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Axonal localization and mitochondrial association of precursor microRNA 338

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Abstract

MicroRNAs (miRNAs) selectively localize to subcompartments of the neuron, such as dendrites, axons, and presynaptic terminals, where they regulate the local protein synthesis of their putative target genes. In addition to mature miRNAs, precursor miRNAs (pre-miRNAs) have also been shown to localize to somatodendritic and axonal compartments. miRNA-338 (miR-338) regulates the local expression of several nuclear-encoded mitochondrial mRNAs within axons of sympathetic neurons. Previous work has shown that precursor miR-338 (pre-miR-338) introduced into the axon can locally be processed into mature miR-338, where it can regulate local ATP synthesis. However, the mechanisms underlying the localization of pre-miRNAs to the axonal compartment remain unknown. In this study, we investigated the axonal localization of pre-miR-338. Using proteomic and biochemical approaches, we provide evidence for the localization of pre-miR-338 to distal neuronal compartments and identify several constituents of the pre-miR-338 ribonucleoprotein complex. Furthermore, we found that pre-miR-338 is associated with the mitochondria in axons of superior cervical ganglion (SCG) neurons. The maintenance of mitochondrial function within axons requires the precise spatiotemporal synthesis of nuclear-encoded mRNAs, some of which are regulated by miR-338. Therefore, the association of pre-miR-338 with axonal mitochondria could serve as a reservoir of mature, biologically active miRNAs, which could coordinate the intra-axonal expression of multiple nuclear-encoded mitochondrial mRNAs.

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Abbreviations

miR:

MicroRNA

Pre-miRNA:

Precursor microRNA

RBPs:

RNA-binding proteins

SCG:

Superior cervical ganglion

COXIV:

Cytochrome C oxidase IV

ATP5G1:

ATP synthase 5 gamma 1

LC/MS:

Liquid chromatography–tandem mass spectrometry

ROS:

Reactive oxygen species

References

  1. Lee Y, Jeon K, Lee J-T et al (2002) MicroRNA maturation: stepwise processing and subcellular localization. EMBO J 21:4663–4670

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Ha M, Kim VN (2014) Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 15:509–524. doi:10.1038/nrm3838

    Article  CAS  PubMed  Google Scholar 

  3. Leung AKL, Sharp PA (2006) Function and localization of microRNAs in mammalian cells. Cold Spring Harb Symp Quant Biol 71:29–38. doi:10.1101/sqb.2006.71.049

    Article  CAS  PubMed  Google Scholar 

  4. Ashraf SI, Kunes S (2006) A trace of silence: memory and microRNA at the synapse. Curr Opin Neurobiol 16:535–539. doi:10.1016/j.conb.2006.08.007

    Article  CAS  PubMed  Google Scholar 

  5. Chiu H, Alqadah A, Chang C (2014) The role of microRNAs in regulating neuronal connectivity. Front Cell Neurosci. doi:10.3389/fncel.2013.00283

    PubMed  PubMed Central  Google Scholar 

  6. Iyer AN, Bellon A, Baudet M-L (2014) MicroRNAs in axon guidance. Front Cell Neurosci 8:78. doi:10.3389/fncel.2014.00078

    Article  PubMed  PubMed Central  Google Scholar 

  7. Olde Loohuis NFM, Kos A, Martens GJM et al (2012) MicroRNA networks direct neuronal development and plasticity. Cell Mol Life Sci. doi:10.1007/s00018-011-0788-1

    PubMed  Google Scholar 

  8. Kaplan BB, Kar AN, Gioio AE, Aschrafi A (2013) MicroRNAs in the axon and presynaptic nerve terminal. Front Cell Neurosci 7:126. doi:10.3389/fncel.2013.00126

    Article  PubMed  PubMed Central  Google Scholar 

  9. Yoo S, van Niekerk EA, Merianda TT, Twiss JL (2010) Dynamics of axonal mRNA transport and implications for peripheral nerve regeneration. Exp Neurol 223:19–27. doi:10.1016/j.expneurol.2009.08.011

    Article  CAS  PubMed  Google Scholar 

  10. Gumy LF, Katrukha EA, Kapitein LC, Hoogenraad CC (2014) New insights into mRNA trafficking in axons. Dev Neurobiol. doi:10.1002/dneu.22121

    PubMed  Google Scholar 

  11. Scott S, Gervasi N, Kaplan BB (2015) Subcellular compartmentalization of neuronal RNAs: an overview. Trends in Cell Mol Bio 10:1–36

    Google Scholar 

  12. Kim E, Jung H (2015) Local protein synthesis in neuronal axons: why and how we study. BMB Rep 48:139–146. doi:10.5483/BMBRep.48.3.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Natera-Naranjo O, Aschrafi A, Gioio AE, Kaplan BB (2010) Identification and quantitative analyses of microRNAs located in the distal axons of sympathetic neurons. RNA. doi:10.1261/rna.1833310

