Dendritic BC1 RNA: functional role in regulation of translation initiation

Abstract

In neurons, local protein synthesis in synaptodendritic microdomains has been implicated in the growth and plasticity of synapses. Prerequisites for local translation are the targeted transport of RNAs to distal sites of synthesis in dendrites and translational control mechanisms to limit synthesis to times of demand. Here we identify dendritic BC1 RNA as a specific repressor of translation. Experimental use of internal ribosome entry mechanisms and sucrose density gradient centrifugation showed that BC1-mediated repression targets translation at the level of initiation. Specifically, BC1 RNA inhibited formation of the 48S preinitiation complex, i.e., recruitment of the small ribosomal subunit to the messenger RNA (mRNA). However, 48S complex formation that is independent of the eukaryotic initiation factor 4 (eIF4) family of initiation factors was found to be refractory to inhibition by BC1 RNA, a result that implicates at least one of these factors in the BC1 repression pathway. Biochemical experiments indicated a specific interaction of BC1 RNA with eIF4A, an RNA unwinding factor, and with poly(A)-binding protein. Both proteins were found enriched in synaptodendritic microdomains. Significantly, BC1-mediated repression was shown to be effective not only in cap-dependent translation initiation but also in eIF4-dependent internal initiation. The results suggest a functional role of BC1 RNA as a mediator of translational control in local protein synthesis in nerve cells.

Fig. 1.

BC1 RNA is a repressor of translation in the submicromolar concentration range. Protein products were labeled by³⁵S-methionine incorporation, using the RRL system, and were visualized by SDS-PAGE and autoradiography. A, Translation of endogenous RRL mRNAs was inhibited by increasing concentrations of BC1 RNA. Relative signal intensities of the major band were quantified by phosphorimaging and are listed for each lane. The signal intensity generated in the absence of BC1 RNA was assigned a relative value of 1. B, Results from three experiments, quantified by phosphorimaging, showed that the signal of the major protein band was reduced by 72% at 320 nm BC1 RNA [one-way ANOVA, p < 0.001; Scheffe's multiple comparison post hoc analysis (comparison with 0 nm BC1 RNA control): **p < 0.01 for 40 nm BC1 RNA, ***p < 0.001 for other groups]. Signal intensities of other protein bands were similarly reduced by 70–80%. Note that the x-axis is exponential. C, No inhibition of translation was observed in the presence of control RNAs, including U4 and U6 RNAs, and tRNAs. D, When capped and polyadenylated α-tubulin mRNA was used as a programming mRNA, translation was similarly inhibited in the same BC1 concentration range. Each experiment shown inC and D was performed at least twice.

Fig. 2.

BC1 RNA inhibits 48S and 80S complex assembly in cap-dependent initiation. A, A schematic diagram summarizes the steps in translation initiation that lead to the successive formation of 48S and 80S complexes. Steps that are targeted by inhibitors GMP-PNP and cycloheximide are indicated byarrows. The heterotrimeric complex eIF4F consists of eIF4A, eIF4E, and eIF4G. The helicase activity of eIF4A is stimulated by eIF4B. In addition, eIF4A is also present in free, monomeric form. [For more detailed diagrams of the translation initiation pathway, seeGingras et al. (1999), Hershey and Merrick (2000), and Dever (2002.)]B, ³²P-labeled capped and polyadenylated α-tubulin mRNA was used as a programming mRNA in the presence of cycloheximide to visualize 80S complexes. At 600 nm BC1 RNA, 80S complex formation was found to be reduced by 61 ± 5% (measured from the slope of the ribonucleoprotein complex peak; 3 experiments). C, Analogously, assembly of 48S preinitiation complexes was visualized by using GMP-PNP. At 600 nm BC1 RNA, 48S complex formation was found to be reduced by 81 ± 5% (measured from the slope of the ribonucleoprotein complex peak; 3 experiments). D, In contrast to BC1 RNA, U4 RNA at the same concen- tration had no effect on 48S complex assembly.E, Formation of 48S complexes on nonadenylated α-tubulin programming mRNA was inhibited in the presence of BC1 RNA to an extent similar to polyadenylated α-tubulin mRNA (compare withC). Assembled complexes were resolved by sucrose density gradient centrifugation. Sedimentation was from right toleft. Fractions from top parts of the gradient have been omitted for clarity. Tub(A) mRNA, Polyadenylated (A₉₈) α-tubulin mRNA; Tub mRNA, nonadenylated α-tubulin mRNA.

