University of Massachusetts Medical School eScholarship@UMMS GSBS Student Publications Graduate School of Biomedical Sciences 2017-07-18 Dynamic Control of Dendritic mRNA Expression by CNOT7 Regulates Synaptic Efficacy and Higher Cognitive Function Rhonda L. McFleder University of Massachusetts Medical School Et al. Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_sp Part of the Biochemistry Commons, and the Molecular and Cellular Neuroscience Commons Repository Citation McFleder RL, Mansur F, Richter JD. (2017). Dynamic Control of Dendritic mRNA Expression by CNOT7 Regulates Synaptic Efficacy and Higher Cognitive Function. GSBS Student Publications. https://doi.org/ 10.1016/j.celrep.2017.06.078. Retrieved from https://escholarship.umassmed.edu/gsbs_sp/2003 Creative Commons License This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License. This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Student Publications by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. Article Dynamic Control of Dendritic mRNA Expression by CNOT7 Regulates Synaptic Efficacy and Higher Cognitive Function Graphical Abstract Authors Rhonda L. McFleder, Fernanda Mansur, Joel D. Richter Correspondence rhonda.mcfl[email protected] (R.L.M.), [email protected] (J.D.R.) In Brief McFleder et al. find that CNOT7, the major deadenylase in eukaryotes, has a specialized role in neurons. CNOT7 regulates the dendritic localization and translation of specific mRNAs and mediates synaptic plasticity and learning and memory in mice. Highlights Accession Numbers d CNOT7 regulates dendritic mRNA transport and local GSE88777 translation d CNOT7 regulates synaptic plasticity in cultured neurons d Dendritic CNOT7 is destroyed during synaptic stimulation d Depletion of CNOT7 from the hippocampus impairs cognitive function McFleder et al., 2017, Cell Reports 20, 683–696 July 18, 2017 ª 2017 The Author(s). http://dx.doi.org/10.1016/j.celrep.2017.06.078 Cell Reports Article Dynamic Control of Dendritic mRNA Expression by CNOT7 Regulates Synaptic Efficacy and Higher Cognitive Function Rhonda L. McFleder,1,2,* Fernanda Mansur,1 and Joel D. Richter1,* 1Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA 2Lead Contact *Correspondence: rhonda.mcfl[email protected] (R.L.M.), [email protected] (J.D.R.) http://dx.doi.org/10.1016/j.celrep.2017.06.078 SUMMARY analysis of RNAs in the mammalian hippocampal neuropil, a region rich in axons and dendrites, there are >2,500 mRNAs Translation of mRNAs in dendrites mediates synaptic localized to neurites (Cajigas et al., 2012). It is almost axiomatic plasticity, the probable cellular basis of learning and that regulation of these mRNAs is necessary to ensure localized memory. Coordination of translational inhibitory and translation in response to synaptic activity (Buxbaum et al., stimulatory mechanisms, as well as dendritic trans- 2015); when this regulation goes awry, autism and other neuro- port of mRNA, is necessary to ensure proper control logical disorders can ensue (Kelleher and Bear, 2008). of this local translation. Here, we find that the deade- Repression of translation is necessary during mRNA transport to dendrites, but, even when localized, silencing must continue nylase CNOT7 dynamically regulates dendritic mRNA until synaptic activity occurs (Costa-Mattioli et al., 2009). This translation and transport, as well as synaptic plas- mRNA masking takes multiple forms and involves repression at ticity and higher cognitive function. In cultured hippo- initiation (Krichevsky and Kosik, 2001) and elongation (Sutton campal neurons, synaptic stimulation induces a rapid et al., 2007; Richter and Coller, 2015). Although the mecha- decrease in CNOT7, which, in the short-term, results nism(s) and/or factors by which these and other processes con- in poly(A) tail lengthening of target mRNAs. However, trol mRNA expression in dendrites is often unclear, it is evident at later times following stimulation, decreased poly(A) that they frequently involve microRNAs (miRNAs) (Ashraf et al., and dendritic localization of mRNA take place, similar 2006; Bicker et al., 2013; Schratt, 2009) or RNA binding proteins to what is observed when CNOT7 is depleted over (Darnell, 2013; Eom et al., 2013; Udagawa et al., 2015). Many of several days. In mice, CNOT7 is essential for hip- these trans-acting factors utilize deadenylation as an initiation pocampal-dependent learning and memory. This step to silence mRNAs (Ashraf et al., 2006; Giorgi et al., 2007; Richter, 2007). Mechanistically, deadenylases repress transla- study identifies CNOT7 as an important regulator of