This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. A link to any extended data will be provided when the final version is posted online. Research Articles: Cellular/Molecular Transcriptomic Analysis of Ribosome-Bound mRNA in Cortical Neurites In Vivo Rebecca Ouwenga1,2,3, Allison M. Lake2,3, David O'Brien2,3, Amit Mogha4, Adish Dani5,6 and Joseph D. Dougherty2,3,6 1Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, MO, USA 2Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA 3Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA 4Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO, USA 5Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA 6Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA DOI: 10.1523/JNEUROSCI.3044-16.2017 Received: 29 September 2016 Revised: 29 June 2017 Accepted: 21 July 2017 Published: 8 August 2017 Author Contributions: RO: Developed and conducted SynapTRAP, data analysis, validation studies, and writing of manuscript; AL: Contributed to data analysis and writing of manuscript; AM: Conducted EM imaging.; DO: Contributed to data analysis; AD: Conducted STORM imaging; JD: Conceived of study, contributed to data analysis, and writing of manuscript. Conflict of Interest: JDD has received royalties related to TRAP in the past. No other authors declare a conflict of interest. We would like to thank M. Wong for Thy-1 mice, K. Monk, C. Weichselbaum and members of the Dougherty lab for helpful comments, N. Pisat, N. Kopp, K. Sakers, and S. Pyfrom for training, advice, and assistance. This work was supported by the CDI (MD-II-2013-269), and NIH (R21DA038458, R21MH099798, R01NS102272). Key technical support was provided by the Genome Technology Resource Center at Washington University (supported by NIH grants P30 CA91842 and UL1TR000448). RO was supported by T32 GM081739. JDD is a NARSAD investigator. Correspondence should be addressed to Contact: Dr. Joseph Dougherty, Dougherty Lab, Department of Genetics, 660 S. Euclid Ave, Campus Box 8232, St. Louis, MO 63110-1093, P: 314-286-0752, F: 314-362-7855, E: [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.3044-16.2017 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2017 the authors 1 Title: Transcriptomic Analysis of Ribosome-Bound mRNA in Cortical Neurites In Vivo 2 3 Running Title: In Vivo Analysis of mRNA in Cortical Neurites 4 5 Authors: Rebecca Ouwenga1-3, Allison M. Lake2,3, David O’Brien2,3, Amit Mogha4, Adish Dani5,6, 6 Joseph D. Dougherty*2,3,6 7 8 1Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. 9 Louis, MO, USA 10 2Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA 11 3Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA 12 4Department of Developmental Biology, Washington University School of Medicine, St. Louis, 13 MO, USA 14 5Department of Pathology and Immunology, Washington University School of Medicine, St. 15 Louis, MO, USA 16 6Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, 17 MO, USA 18 19 Contact: 20 Dr. Joseph Dougherty 21 Dougherty Lab, Department of Genetics 22 660 S. Euclid Ave, Campus Box 8232 23 St. Louis, MO 63110-1093 24 P: 314-286-0752 25 F: 314-362-7855 26 E: [email protected] 27 28 Number of Pages: 29 29 Number of Figures: 10 30 Number of Tables: 8 31 Abstract: 198 words 32 Introduction: 678 words 33 Discussion: 1373 34 35 CONFLICT OF INTEREST 36 37 JDD has received royalties related to TRAP in the past. No other authors declare a conflict of 38 interest. 39 40 ACKNOWLEDGEMENTS 41 42 We would like to thank M. Wong for Thy-1 mice, K. Monk, C. Weichselbaum and members of 43 the Dougherty lab for helpful comments, N. Pisat, N. Kopp, K. Sakers, and S. Pyfrom for training, 44 advice, and assistance. 1 45 ABSTRACT 46 47 Localized translation in neurites helps regulate synaptic strength and development. 48 Dysregulation of local translation is associated with many neurological disorders. However, due 49 to technical limitations, study of this phenomenon has largely been limited to brain regions 50 with laminar organization of dendrites such as the hippocampus or cerebellum. It has not been 51 examined in the cortex, a region of importance for most neurological disorders, where 52 dendrites of each neuronal population are densely intermingled with cell bodies of others. 