Research Focus
We are interested in uncovering RNA regulatory pathways that
control the expression of proteins in cells. A fascinating aspect of cell biology is the selective
localization of mRNA to specific cellular compartments. For example, certain mRNAs are
localized to nuclear and plasma membranes, mitochondria, and to cellular
protrusions. In neurons, specific
mRNAs are enriched in the 1-2 µm-long spines that dot dendrites, and in the
growth cones that cap elongating axons.
These localized mRNAs are translated at these sites, suggesting that
selective RNA localization has a major influence on spatial patterns of protein
expression in cells.
The highly selective localization of many RNAs in cells
raises several important questions.
What advantages are conferred by the use of local translation?
Additionally, why are some signaling pathways dependent on local translation in
order to mediate their effects?
Furthermore, which mRNAs exhibit asymmetric localization, and how is the
localization achieved? Many of
these questions require fundamentally novel tools and techniques in order to
monitor RNA trafficking and translation in specific cellular compartments of
living cells, and to purify mRNA from subcompartments of cells. Our laboratory applies chemical
biology, microfluidic, and proteomic approaches to these key questions in
molecular biology.
A useful cell type to uncover mechanisms that control mRNA
localization and local translation is mammalian neurons. Neurons contain axons and dendrites,
which are morphologically distinct cellular compartments. These compartments are readily
visualized and, as described below, can be isolated for biochemical studies. Additionally, axons undergo readily
assayable morphological changes in response to “guidance cues,” the molecules
that control axon pathfinding during development. For example, axons turn away from a point source of
Semaphorin 3A (Sema3A), and are attracted towards netrin and nerve growth
factor (NGF). We and others showed
that these chemotaxis-like responses are blocked by axonally applied protein
synthesis inhibitors [1-3], indicating a role for intra-axonal translation in
mediating guidance cue responses.
In order to identify the local translation pathways that underlie axonal
responses to guidance cues, we developed microfluidic culturing approaches to
selectively recover dendrites, axons, and growth cones, which has allowed
unprecedented access to the specific transcripts localized to these sites and
the colocalized signaling pathways that control their translation [3-6].
We have identified a network of local translation events
that mediate axonal responses to axon guidance cues. To identify locally translated proteins that are effectors
of axonal signaling pathways, we isolated axons and generated cDNA libraries of
axonal mRNA. Using this approach,
we found that axons are enriched in several classes of transcripts, including
those that encode cytoskeletal regulatory proteins. For example, we found that axons contain transcripts
encoding RhoA, a protein that induces actin depolymerization. Sema3A induces local translation of
RhoA, leading to the actin depolymerization needed for growth cones to turn
away from point sources of Sema3A [2]. In the case of attractive guidance cues,
we found that NGF and netrin induce the translation of axonally localized PAR3
transcripts, which nucleates a multicomponent signaling complex critical for
actin and microtubule polymerization [3].
Local translation of PAR3 induces cytoskeletal polymerization required
for axon growth and attractive turning.
To determine if these transcripts mediate their effects due local
translation within axons, we developed an approach using microfluidic culturing
devices to selectively apply small interfering RNA to axons, inducing
axon-specific RNA interference [3,7].
Intriguingly, many of the axonally enriched transcripts that we
identified are also selectively localized to the leading edge of migrating cells
or to cellular protrusions, suggesting a functionally conserved mechanism for
local translation in different forms of cellular motility. These studies demonstrated the existence of local translation networks that orchestrate axon growth and
guidance.
Why are certain signaling pathways dependent on local
translation? By developing a novel
strategy to perform proteomic analysis of protein ubiquitination, we found that
many of the proteins that are synthesized within axons are also subjected to local
degradation [8,9]. Notably, many
of these proteins were also key effectors of axonal signaling pathways,
suggesting that signaling could be terminated by local ubiquitination and
restored by local translation. In
the case of RhoA, we found that the axonal E3 ubiquitin ligase Smurf 1
selectively degrades RhoA, thereby terminating Sema3A signaling. Local translation of RhoA reconstitutes
this pathway, accounting for the requirement for local translation in Sema3A
signaling.
Our finding that local translation is coupled to local
degradation suggests an intriguing rationale for why local translation may have
evolved as a signaling mechanism.
Local translation frequently has prominent roles in small
subcompartments in neurons, such as spines, which are typically 1 x 2 µm, and
growth cones, which are small specialized endings of axons. The limited space may create a
“molecular crowding” problem if the entire repertoire of signaling proteins
needed to respond to all the various signaling molecules in the extracellular
milieu was present at any given time.
Our data suggest that proteins are synthesized only when needed, and
then degraded shortly thereafter.
This ensures that space is available to accommodate other proteins that
may be required to mediate different signaling pathways. In this manner, the
contents of axons can be readily switched as needed to adapt to the dynamically
changing signaling requirements during axon pathfinding.
Our studies of axonal mRNAs have also revealed unexpected
mechanisms of neuronal signaling.
