Untranslated regions of brain-derived neurotrophic factor mRNA control its translatability and subcellular localization
2023; Elsevier BV; Volume: 299; Issue: 2 Linguagem: Inglês
10.1016/j.jbc.2023.102897
ISSN1083-351X
AutoresIngrid Lekk, Florencia Cabrera-Cabrera, Giorgio Turconi, Jürgen Tuvikene, Eli‐Eelika Esvald, Annika Rähni, Laoise Casserly, Daniel R. Garton, Jaan‐Olle Andressoo, Tõnis Timmusk, Indrek Koppel,
Tópico(s)Nuclear Receptors and Signaling
ResumoBrain-derived neurotrophic factor (BDNF) promotes neuronal survival and growth during development. In the adult nervous system, BDNF is important for synaptic function in several biological processes such as memory formation and food intake. In addition, BDNF has been implicated in development and maintenance of the cardiovascular system. The Bdnf gene comprises several alternative untranslated 5′ exons and two variants of 3′ UTRs. The effects of these entire alternative UTRs on translatability have not been established. Using reporter and translating ribosome affinity purification analyses, we show that prevalent Bdnf 5′ UTRs, but not 3′ UTRs, exert a repressive effect on translation. However, contrary to previous reports, we do not detect a significant effect of neuronal activity on BDNF translation. In vivo analysis via knock-in conditional replacement of Bdnf 3′ UTR by bovine growth hormone 3′ UTR reveals that Bdnf 3′ UTR is required for efficient Bdnf mRNA and BDNF protein production in the brain, but acts in an inhibitory manner in lung and heart. Finally, we show that Bdnf mRNA is enriched in rat brain synaptoneurosomes, with higher enrichment detected for exon I–containing transcripts. In conclusion, these results uncover two novel aspects in understanding the function of Bdnf UTRs. First, the long Bdnf 3′ UTR does not repress BDNF expression in the brain. Second, exon I–derived 5′ UTR has a distinct role in subcellular targeting of Bdnf mRNA. Brain-derived neurotrophic factor (BDNF) promotes neuronal survival and growth during development. In the adult nervous system, BDNF is important for synaptic function in several biological processes such as memory formation and food intake. In addition, BDNF has been implicated in development and maintenance of the cardiovascular system. The Bdnf gene comprises several alternative untranslated 5′ exons and two variants of 3′ UTRs. The effects of these entire alternative UTRs on translatability have not been established. Using reporter and translating ribosome affinity purification analyses, we show that prevalent Bdnf 5′ UTRs, but not 3′ UTRs, exert a repressive effect on translation. However, contrary to previous reports, we do not detect a significant effect of neuronal activity on BDNF translation. In vivo analysis via knock-in conditional replacement of Bdnf 3′ UTR by bovine growth hormone 3′ UTR reveals that Bdnf 3′ UTR is required for efficient Bdnf mRNA and BDNF protein production in the brain, but acts in an inhibitory manner in lung and heart. Finally, we show that Bdnf mRNA is enriched in rat brain synaptoneurosomes, with higher enrichment detected for exon I–containing transcripts. In conclusion, these results uncover two novel aspects in understanding the function of Bdnf UTRs. First, the long Bdnf 3′ UTR does not repress BDNF expression in the brain. Second, exon I–derived 5′ UTR has a distinct role in subcellular targeting of Bdnf mRNA. Brain-derived neurotrophic factor (BDNF) (1Barde Y.A. Edgar D. Thoenen H. Purification of a new neurotrophic factor from mammalian brain.EMBO J. 1982; 1: 549-553Crossref PubMed Scopus (1402) Google Scholar), a member of the neurotrophin family, supports neuronal survival in mammalian development (2Binder D.K. Scharfman H.E. Brain-derived neurotrophic factor.Growth Factors. 