Functionally Diverse Dendritic Mrnas Rapidly Associate with Ribosomes Following a Novel Experience

ARTICLE Received 19 Feb 2014 | Accepted 24 Jun 2014 | Published 29 Jul 2014 DOI: 10.1038/ncomms5510 Functionally diverse dendritic mRNAs rapidly associate with ribosomes following a novel experience Joshua A. Ainsley1, Laurel Drane1, Jonathan Jacobs1, Kara A. Kittelberger1,w & Leon G. Reijmers1 The subcellular localization and translation of messenger RNA (mRNA) supports functional differentiation between cellular compartments. In neuronal dendrites, local translation of mRNA provides a rapid and specific mechanism for synaptic plasticity and memory forma- tion, and might be involved in the pathophysiology of certain brain disorders. Despite the importance of dendritic mRNA translation, little is known about which mRNAs can be translated in dendrites in vivo and when their translation occurs. Here we collect ribosome- bound mRNA from the dendrites of CA1 pyramidal neurons in the adult mouse hippocampus. We find that dendritic mRNA rapidly associates with ribosomes following a novel experience consisting of a contextual fear conditioning trial. High throughput RNA sequencing followed by machine learning classification reveals an unexpected breadth of ribosome-bound dendritic mRNAs, including mRNAs expected to be entirely somatic. Our findings are in agreement with a mechanism of synaptic plasticity that engages the acute local translation of functionally diverse dendritic mRNAs. 1 Department of Neuroscience, Tufts University, Boston, Massachusetts 02111, USA. w Present address: Center for Translational Social Neuroscience, Yerkes National Primate Research Center, Emory University, Atlanta, Georgia 30322, USA. Correspondence and requests for materials should be addressed to L.R. (email: [email protected]). NATURE COMMUNICATIONS | 5:4510 | DOI: 10.1038/ncomms5510 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5510 ubcellular localization of proteins is a highly regulated sought to determine if this also occurs in vivo. To detect process that enables different cellular compartments to activity-induced changes, we collected ribosome-bound mRNA Sperform specialized functions. Local translation of messenger from mice in a resting state and from mice subjected to a novel RNA (mRNA) is an efficient mechanism for precise subcellular experience. Specifically, we collected CA1 tissue punches from localization of proteins. A striking example of local mRNA transgenic mice either resting in their home cage or immediately translation is found in neurons, where mRNA is trafficked to distal after a 500-s contextual fear conditioning trial. Expression of parts of neuronal processes far from their site of transcription in the EGFP-L10a did not affect contextual fear conditioning behaviour nucleus. Translation of mRNA in dendrites and axons is performed (Supplementary Fig. 1a), and EGFP-L10a expression was similar by locally present ribosomes1–3, thereby enabling neurons to between home cage and contextual fear conditioned mice develop and modify their synaptic connections with high spatial (Supplementary Fig. 1b). Tissue punches were placed in either and temporal resolution4. For example, mice with a selective the CA1 layer containing the dendrites of pyramidal neurons depletion of Camk2a mRNA from neuronal dendrites, but not (dendritic punches) or in the CA1 layer containing the soma of soma, have synaptic plasticity and memory deficits5. Accordingly, pyramidal neurons (somatic punches) (Fig. 1f). Accurately placed dendritic mRNA translation has been proposed as a critical dendritic and somatic punches from individual mice were pooled mechanism of memory storage, and has been implicated in the and subjected to an immunoprecipitation protocol optimized for pathophysiology of certain intellectual disability disorders such as low background (Supplementary Fig. 1c–f). This enabled us to Fragile X Syndrome6,7. Despite the widely accepted importance of obtain immunoprecipitate (IP) fractions containing either dendritic mRNA translation, the full set of genes that encode dendritic or somatic mRNA bound to GFP-tagged ribosomes dendritically localized mRNA, and the conditions that drive local (Fig. 1g). The mRNA in both the IP and the supernatant (SN) dendritic translation, are currently unknown. fractions was isolated for further analysis. In agreement with the Here we present a novel approach based on the expression of predicted presence of dendritic mRNA in the IP of dendritic epitope-tagged ribosomes in the dendrites of CA1 pyramidal punches, quantitative PCR (qPCR) analysis showed enrichment neurons in the mouse hippocampus. This approach enables of the known dendritic mRNA Camk2a in the IP of home cage immunoprecipitation of ribosome-bound dendritic mRNA from and