αCaMKII, NG, and ARC RNAs contain similar cis
-acting A2RE sequences that bind to the same trans
-acting factor, hnRNP A2, to mediate dendritic targeting by the A2 pathway. PKMζ RNA also contains an A2RE-like dendritic-targeting element (Muslimov et al., 2006
) that binds to hnRNP A2, suggesting that it follows the same A2 pathway. Thus, the A2 pathway may be a general pathway for multiplexed dendritic targeting of several different A2RE RNAs in hippocampal neurons.
The A2RE dendritic targeting sequences in αCaMKII, NG, and ARC RNAs identified by the microinjection assay are different from previously reported localization elements in these RNAs. One reason may be that previous studies used in situ hybridization to measure steady-state distribution of heterologous RNAs in dendrites, which can be affected by multiple different nuclear and cytoplasmic processes, whereas the microinjection assay specifically measures dendritic targeting while minimizing confounding effects of other processes. This raises two related questions: what specific step(s) in the RNA trafficking pathway is being measured in the microinjection assay and what is the physiological significance of dendritic-targeting elements identified by microinjection compared with localization elements identified by in situ hybridization.
The microinjection assay measures translocation of RNA from perikaryon to dendrites. This could reflect either biased transport of A2RE RNA granules from perikaryon to dendrites or unbiased movement of A2RE RNA granules in dendrites coupled with local immobilization at specific dendritic sites. Because movement of A2RE RNA granules along dendritic microtubules is bidirectional, distinguishing between these two possibilities will require systematic tracking of many individual granules over extended time intervals, which is beyond the scope of this study.
Microinjected RNAs are not translated efficiently because they are not capped or polyadenylated, and they contain fluorophore-conjugated ribonucleotides. In this sense, the injected RNAs represent “artificial” substrates, whose behavior may differ from endogenous RNAs. Despite that they are not capped or polyadenylated the injected fluorescent RNAs are not rapidly degraded in the cell because fluorescence does not accumulate in the nucleus. Furthermore, because translation of RNA is often associated with immobilization, reduced translational efficiency of injected RNAs may allow for more efficient translocation to dendrites by preventing immobilization in the perikaryon. Thus, the artificial properties of the injected RNA may facilitate identification of dendritic targeting elements by facilitating translocation and minimizing confounding effects of translation and RNA degradation. Because the microinjection assay is designed to analyze one specific step (dendritic targeting) in the trafficking pathway, cis-acting elements identified using the microinjection assay likely affect the subcellular distribution of endogenous RNAs but may not account for all aspects of RNA physiology.
The physiological significance of dendritic-targeting elements identified by microinjection is also related to the temporal resolution of the microinjection assay, which measures translocation of A2RE RNAs from perikaryon to dendrites during a relatively short time interval (~30 min) after injection. This is comparable with the time course of changes in synaptic sensitivity that occur during learning and memory. By contrast, in situ hybridization studies measure steady state levels of RNA in dendrites reflecting long-term stability of RNA in dendrites. αCaMKII, NG, ARC, and PKMζ RNAs are required for rapid changes in synaptic sensitivity during learning and memory. Each of these RNAs contain similar A2RE dendritic targeting sequences, identified by the microinjection assay, but they also contain different cis-acting elements, identified by in situ hybridization studies, that mediate steady-state levels in dendrites. This suggests that rapid changes in synaptic sensitivity associated with learning and memory involve multiplexed dendritic targeting of an ensemble of A2RE RNAs by the A2 pathway, whereas steady-state levels of individual A2RE RNAs in dendrites are differentially regulated.
There are three requirements for multiplexed dendritic targeting of A2RE RNAs: a multiplexer (mux) to combine multiple messages (RNA molecules) into a single composite signal (RNA granule), a carrier (microtubules) to transmit the multiplexed signal, and a demultiplexer (demux) (translation machinery) to decompose the multiplexed signal into individual messages. Multiplexing bandwidth is determined by the number of RNA molecules per granule.