2004; 22: 123-131Crossref PubMed Scopus (1018) Google Scholar, 3Bibel M. Barde Y.A. Neurotrophins: key regulators of cell fate and cell shape in the vertebrate nervous system.Genes Dev. 2000; 14: 2919-2937Crossref PubMed Scopus (896) Google Scholar). In the adult brain, BDNF modulates synaptic activity and plays central roles in memory formation and maintenance (4Bramham C.R. Panja D. BDNF regulation of synaptic structure, function, and plasticity.Neuropharmacology. 2014; https://doi.org/10.1016/j.neuropharm.2013.08.012Crossref Scopus (30) Google Scholar, 5Lu B. Nagappan G. Lu Y. BDNF and synaptic plasticity, cognitive function, and dysfunction.Handb Exp. Pharmacol. 2014; 220: 223-250Crossref PubMed Scopus (621) Google Scholar). Dysregulated BDNF has been implicated in depression and other central nervous system disorders (6Wang C.S. Kavalali E.T. Monteggia L.M. BDNF signaling in context: from synaptic regulation to psychiatric disorders.Cell. 2022; 185: 62-76Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). BDNF is a key regulator of food intake and body weight (7Xu B. Xie X. Neurotrophic factor control of satiety and body weight.Nat. Rev. Neurosci. 2016; 17: 282-292Crossref PubMed Scopus (137) Google Scholar), and Bdnf haploinsufficiency has been causally linked to severe hyperphagia in WAGR syndrome (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) patients (8Han J.C. Liu Q.-R. Jones M. Levinn R.L. Menzie C.M. Jefferson-George K.S. et al.Brain-derived neurotrophic factor and obesity in the WAGR syndrome.N. Engl. J. Med. 2008; 359: 918-927Crossref PubMed Scopus (247) Google Scholar). In addition to the nervous system, Bdnf mRNA is highly expressed in the heart and lung (9Maisonpierre P.C. Belluscio L. Friedman B. Alderson R.F. Wiegand S.J. Furth M.E. et al.NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression.Neuron. 1990; 5: 501-509Abstract Full Text PDF PubMed Scopus (1102) Google Scholar). BDNF has been shown to regulate heart development (10Donovan M.J. Lin M.I. Wiegn P. Ringstedt T. Kraemer R. Hahn R. et al.Brain derived neurotrophic factor is an endothelial cell survival factor required for intramyocardial vessel stabilization.Development. 2000; 127: 4531-4540Crossref PubMed Google Scholar) and modulate adult cardiac contractility (11Feng N. Huke S. Zhu G. Tocchetti C.G. Shi S. Aiba T. et al.Constitutive BDNF/TrkB signaling is required for normal cardiac contraction and relaxation.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 1880-1885Crossref PubMed Scopus (6) Google Scholar, 12Fulgenzi G. Tomassoni-Ardori F. Babini L. Becker J. Barrick C. Puverel S. et al.BDNF modulates heart contraction force and long-term homeostasis through truncated TrkB.T1 receptor activation.J. Cell Biol. 2015; 210: 1003-1012Crossref PubMed Scopus (63) Google Scholar). In the lung, BDNF-TrkB signaling promotes regeneration of alveolar epithelial cells after injury (13Paris A.J. Hayer K.E. Oved J.H. Avgousti D.C. Toulmin S.A. Zepp J.A. et al.STAT3-BDNF-TrkB signalling promotes alveolar epithelial regeneration after lung injury.Nat. Cell Biol. 2020; 22: 1197-1210Crossref PubMed Scopus (52) Google Scholar). Owing to its pleiotropic functions, molecular mechanisms governing BDNF expression are of high clinical interest. The Bdnf gene has a complex structure with several promoters preceding alternative 5′ exons that are spliced with a common 3′ exon encoding the BDNF protein (14Aid T. Kazantseva A. Piirsoo M. Palm K. Timmusk T. Mouse and rat BDNF gene structure and expression revisited.J. Neurosci. Res. 2007; 85: 525-535Crossref PubMed Scopus (789) Google Scholar, 15Pruunsild P. Kazantseva A. Aid T. Palm K. Timmusk T. Dissecting the human BDNF locus: bidirectional transcription, complex splicing, and multiple promoters.Genomics. 