Dataset Information

Unbalanced protein expression contributes to pathology in Fragile X Syndrome

ABSTRACT: Dysregulated protein synthesis is a core pathogenic mechanism in Fragile X Syndrome (FX). The mGluR Theory of FX predicts that pathological synaptic changes arise from the excessive translation of mRNAs downstream of mGlu1/5 activation. Here, we use a combination of CA1 pyramidal neuron-specific Translating Ribosome Affinity Purification and RNA-seq (TRAP-seq) and proteomics to identify the overtranslating mRNAs supporting exaggerated mGlu1/5 -induced long-term synaptic depression (mGluR-LTD) in the FX mouse model (Fmr1-/y). Our results identify a robust translation of ribosomal proteins (RPs) upon mGlu1/5 stimulation that coincides with a reduced translation of long mRNAs encoding synaptic proteins. These changes are mimicked and occluded in Fmr1-/y neurons. In addition, proteomics analyses identify increased degradation of multiple proteins at hippocampal synapses, many of which are underexpressed at steady-state.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Miguel A. Gonzalez-Lozano

LAB HEAD: Ka Wan Li

PROVIDER: PXD031932 | Pride | 2022-06-16

REPOSITORIES: Pride

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Publications

Excess ribosomal protein production unbalances translation in a model of Fragile X Syndrome.

Seo Sang S SS Louros Susana R SR Anstey Natasha N Gonzalez-Lozano Miguel A MA Harper Callista B CB Verity Nicholas C NC Dando Owen O Thomson Sophie R SR Darnell Jennifer C JC Kind Peter C PC Li Ka Wan KW Osterweil Emily K EK

Nature communications 20220610 1

Dysregulated protein synthesis is a core pathogenic mechanism in Fragile X Syndrome (FX). The mGluR Theory of FX predicts that pathological synaptic changes arise from the excessive translation of mRNAs downstream of mGlu1/5 activation. Here, we use a combination of CA1 pyramidal neuron-specific TRAP-seq and proteomics to identify the overtranslating mRNAs supporting exaggerated mGlu1/5 -induced long-term synaptic depression (mGluR-LTD) in the FX mouse model (Fmr1-/y</ ...[more]

PMID: 35688821

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Project description:In eukaryotes, regulation of mRNA translation enables a fast, localized and finely tuned expression of gene products. Within the translation process, the first stage of translation initiation is most rigorously modulated by the actions of eukaryotic initiation factors (eIFs) and their associated proteins. These 11 eIFs catalyze the joining of the tRNA, mRNA and rRNA into a functional translation complex. Their activity is influenced by a wide variety of extra- and intracellular signals, ranging from global, such as hormone signaling and unfolded proteins, to specific, such as single amino acid imbalance and iron deficiency. Their action is correspondingly comprehensive, in increasing or decreasing recruitment and translation of most cellular mRNAs, and specialized, in targeting translation of mRNAs with regulatory features such as a 5’ terminal oligopyrimidine tract (TOP), upstream open reading frames (uORFs), or an internal ribosomal entry site (IRES). In mammals, two major pathways are linked to targeted mRNA translation. The target of rapamycin (TOR) kinase induces translation of TOP and perhaps other subsets of mRNAs, whereas a family of eIF2 kinases does so with mRNAs containing uORFs or an IRES. TOR targets translation of mRNAs that code for proteins involved in translation, an action compatible with its widely accepted role in regulating cellular growth. The four members of the eIF2 kinase family increase translation of mRNAs coding for stress response proteins such as transcription factors and chaperones. Though all four kinases act on one main substrate, eIF2, published literature demonstrates both common and unique effects by each kinase in response to its specific activating stress. This suggests that the activated eIF2 kinases regulate the translation of both a global and a specific set of mRNAs. Up to now, few studies have attempted to test such a hypothesis; none has been done in mammals. We use array analysis to determine the global mRNA shift into polysomes following a stress response, and to compare the translational response following activation of GCN2 versus PERK, two of the four eIF2alpha kinases. Experiment Overall Design: Gcn2 wild-type or knockout mouse liver were perfused with complete amino acids media or media lacking methionine for RNA extraction and hybridization of Affymetrix microarrays. RNA was extracted from unfractionated liver samples and polysome fraction of samples separated on sucrose density gradient. To minimize biological variations, we pooled RNA from two perfused liver samples to use in each array analysis. The conditions were total and polysome fraction of Gcn2+/+, +Met or -Met; total and polysome fraction of Gcn2-/-, +Met or -Met. Each array analysis was done in duplicate.

Dataset Information

Unbalanced protein expression contributes to pathology in Fragile X Syndrome

Publications

Excess ribosomal protein production unbalances translation in a model of Fragile X Syndrome.

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