The mux in the A2 pathway performs three functions: selection, linkage, and suppression. Selection distinguishes A2RE RNAs from non-A2RE RNAs, which is accomplished by sequence specific binding of hnRNP A2 to A2RE sequences in different RNAs. Initial binding of hnRNP A2 to endogenous A2RE RNAs probably occurs in the nucleus where the concentration of hnRNP A2 is very high. This marks the RNA for subsequent targeting by the A2 pathway when it reaches the cytoplasm. Binding of hnRNP A2 to exogenous RNAs microinjected into the cytoplasm occurs because the concentration of hnRNP A2 in the cytoplasm still exceeds the KD
for binding to A2RE sequences (Kosturko et al., 2006
). Linkage combines multiple A2RE RNAs into composite granules. This may be accomplished by TOG protein, a granule component that contains multiple binding sites for hnRNP A2 and may function as a multivalent scaffold for coassembly of multiple A2RE RNAs bound to hnRNP A2 molecules into granules (Kosturko et al., 2005
; Carson et al., 2006
). In postmitotic cells such as neurons, TOG is restricted to the cytoplasm so linkage presumably occurs in the cytoplasm rather than in the nucleus. Suppression prevents translation of RNAs in granules during transmission from nucleus to synapse. This may be accomplished by hnRNP E1, which is a granule component that binds to hnRNP A2 and inhibits translation of A2RE RNAs (Kosturko et al., 2006
The demux in the A2 pathway involves translation of individual A2RE RNAs into protein, mediated by protein synthetic machinery associated with each granule. If a single granule is captured at an individual synapse demulitplexing results in local translation of multiple different A2RE RNAs at that synapse. In this way, the ensemble of multiplexed A2RE RNAs in the granule determines the pattern of gene expression at the synapse. Because the A2RE is sufficient for dendritic targeting, virtually any RNA can be targeted to dendrites by inserting an A2RE into its sequence. If heterologous RNA is targeted to dendrites in this way, the encoded protein will be expressed locally at synapses, thereby altering synaptic properties. In this way, A2RE-mediated dendritic targeting of heterologous RNA by the A2 pathway could be used to transform synapses.
In addition to facilitating transmission of genetic information from nucleus to synapse, multiplexing also provides a mechanism for spatial and temporal coordination of gene expression at the synapse. Because αCaMKII and NG have opposing effects on synaptic sensitivity, multiplexed dendritic targeting of these RNAs may play a role in tuning synaptic sensitivity. Granules that contain more αCaMKII RNA may cause increased local synthesis of αCaMKII resulting in increased synaptic sensitivity, whereas granules that contain more NG RNA may cause increased local synthesis of NG, resulting in decreased synaptic sensitivity. Because ARC and PKMζ have opposing effects on synaptic scaling, multiplexed dendritic targeting of these RNAs may be important for scaling synaptic strength during long-term potentiation. Granules with more ARC RNA will result in increased local synthesis of ARC, reducing numbers of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors at the synapse, whereas granules with more PKMζ RNA will result in increased local synthesis of PKMζ increasing numbers of AMPA receptors at the synapse. In this way, random variation in granule composition can contribute to stochasticity in postsynaptic responsiveness.
Because the multiplexing mechanism is saturable, competition among different A2RE RNAs can affect the composition of individual granules. RNAs such as αCaMKII and PKMζ with A2REs that bind to hnRNP A2 with fast on rates may be assembled preferentially into RNA granules. Induction of ARC and PKMζ RNAs after synaptic activity may reduce dendritic targeting of NG and αCaMKII RNAs by competing for the A2 pathway. It may be possible to use competition among A2RE RNAs to systematically identify RNAs targeted by the A2 pathway. RNAs whose dendritic targeting is inhibited by excess A2RE RNA are likely to be targeted to dendrites by the A2 pathway. The rate-limiting component in the A2 pathway may not be hnRNP A2 itself, which is present in very high concentrations in most cells (Kosturko et al., 2006
). A more likely possibility is TOG protein, which is present in lower concentrations in the cytoplasm where it binds to hnRNP A2 and is a component of RNA granules (Kosturko et al., 2005
In summary, multiplexed dendritic targeting of multiple A2RE RNAs by the A2 pathway provides a mechanism for facilitating transmission of genetic information from nucleus to synapse, suppressing translation of dendritic RNAs during transport, coordinating expression of functionally related proteins at synapses, introducing variation in gene expression among synapses, and transforming synapses.