2007; 90: 397-406Crossref PubMed Scopus (550) Google Scholar) (Fig. S1A). The multipromoter arrangement presents a convenient way to fine-tune BDNF expression in a cell type–specific manner and as a response to neuronal activity and other stimuli (16West A.E. Pruunsild P. Timmusk T. Neurotrophins: transcription and translation.Handb Exp. Pharmacol. 2014; 220: 67-100Crossref PubMed Scopus (80) Google Scholar). Promoter IV–driven BDNF has a critical role in the development of cortical inhibition (17Sakata K. Woo N.H. Martinowich K. Greene J.S. Schloesser R.J. Shen L. et al.Critical role of promoter IV-driven BDNF transcription in GABAergic transmission and synaptic plasticity in the prefrontal cortex.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 5942-5947Crossref PubMed Scopus (161) Google Scholar, 18Hong E.J. McCord A.E. Greenberg M.E. A biological function for the neuronal activity-dependent component of Bdnf transcription in the development of cortical inhibition.Neuron. 2008; 60: 610-624Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar) and its disruption in mice causes defects in sensory processing (19Hill J.L. Hardy N.F. Jimenez D.V. Maynard K.R. Kardian A.S. Pollock C.J. et al.Loss of promoter IV-driven BDNF expression impacts oscillatory activity during sleep, sensory information processing and fear regulation.Transl. Psych. 2016; 6: e873Crossref PubMed Scopus (0) Google Scholar). Disruption of promoter I– and II–driven BDNF in mice has been shown to induce aggressive behavior with accompanying expression changes in serotonin signaling pathways (20Maynard K.R. Hill J.L. Calcaterra N.E. Palko M.E. Kardian A. Paredes D. et al.Functional role of BDNF production from unique promoters in aggression and serotonin signaling.Neuropsychopharmacology. 2016; 41: 1943-1955Crossref PubMed Scopus (68) Google Scholar). In addition to allowing transcriptional control over BDNF expression, the multipromoter arrangement generates Bdnf transcripts with alternative 5′ UTRs. Compared to alternative promoter use, the roles of alternative Bdnf UTRs are much less clearly understood. By forming secondary structures and interacting with different RNA-binding proteins, 5′ UTRs can affect several aspects in the fate of an mRNA including its subcellular localization and translation efficiency (21Andreassi C. Crerar H. Riccio A. Post-transcriptional processing of mRNA in neurons: the vestiges of the RNA world drive transcriptome diversity.Front. Mol. Neurosci. 2018; https://doi.org/10.3389/fnmol.2018.00304Crossref PubMed Scopus (17) Google Scholar). The effect of alternative Bdnf 5′ UTRs on subcellular localization in neurons has been thoroughly studied, concluding that exon II– and VI–containing UTRs promote dendritic targeting (22Chiaruttini C. Sonego M. Baj G. Simonato M. Tongiorgi E. BDNF mRNA splice variants display activity-dependent targeting to distinct hippocampal laminae.Mol. Cell Neurosci. 2008; 37: 11-19Crossref PubMed Scopus (136) Google Scholar, 23Chiaruttini C. Vicario A. Li Z. Baj G. Braiuca P. Wu Y. et al.Dendritic trafficking of BDNF mRNA is mediated by translin and blocked by the G196A (Val66Met) mutation.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 16481-16486Crossref PubMed Scopus (179) Google Scholar, 24Colliva A. Tongiorgi E. Distinct role of 5'UTR sequences in dendritic trafficking of BDNF mRNA: additional mechanisms for the BDNF splice variants spatial code.Mol. Brain. 