tion by shortening poly(A) tails and thereby abrogate association RNA transport and translation in dendrites, as well of poly(A) binding protein (PABP), which is important for circular- as higher cognitive function. izing mRNA and recruiting the 40S ribosomal subunit (Richter, 2007). The deadenylase PARN (poly(A) ribonuclease) was pre- INTRODUCTION sumed to be the enzyme responsible for initiating repression for at least a subset of dendritic mRNAs because it interacts Experience-induced modifications of synapses are thought to with the RNA-binding protein CPEB1 (Richter, 2007; Udagawa serve as the molecular basis of learning and memory (Kandel, et al., 2012). In oocytes, CPEB1 regulates translation by recruit- 2001). Synaptic plasticity provides long-lasting alterations in ing both Gld2, a non-canonical poly(A) polymerase, and PARN to neuronal communication that allows memories to be retained specific mRNAs (Richter, 2007). Upon phosphorylation of CPEB, for many years (Costa-Mattioli et al., 2009). Of the several forms PARN is expelled from the ribonucleoprotein complex, which re- of synaptic plasticity, at least three are dependent on protein sults in polyadenylation and subsequently translation of target synthesis: long-lasting neurotrophin-induced enhancement of mRNAs. In the brain, CPEB1 and Gld2 mediate translation in synaptic efficacy (Kang and Schuman, 1996), metabotropic dendrites in response to synaptic stimulation (Wu et al., 1998; glutamate receptor long-term depression (mGluR-LTD) (Huber Zearfoss et al., 2008; Udagawa et al., 2012). However, because and Bear, 2000), and N-methyl-D-aspartate late-phase long- depletion of PARN had no discernable effect on synaptic plas- term potentiation (L-LTP) (Bradshaw et al., 2003; Miller et al., ticity, the deadenylase essential for repressing translation was 2002). The necessity for new protein production in synaptic plas- unclear (Udagawa et al., 2012). We surmised that one or perhaps ticity is independent of transcription and relies upon mRNAs and multiple deadenylases would likely govern poly(A) tail length of translation factors in dendrites (Bradshaw et al., 2003; Kang and several different populations of dendritic mRNAs to impact Schuman, 1996; Martin and Kandel, 1996). Following synaptic learning and memory. stimulation, dendritic mRNAs are translated at postsynaptic The carbon catabolite repression 4 negative on TATA-less sites where their protein products modify synapse structure (CNOT) is a conserved, multisubunit complex that functions as and function (Sutton and Schuman, 2006). Based on sequence the major deadenylase in yeast to humans (Tucker et al., 2001; Cell Reports 20, 683–696, July 18, 2017 ª 2017 The Author(s). 683 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Temme et al., 2004; Schwede et al., 2008). The mammalian analysis of mouse brain lysates revealed CNOT7 to be present CNOT complex consists of four functional deadenylase en- in synaptosomes (Figure S1D), suggesting that it may have a zymes: CNOT6, CNOT6L, CNOT7, and CNOT8 (Lau et al., synaptic function. 2009). Of these, CNOT7 and CNOT8 regulate poly(A) tail length Using fluorescence in situ hybridization (FISH) with labeled on the majority of mRNAs (Schwede et al., 2008). Although oligo(dT), we analyzed poly(A) in cultured hippocampal neurons CNOT7 and CNOT8 are 75% homologous, they have distinct 4 days after infection with a lentivirus expressing either a targets, probably because they are differentially expressed and CNOT7-specific shRNA or a scrambled control (Figure 1B). Sur- associate with different complexes (Lau et al., 2009). CNOT7 prisingly, CNOT7 knockdown (CNOT7KD) resulted in an 50% levels are enriched in neurons relative to CNOT8 (Chen et al., decrease in dendritic poly(A), not the expected increase. This 2011); CNOT7 also associates with both the miRNA machinery result occurred mostly on dendritic mRNA because the cell (Fabian et al., 2009; Piao et al., 2010) and CPEB1 (Ogami et al., body poly(A) FISH signal (Figure 1C) as well as total cellular 2014). These data suggest that CNOT7 might influence transla- poly(A) (Figure S1E) was not significantly affected. The decrease tion in dendrites and as a consequence modify synaptic trans- in dendritic poly(A) signal was replicated with a second shRNA mission and higher cognitive function. targeting CNOT7 (Figures S1F and S1G). Knockdown of Here, we identify CNOT7 as an important enzyme that CNOT8, the CNOT7 paralog, had no effect on dendritic poly(A) regulates local translation, synapse efficacy, and learning and (Figure S1H). We ectopically expressed a catalytically