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Fig. 7
The proposed RNA trafficking sequence (RTS) sequences from mbpa and mbpb do not affect protein translation or mRNA transport. (A) The RTS consensus sequence (the hnRNP A2-binding site, A2RE11, is underlined) and the specific sequences of the rat, mouse and human RTS are shown together with proposed RTS sequences from zebrafish mbpa and mbpb mRNA. Nucleotides differing from the consensus are marked in red. The sequences from mbpa and mbpb with the highest similarity to the consensus are marked in bold. (B) The constructs tested in the translation assay are illustrated. The mbpa and mbpb RTS corresponds to the sequences marked in bold in (A). (C) The constructs shown in (B) were co-transfected with a DsRed-expressing vector into Oli-Neu cells and the translation ratio calculated as described in the “Materials and Methods” section. Data represent the means of four individual experiments ± SD. ns = not significant, **p < 0.01, ***p < 0.001. (D) RNA was extracted from a fixed number of transfected cells and RT-PCR was performed with primers specific for Dendra2 or G6PD (control). (E) The localization of mRNA in larvae with mosaic expression of the indicated constructs was defined as “Cell body” or “Sheath”. Examples of both groups are shown below the chart. Cell bodies are marked with arrowheads, while sheaths are marked with arrows. Bottom micrographs are enlarged views of top images. The relative fraction of cells with each type of mRNA distribution is plotted. For +mbpb RTS, 10 cells were analyzed. For all other constructs, 77–173 cells were analyzed.