Abstract
Transport of neuronal mRNAs into distal nerve terminals and growth cones allows axonal processes to generate proteins autonomous from the cell body. While the mechanisms for targeting mRNAs for transport into axons has received much attention, how specificity is provided to the localized translational apparatus remains largely unknown. In other cellular systems, protein synthesis can be regulated by both cap-dependent and cap-independent mechanisms. The possibility that these mechanisms are used by axons has not been tested. Here, we have used expression constructs encoding axonally targeted bicistronic reporter mRNAs to determine if sensory axons can translate mRNAs through cap-independent mechanisms. Our data show that the well-defined IRES element of encephalomyocarditis virus (EMCV) can drive internal translational initiation of a bicistronic reporter mRNA in distal DRG axons. To test the potential for cap-independent translation of cellular mRNAs, we asked if calreticulin or grp78/BiP mRNA 5′UTRs might have IRES activity in axons. Only grp78/BiP mRNA 5′UTR showed clear IRES activity in axons when placed between the open reading frames of diffusion limited fluorescent reporters. Indeed, calreticulin’s 5′UTR provided an excellent control for potential read through by ribosomes, since there was no evidence of internal initiation when this UTR was placed between reporter ORFs in a bicistronic mRNA. This study shows that axons have the capacity to translate through internal ribosome entry sites, but a simple binary choice between cap-dependent and cap-independent translation cannot explain the specificity for translation of individual mRNAs in distal axons.
Highlights
Eukaryotic cells can temporally and spatially regulate protein composition of subcellular domains through translation of mRNAs transported to these sites
We previously showed that the mRNA encoding the ER chaperone protein calreticulin localizes to the axons of dorsal root ganglion (DRG) and cortical neurons through cis-elements in its 39UTR [18]
We initially examined expression in HEK cells that can be transfected at high efficiency. mCherry fluorescence from the 59 open reading frame (ORF) was clearly visible, but no GFP protein signal was detected from the 39 eGFP cistron (Fig. 1B)
Summary
Eukaryotic cells can temporally and spatially regulate protein composition of subcellular domains through translation of mRNAs transported to these sites. This is relevant to neurons where both the post-synaptic and pre-synaptic processes can be separated from the cell body by long distances. It is appealing to hypothesize that axons maintain multiple levels of translational regulation to temporally match the synthesis of new proteins to the physiological needs of this subcellular domain. For some localizing mRNAs, the same RNA binding protein that is needed for the mRNA’s transport to subcellular sites regulates translation. For other axonal mRNAs, the mechanisms for translational silencing are unknown, and there are several examples of localized mRNAs that are stored until needed. Though the molecular mechanisms are still unclear, these observations indicate that mRNA transport and local translational activation of the mRNA are not always mechanistically linked
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