2021; 14: 10Crossref PubMed Scopus (8) Google Scholar). The effect of Bdnf UTRs on reporter translatability has been studied in SH-SY5Y cells, where the authors identified a differential response pattern upon stimulation of cells with an array of neurotransmitters (25Vaghi V. Polacchini A. Baj G. Pinheiro V.L.M. Vicario A. Tongiorgi E. Pharmacological profile of brain-derived neurotrophic factor (BDNF) splice variant translation using a novel drug screening assay: a "quantitative code".J. Biol. Chem. 2014; 289: 27702-27713Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Previously, we have shown that exon I– and exon IV–containing mRNAs repress BDNF protein translation in primary cortical neurons (26Koppel I. Tuvikene J. Lekk I. Timmusk T. Efficient use of a translation start codon in BDNF exon I.J. Neurochem. 2015; 134: 1015-1025Crossref PubMed Scopus (18) Google Scholar). However, a comparative analysis of all 5′ UTRs in primary neurons is currently missing. In this study, we have for the first time systematically analyzed the effects of all alternative rat Bdnf UTRs on mRNA translatability in rat primary cortical neurons and conclude that commonly used 5′ UTRs repress translation to a similar extent. Use of alternative polyadenylation sites generates Bdnf mRNAs with 3′ UTRs of two different lengths (9Maisonpierre P.C. Belluscio L. Friedman B. Alderson R.F. Wiegand S.J. Furth M.E. et al.NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression.Neuron. 1990; 5: 501-509Abstract Full Text PDF PubMed Scopus (1102) Google Scholar, 27Timmusk T. Palm K. Metsis M. Reintam T. Paalme V. Saarma M. et al.Multiple promoters direct tissue-specific expression of the rat BDNF gene.Neuron. 1993; 10: 475-489Abstract Full Text PDF PubMed Scopus (743) Google Scholar). It has been reported that Bdnf mRNA containing the long, but not short 3′UTR, is targeted to hippocampal dendrites, where it undergoes local translation (28An J.J. Gharami K. Liao G.-Y. Woo N.H. Lau A.G. Vanevski F. et al.Distinct role of long 3' UTR BDNF mRNA in spine morphology and synaptic plasticity in hippocampal neurons.Cell. 2008; 134: 175-187Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar). This finding, however, has been challenged by studies failing to detect Bdnf mRNA (29Will T.J. Tushev G. Kochen L. Nassim-Assir B. Cajigas I.J. tom Dieck S. et al.Deep sequencing and high-resolution imaging reveal compartment-specific localization of Bdnf mRNA in hippocampal neurons.Sci. Signal. 2013; 6: rs16Crossref PubMed Scopus (26) Google Scholar) or protein (30Dieni S. Matsumoto T. Dekkers M. Rauskolb S. Ionescu M.S. Deogracias R. et al.BDNF and its pro-peptide are stored in presynaptic dense core vesicles in brain neurons.J. Cell Biol. 2012; 196: 775-788Crossref PubMed Scopus (243) Google Scholar) in dendrites. Bdnf long 3′ UTR has been reported to act as a translational suppressor at rest, getting derepressed by neuronal activity and allowing BDNF synthesis to proceed (31Lau A.G. Irier H.A. Gu J. Tian D. Ku L. Liu G. et al.Distinct 3'UTRs differentially regulate activity-dependent translation of brain-derived neurotrophic factor (BDNF).Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 15945-15950Crossref PubMed Scopus (175) Google Scholar). Bdnf 3′ UTRs contain multiple miRNA-binding sites and several of these have been experimentally shown to modulate Bdnf translation (32Varendi K. Kumar A. Härma M.-A. Andressoo J.-O. miR-1, miR-10b, miR-155, and miR-191 are novel regulators of BDNF.Cell Mol. Life Sci. 2014; 71: 4443-4456Crossref PubMed Scopus (119) Google Scholar, 33Caputo V. Sinibaldi L. Fiorentino A. Parisi C. Catalanotto C. Pasini A. et al.Brain derived neurotrophic factor (BDNF) expression is regulated by microRNAs miR-26a and miR-26b allele-specific binding.PLoS One. 2011; 6e28656Crossref Scopus (102) Google Scholar, 34Mellios N. Huang H.-S. Grigorenko A. Rogaev E. Akbarian S. A set of differentially expressed miRNAs, including miR-30a-5p, act as post-transcriptional inhibitors of BDNF in prefrontal cortex.Hum. Mol. Genet. 