53 Therefore, we have developed a novel method, SynapTRAP, which combines 54 synaptoneurosomal fractionation with Translating Ribosome Affinity Purification to identify 55 ribosome bound mRNA in processes of genetically defined cell types. We demonstrate 56 SynapTRAP’s efficacy and report local translation in the cortex of mice, where we identify a 57 subset of mRNAs that are translated in dendrites by neuronal ribosomes. These mRNAs have 58 disproportionately longer lengths, enrichment for FMRP binding and G-quartets, and their 59 genes are under greater evolutionary constraint in humans. In addition, we show that 60 alternative splicing likely regulates this phenomenon. Overall, SynapTRAP allows for rapid 61 isolation of cell-type specific localized translation and is applicable to classes of previously 62 inaccessible neuronal and non-neuronal cells in vivo. 63 64 65 SIGNIFICANCE STATEMENT 66 67 Instructions for making proteins are found in the genome, housed within the nucleus of each 68 cell. These are then copied as RNA and exported to manufacture new proteins. However, in the 69 brain, memory is thought to be encoded by strengthening individual connections (synapses) 70 between neurons far from the nucleus. Thus, to efficiently make new proteins specifically 71 where they are needed, neurons can transport RNAs to sites near synapses to locally produce 72 proteins. Importantly, several mutations that cause autism disrupt this process. It has been 73 assumed this process occurs in all brain regions, but has never been measured in the cortex. 74 We applied a newly developed method measure to study, for the first time, local translation in 75 cortical neurons. 76 77 INTRODUCTION 78 79 As all mRNA must come from a single cellular location (the nucleus) there is extensive post- 80 transcriptional regulation of RNA within cells, including localization of mRNA to specific 81 subcellular compartments. Localized translation of mRNA in specific subcellular compartments 82 allows more precise regulation of local protein concentrations, and thus modifies the functional 83 capacity of the compartment. A clear example exists in the nervous system where neurons 84 demonstrate remarkable capacity for regulated local translation with individual mRNAs 85 accumulating near activated synapses (Oswald Steward et al. 2014). Local translation in these 86 dendrites supports synaptic strengthening (Kang and Schuman 1996). While ultrastructural 87 evidence for localized translation in dendrites was first provided over thirty years ago (O. 2 88 Steward and Levy 1982), it is still not clear which mRNAs are translated in cortical neurites nor 89 how this translational profile changes across cell types. 90 91 In addition, several psychiatric diseases have been observed to have perturbations in neuronal 92 local translation. For example, Fragile X syndrome, an Autism Spectrum Disorder (ASD)-related 93 syndrome, and other ASD-associated disorders, are caused by mutations in known local 94 translational regulators (Kelleher and Bear 2008; Ronesi and Huber 2008). Interestingly, it has 95 been shown that the degree of translational perturbation in models of Fragile X can vary across 96 brain regions (Qin et al. 2005); however, it is unclear the extent to which local translation also 97 differs across cell types in response to disease, development, or activity. To study these kinds of 98 perturbations, a method is needed to enrich for both processes-localized and cell-type specific 99 mRNA. 100 101 Previously, studies have utilized cell-culture based methods for examining the RNAs found in 102 neurites in vitro (Poon et al. 2006; Taliaferro et al. 2016) in addition to physical methods (LCM 103 and manual dissection) to isolate processes of certain populations in vivo. In vivo, these 104 isolation techniques were limited to cell types with neurites that grow in a physical layer distant 105 from the cell body, including the CA1 synaptic neuropil of the hippocampus (Cajigas et al. 2012; 106 Poon et al. 2006; Ainsley et al. 2014; Zhong, Zhang, and Bloch 2006), and the Purkinje cell layer 107 of the cerebellum (Kratz et al. 2014). Likewise, a recent study was able to capture translating 108 mRNAs from retinal-geniculate axons because only retinal cells expressed the necessary tag 109 (Shigeoka et al. 2016). While valuable for assessing local translation in these limited neuronal 110 cell types, this approach is unable to assess localization in the intermingled dendrites of 111 neurons found in most regions of the brain, and provides no evidence as to whether the mRNAs 112 are on ribosomes, a prerequisite for local translation. The development of a method to isolate 113 the ribosome bound mRNAs from neurites of densely intermingled cells would allow analysis of 114 local translation across a larger number of cell types in the central nervous system (CNS). 115 116 Here, we describe SynapTRAP, a novel method that permits the harvesting of ribosomes from 117 the intermingled processes of specific cell types in vivo. This method combines subcellular 118 fractionation on a sucrose-percoll gradient with Translating Ribosome Affinity Purification 119 (TRAP) to identify ribosome bound mRNA from neurons in the synaptoneurosomal fraction 120 (SNF).