    PubMed  PubMed Central  Google Scholar 

  14. Sasaki Y, Gross C, Xing L et al (2014) Identification of axon-enriched microRNAs localized to growth cones of cortical neurons. Dev Neurobiol 74:397–406. doi:10.1002/dneu.22113

    Article  CAS  PubMed  Google Scholar 

  15. Lugli G, Torvik VI, Larson J, Smalheiser NR (2008) Expression of microRNAs and their precursors in synaptic fractions of adult mouse forebrain. J Neurochem 106:650–661. doi:10.1111/j.1471-4159.2008.05413.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lugli G, Larson J, Demars MP, Smalheiser NR (2012) Primary microRNA precursor transcripts are localized at post-synaptic densities in adult mouse forebrain. J Neurochem 123:459–466. doi:10.1111/j.1471-4159.2012.07921.x

    Article  CAS  PubMed  Google Scholar 

  17. Kim HH, Kim P, Phay M, Yoo S (2015) Identification of precursor microRNAs within distal axons of sensory neuron. J Neurochem. doi:10.1111/jnc.13140

    Google Scholar 

  18. Hengst U, Cox LJ, Macosko EZ, Jaffrey SR (2006) Functional and selective RNA interference in developing axons and growth cones. J Neurosci 26:5727–5732. doi:10.1523/JNEUROSCI.5229-05.2006

    Article  CAS  PubMed  Google Scholar 

  19. Aschrafi A, Schwechter AD, Mameza MG et al (2008) MicroRNA-338 regulates local cytochrome c oxidase IV mRNA levels and oxidative phosphorylation in the axons of sympathetic neurons. J Neurosci. doi:10.1523/JNEUROSCI.3338-08.2008

    PubMed  PubMed Central  Google Scholar 

  20. Bicker S, Khudayberdiev S, Weiß K et al (2013) The DEAH-box helicase DHX36 mediates dendritic localization of the neuronal precursor-microRNA-134. Genes Dev. doi:10.1101/gad.211243.112

    PubMed  Google Scholar 

  21. Aschrafi A, Kar AN, Natera-Naranjo O et al (2012) MicroRNA-338 regulates the axonal expression of multiple nuclear-encoded mitochondrial mRNAs encoding subunits of the oxidative phosphorylation machinery. Cell Mol Life Sci. doi:10.1007/s00018-012-1064-8

    Google Scholar 

  22. Hillefors M, Gioio AE, Mameza MG, Kaplan BB (2007) Axon viability and mitochondrial function are dependent on local protein synthesis in sympathetic neurons. Cell Mol Neurobiol. doi:10.1007/s10571-007-9148-y

    PubMed  Google Scholar 

  23. Berberich MJ, Kowalak JA, Makusky AJ, Martin D, Vullhorst A, Buonanno A, Markey SP (2011) Development of an on-bead digestion procedure for immunoprecipitated proteins. In: Ivanov AR, Lazarev AV (eds) Sample preparation in biological mass spectrometry. Springer, Netherlands, pp 109–124

    Chapter  Google Scholar 

  24. Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nat Protoc 4(1):44–57

    Article  CAS  Google Scholar 

  25. Huang DW, Sherman BT, Lempicki RA (2009) Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucl Acids Res 37(1):1–13

    Article  Google Scholar 

  26. Zhang X, Zuo X, Yang B et al (2014) MicroRNA directly enhances mitochondrial translation during muscle differentiation. Cell 158:607–619. doi:10.1016/j.cell.2014.05.047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Siegel G, Obernosterer G, Fiore R, Oehmen M, Bicker S, Christensen M et al (2009) A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis. Nat Cell Biol 11:705–716. doi:10.1038/ncb1876

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kye M-J, Liu T, Levy SF, Xu NL, Groves BB, Bonneau R et al (2007) Somatodendritic microRNAs identified by laser capture and multiplex RT-PCR. RNA 13:1224–1234. doi:10.1261/rna.480407

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kosik KS (2006) The neuronal microRNA system. Nat Rev Neurosci 7:911–920. doi:10.1038/nrn2037

    Article  CAS  PubMed  Google Scholar 

  30. Tolino M, Köhrmann M, Kiebler MA (2012) RNA-binding proteins involved in RNA localization and their implications in neuronal diseases. Eur J Neurosci 35:1818–1836. doi:10.1111/j.1460-9568.2012.08160.x

    Article  PubMed  Google Scholar 

  31. Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J et al (2015) STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucl Acids Res 43:D447–D452. doi:10.1093/nar/gku1003

