A HuD‐ZBP1 ribonucleoprotein complex localizes GAP‐43 mRNA into axons through its 3′ untranslated region AU‐rich regulatory element

Abstract

Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β-actin and GAP-43 mRNAs. β-actin 3′UTR has a defined element for interaction with ZBP1, but GAP-43 mRNA shows no homology to this RNA sequence. Here, we show that an AU-rich element (ARE) in GAP-43’s 3′UTR is necessary and sufficient for its axonal localization. Axonal GAP-43 mRNA levels increase after in vivo injury, and GAP-43 mRNA shows an increased half-life in regenerating axons. GAP-43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 coimmunoprecipitate in an RNA-dependent fashion. Reporter mRNA with the GAP-43 ARE competes with endogenous β-actin mRNA for axonal localization and decreases axon length and branching similar to the β-actin 3′UTR competing with endogenous GAP-43 mRNA. Conversely, overexpressing GAP-43 coding sequence with it’s 3′UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP-43’s 3′UTR.

hnRNPs Interacting with mRNA Localization Motifs Define AxoNAl RNA Regulons

“…We used an unbiased proteomics approach to identify axonal RBPs that interact with the localizing motifs of Nrn1, Hmgb1, Actb and Gap43 mRNAs. These four axonal mRNAs are regulated by injury-induced axonal localization, injury-induced intra-axonal translation, neurotrophin-induced transport/translation, and injury-induced transcription-coupled axonal localization, respectively (6,8,22,25,36). We observed a surprising diversity of RBPs showing high specificity binding to these mRNA localization…”

“…The axonal localization and translational regulation of Gap43 and Actb mRNAs are distinct from those of Nrn1 and Hmgb1 (8,39), so we reasoned these mRNAs would provide a rigorous test for potential RBP sharing between motifs. Since known RBPs also purified with the control Nrn1 and Hmgb1 RNA sequences, we used a scrambled oligonucleotide was used as control RNA in the Gap43 and Actb pull downs to determine if those RBPs represented non-specific RNA interactions.…”

“…For example, zip-code binding protein 1 (ZBP1, also called IGF-II mRNA binding [IMP1]) protein was shown to bind to a 56 nucleotide (nt) stem-loop structure in the 3' untranslated region (UTR) of β-actin mRNA (Actb), and this binding is necessary for axonal transport of the mRNA (6,7). Other RBPs implicated in axonal mRNA transport include nucleolin (Ncl), HuD (also called ELAVL4), hnRNP Q, hnRNP R, splicing factor proline and glutamine-rich (SFPQ), fragile X mental retardation (FMRP), Hermes, TRF2-S, and TDP-43 proteins (8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Despite increased insight into RNA transport and translation, including the identification of literally thousands of axonal mRNAs (18,19), relatively few RBPs have been identified in axons.…”

Syntaxin13 Expression Is Regulated by Mammalian Target of Rapamycin (mTOR) in Injured Neurons to Promote Axon Regeneration

“…Interestingly, although GAP43 mRNA and proteins were shown to increase after nerve injury in both DRG cell body and nerve (35, 36), we found that GAP43 levels were mainly increased in the nerve. Recent work showing that only axonally targeted GAP-43 mRNA, but not cell body restricted GAP-43 mRNA, is linked to elongated axonal growth (37,38) suggests that GAP43 axonal translation is controlled by mTOR. Similar to GAP-43, we report here that increased syntaxin13 levels in injured nerves are mediated by mTOR activity.…”

Localization of cofilin mRNA to the leading edge of migrating cells promotes directed cell migration

“…In neurons, a large number of mRNAs are known to be localized in axons during axon elongation, turning and regeneration (Holt and Schuman, 2013). Twiss and colleagues, in a series of elegant experiments, have demonstrated the importance of sufficient ZBP1 (VICKZ1) levels to ensure adequate localization of specific mRNAs into axons; their experiments also demonstrate that the localized translation of these mRNAs is crucial for axon elongation and regeneration following injury (Donnelly et al, 2011;Donnelly et al, 2013;Yoo et al, 2013). The experiments described here, in which the effects of localizing or not localizing mRNA to its intracellular target are compared under conditions where the levels of protein product are similar, extend these previous experiments and emphasize how trafficking mRNAs to particular locations can influence the effect of the protein product on cell behavior.…”