Knock-out mice and HITS-CLIP reveal that the SGs assembly factor G3BP preferentially binds intron-retaining transcripts and controls their stability in the brain

SOPHIE MARTIN; NICOLAS BELLORA; BEN J BLENCOWE; JAMAL TAZI

Davos

Congreso; 18th Annual Meeting of the RNA Society; 2013

International RNA Society

Stress granules (SGs) are non-membranous cytoplasmic foci formed as a cellular protective response toenvironmental stress, such as elevated temperature, oxidative stress, hypoxia, osmotic shock, UV irradiation, glucosedeprivation or viral infection. SGs share common properties with other granules like processing bodies or neuronaltransport granules. G3BP (RasGAP-SH3-domain Binding Protein) is a key factor involved in SGs assembly. To assessthe physiological function of G3BP, we generated viable G3bp1-knockout (KO) mice, which demonstrated behavioraldefects linked to the central nervous system (CNS) associated with ataxia phenotype. Immunohistochemistrypinpointed high expression of G3BP in the cytoplasm of hippocampal neurons and Purkinje cells of the cerebellumof wild-type (WT) mice. Also, electrophysiological measurements revealed that the absence of G3BP1 leads to anenhancement of short-term potentiation (STP) and long-term depression (LTD) in the CA1 area of G3bp1 KO micecompared to WT mice. Consistently, G3BP1-deficiency in neurons leads to an increase in intracellular calcium andcalcium release in response to (S)-3,5-Dihydroxyphenylglycine (DHPG), a selective agonist of group I metabotropicglutamate receptors. HITS-CLIP (High-Throughput Sequencing after Cross-Linking and ImmunoPrecipitation)experiments were carried out on WT and KO mouse brain to identify G3BP-associated RNAs. Surprisingly, manyG3BP?s targets turn out to be non-coding RNA sequences, essentially snoRNAs and intronic sequences. Interestingly,transcripts with retained introns appear to be enriched in the cerebellum compared to the rest of the brain. G3BP1depletion leads to a decrease in the expression of these intronic sequences in the cerebellum. In particular, G3BP1 isessential to repress mature Grm5 (metabotropic glutamate receptor 5) expression in the cerebellum by stabilizationof the intron in its pre-mRNA. This study suggests a new mechanism of gene regulation, important in the cerebellum,based on stabilization of silenced premature RNA transcripts, which might be converted to mature transcripts andtranslated or targeted for degradation upon G3BP depletion.