2008; 17: 3030-3042Crossref PubMed Scopus (223) Google Scholar). Additionally, global perturbation of miRNA processing by CamK2a-Cre conditional knockout of Dicer has been shown to increase BDNF protein levels (35Konopka W. Kiryk A. Novak M. Herwerth M. Parkitna J.R. Wawrzyniak M. et al.MicroRNA loss enhances learning and memory in mice.J. Neurosci. 2010; 30: 14835-14842Crossref PubMed Scopus (247) Google Scholar). Truncation of Bdnf long 3′ UTR in mice displayed reduced Bdnf mRNA in the hypothalamus and developed severe hyperphagic obesity (36Liao G.-Y. An J.J. Gharami K. Waterhouse E.G. Vanevski F. Jones K.R. et al.Dendritically targeted Bdnf mRNA is essential for energy balance and response to leptin.Nat. Med. 2012; 18: 564-571Crossref PubMed Scopus (143) Google Scholar). Taken together, it has remained inconclusive whether Bdnf 3′ UTRs have a repressive or permissive effect on its expression. In this study, we use three different in vitro approaches—luciferase assay, translating ribosome affinity purification(TRAP), and de novo protein synthesis assay—to show that the long Bdnf 3′ UTR does not have repressive effects on protein synthesis. Moreover, by in vivo experiments, using a novel conditional 3′UTR replacement approach (37Mätlik K. Garton D.R. Montaño-Rodríguez A.R. Olfat S. Eren F. Casserly L. et al.Elevated endogenous GDNF induces altered dopamine signalling in mice and correlates with clinical severity in schizophrenia.Mol. Psych. 2022; https://doi.org/10.1038/s41380-022-01554-2Crossref PubMed Scopus (6) Google Scholar, 38Kakoki M. Tsai Y.-S. Kim H.-S. Hatada S. Ciavatta D.J. Takahashi N. et al.Altering the expression in mice of genes by modifying their 3′ regions.Dev. Cell. 2004; 6: 597-606Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), we demonstrate reduced BDNF expression in the brain, indicating that the Bdnf 3′ UTR indeed does not have a repressive effect in this tissue. In contrast, 3′ UTR replacement leads to higher BDNF levels in heart and lungs, suggesting differential 3′UTR–targeted mechanisms of posttranscriptional regulation of Bdnf expression in neuronal and nonneuronal tissues. Finally, we studied subcellular localization of Bdnf mRNA in synaptoneurosomes (SNs). We find that all major Bdnf mRNA isoforms (including long 3′ UTR–containing isoforms) are enriched in SNs, at levels comparable to Camk2a, a known synaptically enriched mRNA. Among Bdnf mRNAs, the highest synaptoneurosomal enrichment was detected for exon I–containing transcripts. Taken together, we demonstrate that in neurons, Bdnf 5′UTRs, but not 3′ UTRs, repress translation and show a previously unknown subcellular enrichment for exon I–containing transcripts. To investigate the effect of Bdnf UTRs on mRNA translatability in primary neurons, we used dual luciferase reporter assay. Cortical neurons were transfected with firefly luciferase constructs where either Bdnf or control 5′ and 3′ UTRs were cloned around the luc2P ORF (Figs. 1A and S1, A and B). For Bdnf 5′ UTRs, we included sequence variants arising from previously determined alternative transcriptional start sites (27Timmusk T. Palm K. Metsis M. Reintam T. Paalme V. Saarma M. et al.Multiple promoters direct tissue-specific expression of the rat BDNF gene.Neuron. 1993; 10: 475-489Abstract Full Text PDF PubMed Scopus (743) Google Scholar, 39Timmusk T. Persson H. Metsis M. Analysis of transcriptional initiation and translatability of brain-derived neurotrophic factor mRNAs in the rat brain.Neurosci. Lett. 1994; 177: 27-31Crossref PubMed Scopus (32) Google Scholar) (Fig. S1B). Seventeen out of the 21 alternative 5′ UTR variants tested displayed a repressive effect on luciferase activity (Fig. S1C), including all most commonly used variants arising from exons I, II(c), IV, and VI (Fig. 1B) (27Timmusk T. Palm K. Metsis M. Reintam T. Paalme V. Saarma M. et al.Multiple promoters direct tissue-specific expression of the rat BDNF gene.Neuron. 