Regulation of axonal trafficking of cytochrome c oxidase IV mRNA

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Abstract

Trafficking and local translation of axonal mRNAs play a critical role in the development and function of this neuronal subcellular structural domain. In this report, we studied cytochrome c oxidase subunit IV (COXIV) mRNA trafficking into distal axons of primary superior cervical ganglia (SCG) neurons, and provided evidence that axonal trafficking and mitochondrial association of the mRNA are mediated by an element located in a 38 bp-long, hairpin-loop forming region within the 3′UTR of the transcript. Our results also suggest that suppression of local translation of COXIV mRNA results in significant attenuation of axonal elongation. Taken together, the results provide the first evidence for the existence of a cis-acting axonal transport element within a nuclear-encoding mitochondrial gene, and demonstrate the importance of the axonal trafficking and local translation of nuclear-encoded mitochondrial mRNAs in axonal growth.

Introduction

Mitochondria in the axons and presynaptic nerve terminals fulfill distinct functions and have been shown to be closely associated with synapses and tethered to vesicle release sites (Zenisek and Matthews, 2000). To date, little is known about the half-life and biogenesis of synaptically localized mitochondria. Evidence that neuronal mitochondrial biogenesis does occur in the axon at a significant distance from the cell body has been recently provided (Amiri and Hollenbeck, 2008). Over the past few years, it has also become widely accepted that a distinct subset of nuclear-encoding mitochondrial mRNAs are selectively transported to the distal structural/functional domains of the neuron, including the axon and presynaptic nerve terminal (Gioio et al., 2001, Gioio et al., 2004, Willis et al., 2007, Taylor et al., 2009). Local proteins synthesized from these mRNAs play a key role, not only for mitochondrial function, but also in the development of the neuron and the function of the axon and nerve terminal. In addition, these studies called attention to the importance of local translation of COXIV mRNA, and its regulation by a brain-specific microRNA, miR-338, that regulates COXIV synthesis locally with distinct consequences on mitochondrial function, such as ATP generation, and axonal function as monitored by neurotransmitter uptake (Aschrafi et al., 2008).

The molecular mechanisms responsible for the transport of COXIV or other nuclear-encoded mitochondrial mRNAs into the axon are unknown. Subcellular mRNA localization and local translation within dendrites and axons are posttranscriptional mechanisms that generally require cis-acting sequences for their localization. These gene sequences are usually found in the 3′ untranslated region (3′UTR) of the transcript, and it was suggested that RNA-binding proteins recognize specific secondary structures in the 3′UTR, forming messenger ribonucleoprotein (mRNP) particles that are subsequently transported along microtubules to specific sites (Bassell and Singer, 1997, Kohrmann et al., 1999).

In this work, we used superior cervical ganglia (SCG) neurons cultured in compartmentalized Campenot cultures to examine the axonal transport and translation of COXIV mRNA. Using green fluorescent protein (GFP)-tagged constructs, we identified a 38 bp-long fragment within the COXIV 3′UTR that was required for mitochondrial targeting and axonal localization of the mRNA. Secondary RNA structure analysis of the 3′UTR suggests that these cis-acting regulating sequences are situated in a hairpin-loop forming region. In addition, our investigations identified local COXIV synthesis as an important determinant of axonal growth, as overexpression or knockdown of axonal COXIV levels significantly increased or attenuated neurite elongation, respectively.

Section snippets

The COXIV 3′UTR is sufficient for axonal mRNA transport

Most of the cis-acting sequences that have been identified as being involved in dendritic or axonal localization are situated in the 3′UTR of the mRNA (Martin and Zukin, 2006). Initial comparative sequence analysis revealed that the 3′UTR of the mammalian COXIV gene is highly conserved (Fig. 1A). In addition, an RNA secondary structure prediction analysis using Mfold (Zuker, 2003) indicated that this 3′UTR consisted of three well-conserved stem–loop structures, that might have functional

Discussion

Asymmetric subcellular distribution of RNA is critical for proper embryonic development, establishing differences in cell fate, and for neuronal function (Chartrand et al., 2001). Recent studies suggested a distinct role of local translation of COXIV mRNA in the modulation of mitochondrial activity and axonal function (Aschrafi et al., 2008). In this report, we provide data on the axonal targeting of COXIV mRNA, and explore the functional importance of the trafficking and local translation of

Neuronal cell cultures

SCG were obtained from 3-day-old Harlan Sprague–Dawley rats, and dissociated neurons plated in the center compartment of compartmentalized Campenot culture dishes as previously described (Hillefors et al., 2007). Cells were cultured in serum-free medium containing 20 mM KCl, NGF (50 ng/ml), and 20 U/ml Penicillin/20 µg/ml Streptomycin (Hyclone) for 3–10 days prior to use, with media changes every 3–4 days. The complete culture media, including NGF, was present in both the central and side

Acknowledgments

This work was supported by the Division of Intramural Research Programs of the National Institute of Mental Health.

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