    Article  PubMed  Google Scholar 

  32. Campbell PD, Shen K, Sapio MR et al (2014) Unique function of Kinesin Kif5A in localization of mitochondria in axons. J Neurosci 34:14717–14732. doi:10.1523/JNEUROSCI.2770-14.2014

    Article  PubMed  PubMed Central  Google Scholar 

  33. Matsumoto K, Wolffe AP (1998) Gene regulation by Y-box proteins: coupling control of transcription and translation. Trends Cell Biol 8:318–323

    Article  CAS  PubMed  Google Scholar 

  34. Barrey E, Saint-Auret G, Bonnamy B et al (2011) Pre-microRNA and mature microRNA in human mitochondria. PLoS One. doi:10.1371/journal.pone.0020220

    PubMed  PubMed Central  Google Scholar 

  35. Kar AN, Sun CY, Reichard K et al (2014) Dysregulation of the axonal trafficking of nuclear-encoded mitochondrial mRNA alters neuronal mitochondrial activity and mouse behavior. Dev Neurobiol. doi:10.1002/dneu.22141

    PubMed  Google Scholar 

  36. Gehrke S, Wu Z, Klinkenberg M et al (2015) PINK1 and Parkin control localized translation of respiratory chain component mRNAs on mitochondria outer membrane. Cell Metab 21:95–108. doi:10.1016/j.cmet.2014.12.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Huang L, Mollet S, Souquere S et al (2011) Mitochondria associate with P-bodies and modulate microRNA-mediated RNA interference. J Biol Chem 286:24219–24230. doi:10.1074/jbc.M111.240259

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Das S, Ferlito M, Kent OA et al (2012) Nuclear miRNA regulates the mitochondrial genome in the heart. Circ Res 110:1596–1603. doi:10.1161/CIRCRESAHA.112.267732

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Bandiera S, Matégot R, Girard M et al (2013) MitomiRs delineating the intracellular localization of microRNAs at mitochondria. Free Radic Biol Med. doi:10.1016/j.freeradbiomed.2013.06.013

    PubMed  Google Scholar 

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Acknowledgments

The work was supported by the Division of Intramural Research Programs of the National Institute of Mental Health (MH002768). The authors would like to thank Mrs. Ching-Yu Sun, Ms. Miranda Tompkins, Dr. Noreen Gervasi, Mr. Shane Scott, and Ms. Anna-Leigh Brown for their scientific insights and engaging conversations, which contributed to the fruition of this work.

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Authors and Affiliations

  1. Section on Neurobiology, Laboratory of Molecular Biology, Division of Intramural Research Programs, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, 20892-4411, USA

    Jose Norberto S. Vargas, Amar N. Kar, Jenna R. Gale, Armaz Aschrafi, Cai-Yun Chen, Anthony E. Gioio & Barry B. Kaplan

  2. NINDS/NIMH Clinical Proteomics Unit, Division of Intramural Research Programs, National Institutes of Health, Bethesda, MD, USA

    Jeffrey A. Kowalak

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  1. Jose Norberto S. Vargas
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Correspondence to Barry B. Kaplan.

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Electronic supplementary material

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18_2016_2270_MOESM1_ESM.pptx

Fig. S1. A schematic of the RNA affinity purification protocol to identify pre-miR-338 interacting proteins. Biotinylated pre-miR-338 was incubated with P4 rat brain cytosolic extracts. The formation of RNP granules/complexes was monitored by EMSA gel shift assay. After incubation, the RNA-protein complexes were isolated using streptavidin beads. The identity of the pre-miR-338 binding proteins was determined by mass spectrometry and validated by Western analyses (pptx 178 kb)

18_2016_2270_MOESM2_ESM.pdf

Fig. S2. Full blot images of Western analyses performed to confirm pre-miR-338 RNP identified via LC/MS. RNA affinity pulldowns were performed with the biotinylated pre-miR-338 oligomer and P4 brain cytosolic lysates or cytosolic extracts isolated from distal axons present in the side-compartments of Campenot cultures (see Fig. 6). Arrows indicate bands corresponding to protein of interest. Asterisks indicate non-specific protein band (pdf 150 kb)

18_2016_2270_MOESM3_ESM.xlsx

Table S1. Pre-miR-338 interacting protein list. Proteins identified by LC/MS to be enriched in pre-miR-338 pulldowns (XLSX 47 kb)

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Vargas, J.N.S., Kar, A.N., Kowalak, J.A. et al. Axonal localization and mitochondrial association of precursor microRNA 338. Cell. Mol. Life Sci. 73, 4327–4340 (2016). https://doi.org/10.1007/s00018-016-2270-6

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  • DOI: https://doi.org/10.1007/s00018-016-2270-6

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