1993; 10: 475-489Abstract Full Text PDF PubMed Scopus (743) Google Scholar, 39Timmusk T. Persson H. Metsis M. Analysis of transcriptional initiation and translatability of brain-derived neurotrophic factor mRNAs in the rat brain.Neurosci. Lett. 1994; 177: 27-31Crossref PubMed Scopus (32) Google Scholar), and none of the 5′ UTRs showed increased translation compared to the control 5′ UTR (Figs. 1B and S1C). The strongest repressive effect was observed for the longest exon VI 5′ UTR (VI-1, > 100-fold repression), exon VIII variants (∼6-fold), exon I variants (∼3-fold), and the main variant of exon IIc (∼3-fold) (Fig. S1C). The hyper-repressive effect of VI-1 is in line with previous results (25Vaghi V. Polacchini A. Baj G. Pinheiro V.L.M. Vicario A. Tongiorgi E. Pharmacological profile of brain-derived neurotrophic factor (BDNF) splice variant translation using a novel drug screening assay: a "quantitative code".J. Biol. Chem. 2014; 289: 27702-27713Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) and arises from using an upstream ATG that generates an ORF out of frame with the luciferase ORF (Fig. S1, E and F). When analyzing the effect of 3′ UTRs, we found that the long, but not short 3′ UTR isoform, caused a decrease in luciferase signal (Fig. 1B). As alternative UTRs could also affect RNA stability (40Fukuchi M. Tsuda M. Involvement of the 3′-untranslated region of the brain-derived neurotrophic factor gene in activity-dependent mRNA stabilization.J. Neurochem. 2010; 115: 1222-1233Crossref PubMed Scopus (35) Google Scholar), we measured the levels of reporter RNA by quantitative reverse transcription PCR and recalculated the translatability scores in Figures 1B and S1C as normalized by RNA levels (Figs. 1C and S1D). After RNA amounts were accounted for, all 5′ UTRs still exerted a repressive effect on luciferase reporter translation, with a more pronounced repression detected for exon IIc UTR (the longest alternative splicing variant of exon II). Overall, normalization with mRNA levels did not change the outcome of luciferase assay results for the analysis of 5′ UTRs. In contrast, normalization completely changed the interpretation for 3′ UTRs: long and short Bdnf 3′ UTR variants both displayed similar translatability after normalization (Fig. 1C), suggesting that in cortical neurons, the long 3′ UTR does not have a direct repressive effect on protein synthesis but decreases RNA stability. Next, we used TRAP (41Heiman M. Schaefer A. Gong S. Peterson J.D. Day M. Ramsey K.E. et al.A translational profiling approach for the molecular characterization of CNS cell types.Cell. 2008; 135: 738-748Abstract Full Text Full Text PDF PubMed Scopus (828) Google Scholar) to test translatability of endogenous Bdnf mRNA. Primary hippocampal neurons were transduced with adeno-associated viruses (AAVs) carrying Rpl10a-EGFP driven by the neuron-specific human Synapsin I promoter (Syn) (Fig. 1D). A ratio between immunoprecipitated ribosome-associated RNA and total RNA used for input samples was used to calculate a relative translatability index for the comparison of different RNAs. RT-qPCR analysis showed that transcripts containing Bdnf exon IIc were significantly less associated with translating ribosomes than total Bdnf mRNA (detected with primers against the coding sequence), in agreement with our dual luciferase assay results. Ribosome recruitment of exon I, IV, VI, or Bdnf long 3′ UTR–containing RNAs was not significantly different from that of total Bdnf mRNA (Fig. 1E). As a reference, we quantified Camk2a, a known dendritically localized mRNA (42Burgin K.E. Waxham M.N. Rickling S. Westgate S.A. Mobley W.C. Kelly P.T. In situ hybridization histochemistry of Ca2+/calmodulin-dependent protein kinase in developing rat brain.J. Neurosci. 