The Journal of Neuroscience, December 20, 2006, ():
Identification of Process-Localized mRNAs from Cultured Rodent Hippocampal Neurons
J. Neurosci. Poon et al.
26: 13390
Supplemental Data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure 1: Analysis of the Process to Whole Cell (P/T) ratio frequencies indicates that there are two or more populations of transcripts. A, P/T ratios were grouped into 0.05 increment bins and plotted versus frequency of occurrence. B, Approximate curve fitted using a mixture-distribution modeler (http://socr.stat.ucla.edu) set to estimate based on 2 populations. The predicted curves intersect at P/T=0.926, suggesting a threshold for process up- and down-regulated transcripts. If calculated for 3 populations, thresholds at P/T=0.947 and 2.28 are observed. If calculated for 4 populations, thresholds are observed at P/T=0.959, 1.78 and 2.34. By in situ hybridization, we have shown that the lowest P/T threshold likely delineates process up- and down-regulated transcripts.
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Supplementary Figure 2: In situ hybridization of additional genes identified by the microarray. Panels from left to right, 1) ISH with antisense riboprobe (green); 2) 4x zoom of a linearized region of dendrite >20μm from soma; 3) double label MAP2 immunoreactivity (red) in the antisense sample; 4) ISH with sense control riboprobe (green); and 5) double label MAP2 immunoreactivity in the sense sample (red). Abbreviations, A, hnRNPA/B: heteronuclear ribonuclear protein A/B; Ftl1: ferritin light chain 1; Rip3: rho interacting protein 3; CathD: cathepsin D; NNAT: neuronatin. B, H2AZ: H2A histone family, member Z; Impβ: importin β1; VILIP1: visinin-like protein 1. C, RNAse pretreatment control using eIF4γ2 riboprobe. Left, antisense riboprobe (green), right, corresponding anti-MAP2 immunocytochemistry (red). Scale bar: 10μm.
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Supplemental Figure 3. Model for the regulation of synapse-specific changes by local translation. 1, Synaptic stimulation that is of sufficient strength to initiate long-lasting plasticity a) generates a signal (blue trapezoid) that travels back to the nucleus to initiate transcription and b) activates translation of localized mRNAs. 2, Since many of the localized mRNAs encode translation factors, the translational capacity of stimulated synapses is increased as a result of local translation. Thus, when the products of new gene transcription are delivered throughout the cell, they are most efficiently translated at the previously stimulated synapse. In this manner, local synthesis of translational machinery generates a “translational sink,” which captures the products of transcription so that they are expressed at specific, stimulated synapses.
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Supplementary Table 1: List of genes identified by microarray analysis. Genes were screened using a p-value of 0.01 without false discovery rate (0.01noFDR) or a p-value of 0.05 with false discovery rate (0.05FDR). All transcripts in the 0.05FDR list were present in the 0.01noFDR list.
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Supplementary Table 2: Genes selected for confirmation by in situ hybridization. Abbreviations and definitions: P/T: process to whole cell ratio after normalization to reference sample, Raw Signal Intensity: Signal intensity as detected on the microarray.
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Supplementary Table 3: Primer sequences and clones used for generating riboprobes and performing quantitative PCR.
