, 2001) using forward primers at the 3′ end of the Importin β1 open reading frame (ORF) and a mixture of three reverse primers anchored on polyA sequences ( Figure 1A). Two major 3′ UTR variants of Importin

β1 were obtained and sequenced, comprising a short (134 bases) isoform more prominent in cell bodies and a long (1,148 bases) isoform overlapping with the short form and more prominent in axons ( Figures VX770 1A, 1B, and see S1A available online). These two Importin β1 UTRs arise from differential usage of polyadenylation sites ( Figure S1A), a widespread mechanism for defining different 3′ UTRs ( Proudfoot, 2011). The Importin β1 UTR sequence is highly conserved in the vertebrate lineage, with 95% sequence identity between rat and mouse and 86% identity with humans and other primates ( Figure 1C). Despite the high sequence

conservation, we did not detect any known localization motifs in the Importin β1 UTR sequences and therefore set out to test their capacity to induce axonal localization of reporter genes. We first generated fusion constructs of Importin β1 3′ UTR segments and deletions thereof ( Figure 1A) with a myristylated green fluorescent protein (GFP) ORF ( Aakalu et al., 2001) and this website examined localization of GFP transcripts by fluorescence in situ hybridization (FISH) on transfected DRG neurons in culture. Note that all the processes extended by adult DRG neurons in culture were previously shown to be axonal in nature ( Vuppalanchi et al., 2010; Zheng et al., 2001). Axonal localization of aminophylline GFP transcript was observed for the long UTR isoform, but not for the short UTR ( Figure 1D). Two deletion constructs comprising the central and 3′ terminal segments of the long UTR (Δ1 and Δ2, Figure S1B) were then tested

for axon-localizing capacity of the GFP reporter. In situ hybridization on neurons transfected with these two constructs support the existence of an axonal localization motif only within the Δ2 region, hence toward the 3′ terminal segment of the long Importin β1 UTR ( Figure 1D). In order to further test axonal localization by a different approach, we carried out fluorescence recovery after photobleaching (FRAP) experiments on axon terminals of cultured neurons transfected with Importin β1 3′ UTR-myrGFP fusion constructs. The myristylation domain limits diffusion for this reporter in both dendrites ( Aakalu et al., 2001) and axons ( Yudin et al., 2008), enabling precise visualization of local translation events. Fluorescence recovery was observed only for constructs containing the long form of importin β1 3′ UTR or the 3′ end Δ2 segment, but not for the short UTR variant or the Δ1 construct ( Figures 2A, 2B, and S2). The translation inhibitor anisomycin blocked fluorescence recovery, further confirming that the reporter signal arose from locally translated axonal transcript.