Co-transport of the nuclear-encoded Cox7c mRNA with mitochondria along axons occurs through a coding-region-dependent mechanism.

Nuclear-encoded mitochondrial protein mRNAs have been found to be localized and locally translated within neuronal processes. However, the mechanism of transport for those mRNAs to distal locations is not fully understood. Here, we describe axonal co-transport of Cox7c with mitochondria. Fractionation analysis and single-molecule fluorescence in situ hybridization (smFISH) assay revealed that endogenous mRNA encoding Cox7c was preferentially associated with mitochondria in a mouse neuronal cell line and within mouse primary motor neuron axons, whereas other mRNAs that do not encode mitochondrial protein were much less associated. Live-cell imaging of MS2-tagged Cox7c mRNA further confirmed the preferential colocalization and co-transport of Cox7c mRNA with mitochondria in motor neuron axons. Intriguingly, the coding region, rather than the 3' untranslated region (UTR), was the key domain for the co-transport. Our results reveal that Cox7c mRNA can be transported with mitochondria along significant distances and that its coding region is a major recognition feature. This is consistent with the idea that mitochondria can play a vital role in spatial regulation of the axonal transcriptome at distant neuronal sites.

Localization and RNA Binding of Mitochondrial Aminoacyl tRNA Synthetases.

Mitochondria contain a complete translation machinery that is used to translate its internally transcribed mRNAs. This machinery uses a distinct set of tRNAs that are charged with cognate amino acids inside the organelle. Interestingly, charging is executed by aminoacyl tRNA synthetases (aaRS) that are encoded by the nuclear genome, translated in the cytosol, and need to be imported into the mitochondria. Here, we review import mechanisms of these enzymes with emphasis on those that are localized to both mitochondria and cytosol. Furthermore, we describe RNA recognition features of these enzymes and their interaction with tRNA and non-tRNA molecules. The dual localization of mitochondria-destined aaRSs and their association with various RNA types impose diverse impacts on cellular physiology. Yet, the breadth and significance of these functions are not fully resolved. We highlight here possibilities for future explorations.

Localized translation near the mitochondrial outer membrane: An update.

Local synthesis of proteins near their activity site has been demonstrated in many biological systems, and has diverse contributions to cellular functions. Studies in recent years have revealed that hundreds of mitochondria-destined proteins are synthesized by cytosolic ribosomes near the mitochondrial outer membrane, indicating that localized translation also occurs at this cellular locus. Furthermore, in the last year central factors that are involved in this process were identified in yeast, Drosophila, and human cells. Herein we review the experimental evidence for localized translation on the cytosolic side of the mitochondrial outer membrane; in addition, we describe the factors that are involved in this process and discuss the conservation of this mechanism among various species. We also describe the relationship between localized translation and import into the mitochondria and suggest avenues of study that look beyond cotranslational import. Finally we discuss future challenges in characterizing the mechanisms for localized translation and its physiological significance.

DC3-decorated polyplexes for targeted gene delivery into dendritic cells.

Dendritic cells (DCs) are a family of specialized antigen presenting cells (APCs) that detect antigens and initiate a wide spectrum of immune responses against them. These characteristics make them promising candidates for immunotherapy manipulations. In this study we designed and synthesized DC-targeted block copolymers composed of linear polyethylenimine (PEI) conjugated to hydrophilic polyethylene glycol (PEG) installed with a DC-targeting peptide (DC3, primary sequence FYPSYHSTPQRP). Two different conjugation procedures (basic and modified) were employed to synthesize the DC3-PEG-b-PEI and the control SCRM-PEG-b-PEI (with a scrambled DC3 peptide sequence) by one-pot synthesis, in two steps. In the first, basic conjugation procedure, PEG with N-hydroxysuccinimide (NHS) ester and maleimide (MAL) groups (NHS-PEG-MAL, 3.5 kDa) was first coupled to linear PEI (25 kDa) via the NHS group to yield the intermediate MAL-PEG-b-PEI, that was then conjugated to N-terminus-cysteine harboring peptides DC3 or SCRM via the MAL double bond to yield the final DC3-PEG-b-PEI or SCRM-PEG-b-PEI copolymers, respectively. In the second, modified conjugation procedure, Fmoc-cysteine harboring DC3 or SCRM peptides were first conjugated to NHS-PEG-MAL via the MAL group followed by coupling to linear PEI via the NHS functional group. Fmoc cleavage yielded the same final product as in the basic procedure. The modified conjugation procedure was capable of yielding block copolymers richer with peptides, as determined by (1)H NMR analysis. Self-assembly of DC3-PEG-b-PEI copolymers and DNA molecules yielded nanosized polyion complexes (polyplexes), with lower surface charge and limited cytotoxicity when compared to the PEI building block. Significant transfection efficiency of the DC-targeted polyplexes by murine dendritic DC2.4 cells was observed only in DC3-PEG-b-PEI/DNA polyplexes synthesized by the modified conjugation procedure. These polyplexes, with higher peptide-load, showed greater transfection capability in DC2.4 cells relative to the control nontargeted SCRM-PEG-b-PEI/DNA polyplexes, but not in endothelial cells. The transfection efficiency was comparable to or higher than that of the PEI/DNA positive control. The results indicate that PEGylated-PEI polyplexes show significant transfection efficiency into DCs when decorated with DC3 peptide, and are attractive candidates for immunotherapy via DCs.