The final step in the multiplexing process is demultiplexing, whereby individual A2RE RNA molecules in the granule are translated into proteins in the myelin compartment in oligodendrocytes or at dendritic spines in neurons. Since each granule contains multiple different A2RE RNAs one might expect that all the RNAs in the granule are translated simultaneously. However, careful serial section electron microscopic studies reveal that sites of myelin synthesis in oligodendrocytes and dendritic spines in neurons generally contain only one or at most a few polyribosomes [37
]. Since each polyribosome represents a single translationally active RNA molecule this indicates that A2RE RNAs in granules are translated one at a time. Furthermore, since formation of a polyribosome requires repetitive re-initiation of translation on the same RNA molecule this implies that translation initiation factors and ribosomal subunits engaged on one RNA are continually recycled back onto the same RNA. However, since each of the RNAs in the granule is presumably translated eventually, initiation factors and ribosomal subunits engaged on one RNA must occasionally migrate to engage different RNAs in the same granule. The temporal pattern of translation of different A2RE RNAs in each granule may be determined by cis
-acting translational regulatory elements in individual RNAs.
Both MBP RNA in oligodendrocytes and αCaMKII RNA in neurons contain similar cis
-acting elements that may regulate translation () [39
]. The first is the cytoplasmic polyadenylation element (CPE). RNAs containing CPEs generally have short poly(A) tails, which is correlated with poor translation efficiency [40
]. CPE is bound by the cytoplasmic polyadenylation element binding protein (CPEB). CPEB also binds maskin, which in turn binds the translation elongation factor eIF4E, inhibiting translation because eIF4E cannot bind eIF4G, which is an essential step for initiation of translation. Activation of the kinase Aurora A results in phosphorylation of CPEB, which causes it to recruit the cleavage and polyadenylation specificity factor (CPSF), which in turn recruits poly(A) polymerase that elongates the poly(A) tail [41
]. Phosphorylation of CPEB also results in dissociation of maskin and eIF4E, which binds to eIF4G allowing translation initiation to proceed. Thus, elongation of the poly(A) tail is coincident with, and possibly related to translation activation [42
]. The second translational regulatory element found in both MBP RNA and αCaMKII RNA is the A2RE, which binds to hnRNP A2, resulting in suppression of translation in the presence of hnRNP E1 [33
] or stimulation of translation in the absence of hnRNP E1 () [44
]. Binding of hnRNP A2 to TOG protein also appears to stimulate translation of A2RE RNAs because reducing expression of TOG with RNAi results in inhibition of translation of MBP RNA in oligodendrocytes. The reason why TOG stimulates translation of A2RE RNAs is not known although several possible explanations have been suggested [32
]. One possible explanation is based on the function of TOG in linking multiple A2RE RNAs together in the same granule. By maintaining linkage among different A2RE RNAs in the same granule TOG may facilitate migration of translational initiation factors and ribosomal subunits among different A2RE RNAs resulting in enhanced translational output from each granule. In this way TOG may enhance translation of multiplexed A2RE RNAs in the myelin compartment of oligodendrocytes and in dendritic spines of neurons. Although CPE and A2RE are known to regulate translation of heterologous reporter RNAs in certain cell types it is not known if these cis
-acting elements regulate translation of MBP RNA in oligodendrocytes or of αCaMKII RNA in neurons.
Cis/trans determinants for translational regulation in MBP and αCaMKII RNAs