Copb1-facilitated axonal transport and translation of kappa opioid-receptor mRNA

Abstract

mRNA of kappa opioid receptor (KOR) can be transported to nerve fibers, including axons of dorsal root ganglia (DRG), and can be locally translated. Yeast three-hybrid screening identifies Copb1 as a kor mRNA-associated protein that form complexes with endogenous kor mRNA, which are colocalized in the soma and axons of DRG neurons. Axonal transport of kor mRNA is demonstrated, directly, by observing mobilization of biotin-labeled kor mRNA in Campenot chambers. Efficient transport of kor mRNA into the side chamber requires Copb1 and can be blocked by a drug that disrupts microtubules. The requirement for Copb1 in mobilizing kor mRNA is confirmed by using the MS2-GFP mRNA-tagging system. Furthermore, Copb1 also facilitates the translation of kor mRNA in the soma and axons. This study provides evidence for a microtubule-dependent, active axonal kor mRNA-transport process that involves Copb1 and can stimulate localized translation and suggests coupling of transport and translation of mRNAs destined to the remote areas such as axons.

Fig. 1.

In vivo association of Copb1 with kor mRNA, HuR, and kinesin. (A) RNA immunoprecipitation (Upper) and protein coimmunoprecipitation (Lower) of anti-Copb1 precipitated complexes from cortical neurons. In A Upper, RT-PCR was used to detect kor transcript in Copb1 complex with cellular retinoic-acid-binding protein (CRABP) mRNA as a negative control. In A Lower, Western blot (WB) was used to detect proteins associated with Copb1 and the input (actin). (B) kor mRNA immunoprecipitated with Copb1 (Flag-Copb1) (Top) and HuR (Middle) from P19 cells. Input (actin) is also shown. For the graph (Bottom), a reporter assay was used to show the effect of HuR on translation of kor mRNA in P19. *, P <0.05 comparing with and without HuR overexpression in pGL3 luciferase with 5′- and 3′-UTR.

Fig. 2.

Colocalization of Copb1 with kor mRNA in DRG neurons. The constructs of the GFP–MS2-tagging system are shown at the top. NSL, nuclear localization signal. (A) The left sides, of the gels show the culture harboring the intact kor mRNA tagged with GFP–MS2, 5′k3′k. GFP (green) signifies the location of kor mRNA, whereas Copb1 (red) depicts the distribution of endogenous Copb1. Overlapped images are shown at the bottom. The right sides of the gels are enlarged images of two boxed areas containing axons. (B) Images of the culture harboring the negative control 5′tk3′SV40. a and b are enlarged ×3. (Scale bars, 100 μm.)

Fig. 3.

The role of Copb1 in kor mRNA transport in DRG neurons. (A) Biotin-labeled kor mRNA was introduced into the central compartment of the Campenot slide, and its distribution in the central and the side chambers was monitored (two leftmost panels). The expression of endogenous Copb1 was monitored by anti-Copb1 immunohistochemistry (rightmost panel). (B) A similar experiment as that described for A that was conducted in the culture transfected with Copb1 for 24 h. The elevation of Copb1 was confirmed by immunohistochemistry in the rightmost panel. (C) A similar experiment as that described for A was conducted in the culture receiving Copb1 siRNA for 24 h. Knockdown of Copb1 was also monitored by immunohistochemistry (rightmost panel). Both biotin and anti-Copb1 signals are shown in red. BF, bright field; TF, transfection. (D) A statistical analysis of the amounts of biotin-labeled RNA in the central and the side chambers (n = 3; *, P < 0.025 for comparing to the control; #, P < 0.025 for comparing the Copb1–siRNA culture to the Copb1-transfected culture). (E) The graph shows the statistical analysis of the intensity of Copb1 for each group (n = 5; *, P < 0.025 for comparing with the control; #, P < 0.025 for comparing the Copb1–siRNA culture with the Copb1-transfected culture). The blot shows Western blot results detecting Copb1 and actin expression. (Scale bars, 50 μm.)

Fig. 4.

kor mRNA transport is microtubule dependent. (A) Micrographs show the mobilization of MS2–GFP-tagged kor mRNA (Left) and the negative control (Right) in DRG neurons with (Lower) or without (Upper) the microtubule disruption drug colchicines. (B) Micrographs show mobilization of biotin-labeled kor mRNA (Left) from the central to the side chambers of the Campenot cultures with (Lower) or without (Upper) colchicines after Copb1 transfection for 24 h to stimulate the transport. Only the side chambers are shown. GFP signals are in green, and biotin-RNA signals are in red. (Scale bars, 25 μm.) Statistical analyses is shown in the graphs (for both, n = 3; *, P < 0.025 for comparing with the control). BF, bright field.

Fig. 5.

Copb1-stimulated kor mRNA translation. (A) The construct of kor template (KOR–GFP) with its coding sequence replaced, in-frame, with the GFP coding sequence is shown above the micrographs. The micrographs show in vitro synthesized, capped, and polyadenylated KOR–GFP transcript introduced into primary cortical neurons of a control culture (Top), a Copb1-transfected culture (Middle), and a culture receiving Copb1 siRNA (Bottom). GFP signals were monitored at 4 and 24 h, and the statistical analyses are shown in the graphs. (B) The diagram above the photographic images represents the central and side compartments of a Campenot chamber. Micrographs show KOR–GFP transcript introduced into the central compartment of the Campenot culture of a control (Top), a culture receiving supplemented Copb1 (Middle), or a culture receiving Copb1 siRNA (Bottom). Only the side chambers are shown. The GFP signals in the side chamber were monitored at 24 h, as shown, and the statistical analysis is shown in the graph. GFP signals are in green (n = 5, *, P < 0.025 for comparing with the control; #, P < 0.025 for comparing Copb1 siRNA with Copb1 transfection). (Scale bars, 50 μm.)