The Journal of Neuroscience, April 27, 2005, ():
A Novel RNA-Binding Protein in Neuronal RNA Granules: Regulatory Machinery for Local Translation
J. Neurosci. Shiina et al.
25: 4420
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure 1. In situ hybridization of A6 cells with poly(dT) for mRNA detection. In ~15% of the cells, mRNA was concentrated more heavily in the nucleus than in the cytoplasm (arrow), whereas its distribution was rather homogeneous throughout the cell in most cells, although the reason for the difference are not known. The right panel is a DIC image. Bar, 10 ?m.
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Supplementary Figure 2. Expression of XRNG105-GFP affects polysome formation in A6 cells. Extracts from A6 cells expressing XRNG105-GFP (blue) or control A6 cells expressing GFP (orange) were fractionated by sucrose density gradient centrifugation. Elution profiles of ribosomes and EF-1? are shown. Ribosomes and EF-1? in the polysome fractions were reduced by expression of XRNG105-GFP in the cells. Increase of ribosomes in the fractions heavier than the polysome fractions may be due to the formation of ribosome-containing granules by XRNG105-GFP expression in the cells.
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Supplementary Figure 3. Local translation in transected dendrites. A, GFP fluorescence images of cultured hippocampal neurons before (left) and after (right) transection of the dendrite. An arrow denotes the point of the transection. Bar, 10 ?m. B, Cultured hippocampal neurons were transfected both with GFP reporter fused to the CaMKII? 3’UTR and RNG105-mRFP1. The dendrites were transected and treated with or without 10 ?g/ml cycloheximide (CHX). After 10 min, they were treated with BDNF. Time-lapse images of GFP and RNG105-mRFP1 proteins in the transected dendrites after BDNF stimulation are shown. Arrowheads denote positions of RNG105-localizing RNA granules in which GFP signals were increased after BDNF stimulation. Some RNG105-localizing RNA granules did not show any GFP signals, which may be because the amount of GFP-CaMKII? 3’UTR mRNA or translational activity might be different among the granules. In the CHX-treated dendrites, GFP signals were detected in some RNG105-localizing RNA granules, but not significantly increased (arrows). Bar, 5 ?m. C, Profile of GFP fluorescence changes during the experiment in B. Plotted are the changes in GFP fluorescence intensities near the RNG105-localizing RNA granules between the indicated time points and t= 5 min. Data are means and standard errors from 16 granules of two independent experiments. The fluorescence intensity of GFP was measured as described in Materials and methods, and the mean fluorescence intensity was calculated within FWHMt=5 of RNG105-mRFP1 in the same granule. ?F is shown as {(Fg–Fb)t–(Fg–Fb)t=5}/(Fg–Fb)t=5.
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Supplementary Figure 4. Model for the role of RNG105 in the RNA granule in neuronal dendrites. RNG105 binds to and represses translation of mRNAs. Although the mRNA binding of RNG105 is sequence-independent, the association of RNG105 with the granule would restrict its binding to mRNAs specifically localized in the granule. The N and C termini of RNG105 are responsible for the association with the RNA granule, and the N terminus, for the mRNA-binding and translation repression. Upon synaptic stimulation by BDNF, the affinity of RNG105 to the granule is reduced, leading to the release of RNG105 from the granule and the mRNAs, which is concomitant with increase of local translation near the granule.
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