Fig. 3.

BC1 RNA inhibits translation initiated by the EMCV IRES. A, The programming mRNA encoded GFP, contained an EMCV IRES in the 5′ untranslated region, and was used uncapped.B, Results from six experiments, quantified by phosphorimaging, showed that translation was repressed by 79% at 320 nm BC1 RNA [one-way ANOVA, p < 0.001; Scheffe's multiple comparison post hoc analysis (comparison with 0 nm BC1 RNA control): ***p < 0.001 for all groups]. C, As a control, the same mRNA was translated in the presence of U4 RNA.D, Both cap-initiated and IRES-initiated translation from a dicistronic programming mRNA were repressed by BC1 RNA. The first, cap-dependent cistron encoded blue fluorescent protein (BFP). An EMCV IRES preceded the second, GFP-encoding cistron.

Fig. 4.

Translation and 48S complex formation mediated by the CSFV IRES are refractory to repression by BC1 RNA. The uncapped but polyadenylated programming mRNA encoded a truncated version of the influenza virus nonstructural protein (NS′).A, B, Translation efficiency was not significantly altered by increasing concentrations of BC1 RNA (one-way ANOVA, p = 0.9694; n = 5).C, Nuclear U4 RNA also failed to affect translation initiated from the CSFV IRES. D, Assembly of 48S complexes mediated by the CSFV IRES was refractory to inhibition by BC1 RNA (3 experiments). 48S complexes were assembled in the presence of GMP-PNP and resolved by sucrose density gradient centrifugation as described above (see also Fig. 2).

Fig. 5.

BC1 RNA binds to translational factors eIF4A and PABP. EMSA experiments were performed with ³²P-labeled BC1 RNA. A, BC1 RNA was incubated with eIF4A in the absence or presence of unlabeled competitor RNAs. Unlabeled BC1 RNA, but not unlabeled random sequence (RS) RNA or tRNAs, competed for binding to eIF4A and effectively abolished the mobility shift.B, BC1 RNA produced a band shift with full-length PABP. Effective competition was seen with unlabeled BC1 RNA but not with unlabeled U4 RNA or U6 RNA. C, Simultaneous incubation of BC1 RNA with eIF4A and PABP (N-terminal segment) produced a more substantial mobility shift than incubation with either protein alone.D, In rat brain extracts, BC1 RNA was observed to be shifted to two bands of lower mobility (lane 1). An antibody specific for PABP (lane 2), but not a control antibody against GST (lane 3), produced a supershift with BC1 RNA. Conversely, the regular mobility shift of BC1 RNA was reduced in brain extracts that had been immunodepleted of PABP; note the reduction in intensity of the major BC1 RNA complex bands and the appearance of a band at higher mobility (lane 5).BE, Brain extract; ID BE, PABP-immunodepleted brain extract.

Fig. 6.

Factors eIF4A, eIF4G, and PABP are enriched in synaptodendritic microdomains of hippocampal neurons in culture. Neurons were labeled (red fluorescence) for eIF4G (A), for PABP (B), or for eIF4A (C). Cells were double labeled with an antibody against synaptophysin (green fluorescence). Boxed dendritic segments are shown at three times higher magnification in insets. Note the clustered appearance of dendritic labeling signals for all three factors. Such clusters were often but not always observed in apposition to synaptophysin puncta. D, Control experiments were performed in an identical manner except that incubation with primary antibodies was omitted. Scale bar, 10 μm.