One of the most surprising classes of axonal transcripts that we
identified were mRNAs encoding transcription factors, including CREB [5]. As the sensory neuron axons grow
towards target tissues, they encounter NGF, which triggers the translation of
CREB within distal axons. CREB is
then retrogradely trafficked to cell bodies, where it induces neuronal survival
gene pathways [5]. These studies
demonstrate a fundamentally novel mechanism by which the effect of NGF at the
end of an axon can induce transcription in the nucleus, which can be
centimeters away. After our
initial studies, other groups have shown that this mechanism is important in
axon regeneration after injury [10], supporting the idea that local translation
and retrograde trafficking of axonal transcription factors is a recurrent
feature of neuronal signaling.
A major impediment to studying RNA pathways in cells is the
absence of simple methods to image RNA trafficking in living cells. To address this problem, we developed a
novel class of RNAs that mimic GFP in cells and enable simple and robust
genetic encoding of fluorescently tagged RNAs [11]. These RNAs bind fluorophores resembling the fluorophore in
GFP. Upon binding the fluorophores,
the RNAs “switch on” these otherwise nonfluorescent molecules, resulting in
fluorescence specifically associated with the tagged RNA. We developed a palette of
RNA-fluorophore complexes that span the visible spectrum. An RNA-fluorophore complex resembling
enhanced GFP, termed Spinach, emits a green fluorescence comparable in
brightness to fluorescent proteins.
Spinach is markedly resistant to photobleaching, and Spinach-fusion RNAs
can be imaged in living cells [11]. These RNA mimics of GFP provide an approach
to genetically encode fluorescent RNAs.
Using Spinach, we have tagged small noncoding RNAs to study their
localization in response to cellular signaling. For example, we tagged 5S, a small RNA component of
ribosomes to monitor the formation of RNA granules that form in response to
various types of cellular stress.
The Spinach fluorescent RNA system opens the door to
fundamentally new approaches to explore RNA biology in cells. We are currently using Spinach to
monitor molecular biology processes in living cells. For example, we are using Spinach to monitor splicing
reactions in living cells such that the splicing reaction results in the
formation of an intact Spinach aptamer.
Additionally, Spinach enables RNA-protein FRET studies, which we are
using to monitor the binding of axonal RNAs with P-body proteins in response to
guidance cue signaling.
Furthermore, we are currently using Spinach, as well as newer red and
orange fluorescent RNA-fluorophore tags that we have developed, Carrot and Radish,
to simultaneously image mRNAs and noncoding RNAs in living neurons. New tools, including a DNA-based
version of Spinach, have recently been designed. We expect that these novel chemical biology tools will have
a an important role in studies of RNA processing in living cells.
REFERENCES
1.
Campbell DS, Holt CE. Chemotropic responses of retinal growth cones mediated by
rapid local protein synthesis and degradation. Neuron, 32:1013-26, 2001.
2.
Wu KY, Hengst U, Cox LJ, Macosko EZ, Jeromin A, Urquhart ER, Jaffrey SR. Local
translation of RhoA regulates growth cone collapse. Nature, 436:1020-4, 2005.
3.
Hengst U, Deglincerti A, Kim HJ, Jeon NL, Jaffrey SR. Axonal elongation
triggered by stimulus-induced local translation of a polarity complex protein.
Nat Cell Biol, 11:1024-30, 2009.
4.
Hengst U, Cox LJ, Macosko EZ, Jaffrey SR. Functional and selective RNA
interference in developing axons and growth cones. J Neurosci, 26:5727-32,
2006.
5.
Cox LJ, Hengst U, Gurskaya NG, Lukyanov KA, Jaffrey SR. Intra-axonal
translation and retrograde trafficking of CREB promotes neuronal survival.
Nature Cell Biology, 10:149-59, 2008.
6.
Cohen MS, Bas Orth C, Kim HJ, Jeon NL, Jaffrey SR. Neurotrophin-mediated
dendrite to nucleus signaling revealed by microfluidic compartmentalization of
dendrites. Proc Natl Acad Sci U S A, 108:11246-11251, 2011.
7.
Hengst U, Jaffrey SR. Function and translational regulation of mRNA in
developing axons. Semin Cell Dev Biol, 18:209-15, 2007.
8.
Xu G, Paige JS, Jaffrey SR. Global analysis of lysine ubiquitination by
ubiquitin remnant immunoaffinity profiling. Nat Biotechnol, 28:868-73, 2010.
9.
Deglincerti A, Hengst U, Xu G, Jaffrey SR. Homeostatic control of intra-axonal
translation by local ubiquitination. In preparation, 2011.
10.
Yan D, Wu Z, Chisholm AD, Jin Y. The DLK-1 kinase promotes mRNA stability and
local translation in C. elegans synapses and axon regeneration. Cell,
138:1005-18, 2009.
11.
Paige JS, Wu KY, Jaffrey SR. RNA mimics of green fluorescent protein. Science,
333:642-646, 2011.
Research Topics
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