Skip to main content
European Commission logo print header

“Linking dendritic mRNA metabolism to neuronal functions and disorders”

Final Report Summary - DENDRITIC MRNAS (“Linking dendritic mRNA metabolism to neuronal functions and disorders”)

Project objectives
The aim of this project was two fold; the identification of pathways controlling dendritic mRNAs expression and the development of a protocol for the in vivo characterization of mRNPs. The model mRNA analyzed in my studies is the dendritic mRNA Arc, whose protein is essential in all forms of plasticity and for memory consolidation and maintenance. Also, recently Arc was found to be altered in Alzheimer patients and to be involved in the overproduction of beta amyloid.
Arc's 3'UTR is the focus of this study, as it contains several regulatory elements, including two introns which, as I have shown previously, modulate Arc expression via the Nonsense Mediated Decay (NMD) pathway. The specific objectives of this project were to biochemically isolate the ribonucleoprotein particle associated with Arc mRNA 3'UTR in vivo and to investigate how cis- and trans- acting factors associated to its 3'UTR cooperate in controlling Arc protein expression upon synaptic activity.

Main results and impact
To characterize the proteins bound to Arc mRNA 3'UTR in vivo, I have designed a purification strategy in which a reporter mRNA harboring Arc 3'UTR is co-expressed with a tagged bait that mediates binding of the mRNA ribonucleoprotein complex with a resin. Co-expression of these two genes was to be achieved by infecting cells with a lentivirus providing dual gene transfer and encompassing the reporter and bait genes. The lentiviruses have been cloned and the resulting viruses have been utilized to infect PC12 cells. Extracts of the infected cells have been used in preliminary immunopurification experiments , however the bait is expressed at levels in the infected cellsthat are too low, not allowing efficient immunopurification of the reporter-associated ribonucleoprotein complex. Since the low expression profile is likely due to the type of lentiviral vector utilized in this first attempt, I have thus decided to switch to a different strategy, achieving co-expression of the two (the reporter and the bait) by cloning these elements in two separate lentiviral plasmids and proceed to co-infect cells with both viral particles. At this stage I have terminated the cloning of the new plasmids and have achieved good expression levels of both elements upon infection. Yet, given these time consuming technical difficulties, I have concentrated my efforts towards the characterization of cis- and trans-acting elements controlling the expression of Arc mRNA in neurons through an alternative approach. I have generated several mammalian expression plasmids harboring different versions of Arc 3'UTR downstream of a pRL-TK gene to be analyzed in a dual-luciferase reporter assays. Several elements potentially controlling Arc mRNA expression and stability were either mutated or deleted including its 3'UTR introns, Arc's specific polyadenylation sequence and potential miRNA binding sites.
These plasmids were transfected in Hela and in neuronal cells in culture, to assess the contribution of each element to the modulation of Arc mRNA expression.

The resulting data indicate that I can recapitulate Arc modulation by the Nonsense Mediated Decay (NMD) triggered by the presence of Arc's introns in its 3'UTR.
Additionally, experiments in HeLa suggest that splicing of Arc mRNA’s UTR influences targeting by miRNA 19. A major result obtained is that in neurons, upon BDNF induction, the luciferase constructs harbouring Arc 3’UTR with introns are subjected to a 2.7 upregulation in their expression levels compared to the ones lacking introns (fig1B).

Multiple analyses show that this induction occurs at the translational level, as mRNA levels of the reporter are unchanged and this effect is inhibited by the translation inhibitor anisomycin. I have thus performed a panel of experiments to characterize the trans-acting factors involved in this regulatory cascade. By pre-incubating neuronal cells with a series of compounds, each specifically inhibiting regulatory proteins involved in BDNF signalling or in Arc translation, I have been able to characterize the signalling cascade that elicits this splicing-dependent translational up-regulation.


In particular I found that eIF2 dephosphorylation and PKA activity are crucial to this process. Both factors play important roles in long-term synaptic plasticity. PKA involvement in Arc translation had been previously reported, however that splicing of Arc’s UTR is required in this process is a novel contribution of this work. Even more significantly, eIF2 dephosphorylation, a critical step for the switch from short to long-term synaptic plasticity, was previously reported to match the time window of Arc translation upon LTP induction. However, here I show the requirement of eIF2 dephosphorylation in the splicing dependent upregulation of Arc translation.
These data thus represent an important contribution to the understanding of the complex molecular cascade regulating synaptic plasticity by modulation of Arc expression levels at synapses. First of all, by transfecting constructs with Arc 3’UTR, I was able to examine how this uniquely structured regulatory region of the mRNA plays into the complex regulatory cascade that controls Arc mRNA and protein levels upon synaptic plasticity. Secondly, a major finding of this project was that splicing of Arc 3’UTR exerts a dual and unique effect in fine-tuning Arc expression upon synaptic signalling. On one hand it triggers mRNA decay, limiting the time window in which this essential protein is made in response to stimuli, on the other it elicits translation of the fast decaying mRNA. This apparent paradox likely serves to achieve a rapid yet limited burst of expression, a feature of Arc expression which potentially underlies its role as a key sensor in synapse-specific homeostatic plasticity.

Overall, while the critical objective of “biochemically isolate the ribonucleoprotein particle associated with Arc mRNA 3'UTR in vivo” was not completed in time, its intended purpose to “identify proteins and RNA trans-acting factors whose binding is connected to synaptic activity and splicing of the mRNA.” was nonetheless addressed by an alternative strategy. The analysis via luciferase constructs in cultured neurons has indeed unveiled a unique mechanism by which splicing of Arc UTR , its expression and synaptic activity are interconnected, also identifying trans acting factors key to this regulatory process.
These results significantly add to the overall understanding of the complex regulation of Arc expression upon synaptic activity. This process is of particular importance as arc expression levels are key in establishing long term memories and participate in altering the physiological responses of synapses in neurodegenerative diseases such as Alzheimer.