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Pumilio2 and Staufen2 selectively balance the synaptic proteome

ABSTRACT: Cells, in particular neurons, have the capacity to adapt to environmental stimuli, a phenomenon termed cellular plasticity. The underlying processes are controlled by a network of RNA-binding proteins (RBPs). Their impact on cellular plasticity, however, is largely unknown. To address this important question, we chose Pumilio2 (Pum2) and Staufen2 (Stau2) that both regulate synaptic transmission. Importantly, even though both RBPs dynamically interact with each other in neurons, their respective impact on the transcriptome and proteome is highly selective. While Pum2 deficiency leads to reduced translation and protein expression, Stau2 depletion preferentially impacts RNA levels and increases protein abundance. Furthermore, Pum2 activates expression of key GABAergic synaptic components, e.g. the GABAA receptor scaffold protein Gephyrin. Consequently, Pum2 depletion selectively reduced the amplitude of miniature inhibitory postsynaptic currents. Together, our data demonstrate that RBPs are needed to maintain proteostasis in order to control distinct signaling pathways which critically balance synaptic transmission.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Brain, Neuron

SUBMITTER: Ignasi Forne

LAB HEAD: Prof Michael Kiebler

PROVIDER: PXD025738 | Pride | 2021-06-23

REPOSITORIES: Pride

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Project description:Background: Synaptic transmission is required for functional maturation of the CNS and to induce gene transcription involved in neuronal plasticity. Our aim is to identify genes that are transcriptionally regulated by synaptic transmission during development. cDNA microarrays will be used to compare gene expression in normal embryos and embryos with no synaptic transmission. Using the Gal4 UAS system, the tetanus toxin light fragment (TET) will be expressed throughout the nervous system. The effects of TET are well characterised: the synaptic protein n-Synaptobrevin is cleaved, blocking evokes vesicle fusion in TET expressing neurons and as a result synaptic transmission is blocked. Embryos expressing an inactive, mutant version of TET (iTET) show no detectable phenotype. Thus a comparison of gene expression in iTET expressing embryos and TET expressing embryos focuses our screen cleanly and specifically on genes that are regulated by synaptic transmission. At the end of larval life, during metamorphosis, there is a second phase of nervous system development with the same requirement for neural circuit differentiation and maturation as in the embryo. We will compare gene expression in TET expressing and iTET expressing pupae to identify genes which are regulated by synaptic transmission in this second phase of nervous system development. We will focus our analysis on genes which are similarly regulated in both systems. Plan: elav Gal4 (expressed in all the neurons) for the embryos and Heat Shock Gal4 (which allows a temporally controlled expression) for the pupae are used to drive UAS TET and UAS iTET throughout the nervous system. Crosses are set up with flies homozygous for Gal4 or UAS transgenes, so that all the progeny have the same genotype. Total RNA is extracted from 500 carefully staged embryos that have been collected at the time of hatching. Pupae are heat shocked at 24 hours after puparium formation, and heads are then collected 5 days after puparium formation. TET expressing pupae are paralysed but morphologically normal whereas iTET expressing pupae emerge as normal adults. Total RNA will be extracted from 250 heads of each genotype. Probes made from TET and iTET expressing embryos extracts will be hybridised together to the chip and so will be probes made from TET and iTET expressing pupae extracts. Genes regulated in both systems will be compared together.

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Pumilio2 and Staufen2 selectively balance the synaptic proteome

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