contextual fear conditioned mice, while the glial gene Gfap the intact brain, thereby resolving two challenges that hampered was enriched in the SN of both groups, illustrating the specificity previous studies. First, because of the inability to physically of the immunoprecipitation (Fig. 1h). separate dendrites from intact brain tissue, previous in vivo studies relied on samples containing a mix of dendritic and non- dendritic mRNA without robust means to distinguish these two Dendritic mRNAs rapidly bind ribosomes upon novel experi- sources8,9. Our approach circumvents the challenge of physically ence. We used high throughput RNA sequencing (RNA-Seq) to separating dendrites from the intact brain, which enabled us to perform genome-wide characterization of dendritic and somatic identify 1,890 dendritically localized mRNAs that include many mRNA bound to GFP-tagged ribosomes (Supplementary mRNAs assumed to be restricted to the soma. Second, previous Table 1). Analysis of sequencing read distribution revealed that genome-wide studies relied on total mRNA samples with a smaller number of sequencing reads aligned to the 30 unknown ribosome-binding status, making their functional untranslated regions (30UTR) in IP samples compared with SN relevance unclear10. Here we report that a novel experience samples (Fig. 2a). Further characterization of this 30UTR deple- increases the association of a large number of dendritic mRNAs tion revealed a decrease in IP read coverage B200 nucleotides with ribosomes, thereby providing strong support for the after the stop codon with a concomitant increase in SN read functional relevance of these dendritic mRNAs. Our findings coverage (Fig. 2b). This loss of 30UTR reads in the IP can be provide the first in vivo evidence for broad activity-induced explained by RNA fragmentation that occurred during the tissue changes in ribosome binding of dendritic mRNA. processing steps and immunoprecipitation. Because ribosomes bind to the 50UTR and coding sequence (CDS) of transcripts and fall off at the stop codon preceding the 30 UTR, random RNA Results fragmentation would produce distal 30UTR fragments that are not Collecting ribosome-bound mRNA from in vivo dendrites.We associated with GFP-tagged ribosomes and therefore end up in developed a strategy to collect in vivo dendritic mRNA from adult the SN (Fig. 2c). The depletion of 30UTR reads observed in the IP mouse brains using ribosome immunoprecipitation. Specifically, samples therefore supports the predicted ribosome-bound state of we created a transgenic mouse in which the Camk2a promoter the mRNAs in the IP samples. drives expression of the epitope-tagged ribosomal protein (EGFP- The RNA-Seq data enabled us to further test the expected L10a) through the tetracycline transactivator (tTA)–tetO sys- enrichment of dendritic mRNA in the IP samples by looking at a tem11 (Fig. 1a). Transgenic mice express EGFP-L10a at high larger set of control genes than would be practical with qPCR levels in the striatum and in CA1 region of the hippocampus analysis. For this, we used a positive-control set of 74 genes that (Fig. 1b,c). The interneuron marker Gad1 did not overlap with within the CA1 are exclusively expressed in the pyramidal the EGFP-L10a expression, confirming that within the CA1 neurons ( þ pyr; for example, Camk2a and Ddn; Fig. 3a), and a region the transgene expression is specific to excitatory pyramidal negative-control set of 124 genes that within the CA1 are neurons (Fig. 1d). EGFP-L10a expression was seen in the exclusively expressed outside of the pyramidal neurons ( À pyr; dendrites of CA1 pyramidal neurons (Fig. 1e), in agreement for example Gad1 and Gfap; Fig. 3b). The þ pyr and À pyr lists with previous reports of dendritic ribosome localization1–3. Since were created by manually curating the in situ images of 1,238 previous studies demonstrated the functional incorporation of genes available through the Allen Mouse Brain Atlas EGFP-L10a into functional translating ribosomes12–14, the (Supplementary Data 1). The levels of þ pyr and À pyr mRNAs observed dendritic expression pattern of EGFP-L10a suggested were quantified as Fragments Per Kilobase of gene length per the possibility of collecting dendritic mRNA by immuno- Million mapped reads (FPKM) values (see Methods for details). precipitating dendritically localized green fluorescent protein Because of the decreased 30UTR read coverage starting B200 (GFP)-tagged ribosomes. nucleotides after the stop codon in the IP samples (Fig. 2b), we As previous in vitro studies found that ribosome association of calculated mRNA levels using a FPKM value based on the RNA- dendritic mRNA changes after neuronal activation15–21,we Seq reads that mapped upstream of 200 nucleotides

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