knockdown cells compared to control could be normalized and quantified by the appropriate
imaging software, though it’s entirely possible that no significant increase will be detected. Such
a result merely indicates that the knockdown proteins were not involved in translational
regulation.
Caveats:
Hirokawa and colleagues cited a low transfection rate of hippocampal neurons, which
have a reputation for resistance to siRNA (Kanai, Dohmae et al. 2004). Therefore, it is entirely
possible that the knockdown will fail or will not transfect in sufficient quantities. It’s possible
that treatment with lentiviruses (Janas, Skowronski et al. 2006) or electroporation could
overcome this. Barring experimental difficulties, however, the results could appear misleading if
proteins in the RNA-transport granule perform similar or redundant functions. To test this
possibility, double siRNA knockdowns should be constructed, or alternately, a siRNA
knockdown of one protein with a dominant negative of a second protein. This sort of
combinatorial elimination of proteins could help demonstrate interactions between granule
proteins that may not be evident otherwise.
Aim II: FRET assay for RISC complex association/dissociation at mammalian synapses.
Fluroescence Resonance Energy Transfer, or FRET, is a biochemical technique to assay
for the association of two proteins. Two proteins are tagged with fluorophores with different
emission wavelengths. When the proteins are not in direct contact, these fluorophores remain
distinct colors. Once the tagged proteins bind to each other, the fluorophores shift their emitted
wavelength of light to a third, distinct wavelength that can be detected as a new color. Ideally,
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this experiment would be performed in mice in vivo, since previous experiments with the RISC
complex have been done completely in Drosophila neurons.
The FRET construct, consisting of two RISC genes each fused to a fluorophore-encoding
sequence, would be incorporated into the mouse genome through homologous recombination.
During homologous recombination, a synthetic sequence of DNA such as the FRET construct is
flanked by several kilobases of cloned genomic DNA. Through a mechanism that is still poorly
understood, the arms of genomic DNA in the synthetic construct align with the homologous
sequence in the endogenous genome, and the two are swapped.
Once the FRET construct is incorporated into a mouse, slices of adult mouse
hippocampus should be examined for fluorescence. Then, a specific neuron should be provided
with an artificial stimulus at its dendritic synapses and any change in FRET wavelength should
be recorded.
Expected Results, Interpretations:
Because translational suppression by miRNA is a widely conserved mechanism, I would
expect to observe some fluorescence in the sample. If the mammalian system is similar to that of
Drosophila, then we should see RISC proteins in the bound state throughout the cell body and
near dendritic synapses. It’s possible that I would observe some RISC proteins in the unbound
state, as two separate wavelengths. However, if only unbound RISC proteins are observed, I
would suspect that something went wrong in the experimental design (see Caveats).
When examining individual neurons provided with an artificial stimulus, I would expect
RISC proteins to change from a predominantly bound form, with only one wavelength, to a
predominantly unbound state, with two separate wavelengths. This would indicate that the
mammalian homologue to Armitage is also degraded upon synapse remodeling, causing the
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RISC complex to dissociate. If there is no change in the light wavelength, it’s possible that the
RISC complex is simply not degraded in mammalian neurons.
Caveats:
Both FRET and homologous recombination are experimentally complex systems with
many opportunities for problems during implementation. However, assuming that both systems
were successfully combined in the proposed experiment, there are other possible caveats. For
one, it’s possible that the fusion between RISC genes and the fluorophore affected the normal
dynamics of the RISC protein. The fluorophores are bulky and could impede binding because of
sterics, a problem that would be exacerbated by the size and intricacy of the RISC complex.
Importantly, the precise configuration of the RISC complex is not known. Because of its
size, it’s possible that two unlucky RISC proteins would never come into close enough contact
for the fluorescent FRET tags to interact. If this were the case, all fluorescent tags would appear
to be in the unbound state, always. Both of these problems have no obvious solution, but may
require selecting different fluorophore tags or different members of the RISC complex.
The molecular mechanisms behind synaptic plasticity have not been completely
characterized, and local protein synthesis in dendrites appears to play an important role. These
proposed experiments could help define the cellular machinery behind local protein synthesis by
exploring the ways that mRNA is transported and regulated at the synapse. With this new
information, we would better understand the most fundamental components behind the complex
processes of learning and memory.
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