1990; 10: 1788-1798Crossref PubMed Google Scholar) that is basally repressive and undergoes neuronal activity–dependent translation by a cytoplasmic polyadenylation-dependent mechanism (43Wu L. Wells D. Tay J. Mendis D. Abbott M.A. Barnitt A. et al.CPEB-mediated cytoplasmic polyadenylation and the regulation of experience-dependent translation of alpha-CaMKII mRNA at synapses.Neuron. 1998; 21: 1129-1139Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). Camk2a showed significantly lower translatability than Bdnf mRNA (Fig. 1E). Finally, we labeled newly synthesized proteins as described in (44Forester C.M. Zhao Q. Phillips N.J. Urisman A. Chalkley R.J. Oses-Prieto J.A. et al.Revealing nascent proteomics in signaling pathways and cell differentiation.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 2353-2358Crossref PubMed Scopus (35) Google Scholar) to study the effect of the long Bdnf 3′ UTR on de novo synthesis of the firefly luciferase reporter protein. To this end, we transfected HEK293 cells with the control and Bdnf 3′ UTR luciferase constructs used for luciferase assays above (see Fig. 1A), labeled newly synthesized proteins with O-propargyl puromycin (OPP), click-conjugated labeled proteins with biotin azide, and isolated biotinylated proteins by streptavidin pulldown as described in (44Forester C.M. Zhao Q. Phillips N.J. Urisman A. Chalkley R.J. Oses-Prieto J.A. et al.Revealing nascent proteomics in signaling pathways and cell differentiation.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 2353-2358Crossref PubMed Scopus (35) Google Scholar) (Fig. 2A). Western blot analysis of de novo–synthesized firefly luciferase protein (pulldown) and steady state luciferase protein (input) showed a higher pulldown to input ratio for long 3′ UTR constructs than the short 3′ UTR and control (SV40) 3′ UTR constructs, suggesting a facilitating effect on translation by the long Bdnf 3′ UTR (Fig. 2, B–D). Focusing on the regulation of Bdnf by the 3′ UTR, we next studied BDNF expression levels in a conditional 3′ UTR replacement knock-in mouse model (Fig. 3A). To this end, we generated mice with targeted insertion of inverted floxed bovine growth hormone (bGH) 3′ UTR between the Bdnf stop codon and its native 3′ UTR (Figs. 3A and S2A). We chose the bGH 3′ UTR since, to the best of our knowledge, it ensures the highest levels of transgene expression by promoting mRNA stability (38Kakoki M. Tsai Y.-S. Kim H.-S. Hatada S. Ciavatta D.J. Takahashi N. et al.Altering the expression in mice of genes by modifying their 3′ regions.Dev. Cell. 2004; 6: 597-606Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Cre-dependent inversion of the the bGH cassette using FLEX sequence (45Schnütgen F. Doerflinger N. Calléja C. Wendling O. Chambon P. Ghyselinck N.B. A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse.Nat. Biotechnol. 2003; 21: 562-565Crossref PubMed Scopus (278) Google Scholar) results in the transcription of Bdnf mRNAs containing the bGH 3′ UTR instead of the endogenous 3′ UTR sequences (Fig. 3A). Mice carrying the conditional Bdnf 3′ UTR replacement to bGH 3′UTR (Bdnf c3′ R) allele were crossed with a deleter-Cre line (PGK-Cre; (46Lallemand Y. Luria V. Haffner-Krausz R. Lonai P. Maternally expressed PGK-Cre transgene as a tool for early and uniform activation of the Cre site-specific recombinase.Transgenic Res. 1998; 7: 105-112Crossref PubMed Scopus (303) Google Scholar)), which results in ubiquitous recombination detectable in tail DNA (data not shown). Offspring was used to compare Bdnf expression in littermates. Western blot analysis of brain tissue in mice revealed a broad decrease in BDNF protein across different brain regions in an allele dose-dependent manner in mice where native Bdnf 3′ UTR was replaced with the bGH 3′ UTR (Fig. 3, B and C). Strikingly, we observed the opposite effect in heart and lung (Fig. 3, B and C), two non-neuronal tissues expressing BDNF (9Maisonpierre P.C. Belluscio L. Friedman B. Alderson R.F. Wiegand S.J. Furth M.E. et al.NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression.Neuron. 1990; 5: 501-509Abstract Full Text PDF PubMed Scopus (1102) Google Scholar, 27Timmusk T. Palm K. Metsis M. Reintam T. Paalme V. Saarma M. et al.Multiple promoters direct tissue-specific expression of the rat BDNF gene.Neuron. 1993; 10: 475-489Abstract Full Text PDF PubMed Scopus (743) Google Scholar). Bdnf mRNA analysis by RT-qPCR showed similarly decreased levels in the brain regions of homozygous Bdnf c3′ R mice (Fig. S2B), suggesting that decreased mRNA stability contributes to reduced BDNF protein levels. Neuronal activity–dependent activation of BDNF translation has been reported using rat hippocampal cultures and the KCl depolarization model (47Baj G. Pinhero V. Vaghi V. Tongiorgi E. Signaling pathways controlling activity-dependent local translation of BDNF and their localization in dendritic arbors.J. Cell Sci. 2016; 129: 2852-2864PubMed Google Scholar). That study also showed KCl depolarization-induced dendritic localization and translation of a GFP reporter RNA containing both rat Bdnf exon VI 5′UTR and long 3′UTR (47Baj G. Pinhero V. Vaghi V. Tongiorgi E. Signaling pathways controlling activity-dependent local translation of BDNF and their localization in dendritic arbors.J. Cell Sci. 2016; 129: 2852-2864PubMed Google Scholar). Therefore, we were interested in assessing whether neuronal activity can modulate the effect on translatability in cultured neurons by any of the alternative Bdnf UTRs. To test this, we transfected rat cortical or hippocampal neurons with UTR-luc constructs (same as in Fig. 1, A and B) and stimulated the cells with KCl depolarization (55 mM) for 15 min or 3 h before measuring luciferase activity (Fig. 4, A and B). The time points were selected based on reported KCl-induced BDNF protein elevation and induced luciferase from Bdnf VI 5′ UTR/3′ UTR long reporter (47Baj G. Pinhero V. Vaghi V. Tongiorgi E. Signaling pathways controlling activity-dependent local translation of BDNF and their localization in dendritic arbors.J. Cell Sci. 2016; 129: 2852-2864PubMed Google Scholar). This analysis revealed a modest activity-dependent induction of luciferase translation from Bdnf exon-II–containing mRNA in cortical neurons at 3h but not at 15 min of depolarization. In hippocampal neurons, we detected a small increase in translation from reporters containing exon VI UTR, short 3′ UTR and exon VI UTR in combination with 3′ UTR long. Taken together, no single UTR nor the combination of 5′ UTR VI and or 3′ UTR long showed robust and reproducible activity-dependent regulation in both culture systems analyzed in our study. Next, we tested the regulation of BDNF translation at the endogenous protein level in cultured neurons. We treated matured cortical neurons (DIV 7) with 55 mM KCl or with 100 μM glutamate for 5 to 15 min and quantified BDNF protein by immunoblotting (Fig. 4, C and D). We focused on these short treatment periods for two main reasons: first, Bdnf transcription is strongly regulated by neuronal depolarization (16West A.E. Pruunsild P. Timmusk T. Neurotrophins: transcription and translation.Handb Exp. Pharmacol. 2014; 220: 67-100Crossref PubMed Scopus (80) Google Scholar) and increases in protein levels at time points longer than 30
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