Argonaute2 turnover is regulated by the ubiquitin proteasome pathway Argonaute (AGO) proteins play a central role in the RNA interference (RNAi) pathway, which is a cytoplasmic mechanism important for post-transcriptional regulation of gene expression. In Drosophila, AGO2 also functions in the nucleus to regulate chromatin insulator activity and transcription. Although there are a number of studies focused on AGO2 function, the regulation of AGO2 turnover is not well understood. We found that mutation of T1149 or R1158 in the conserved PIWI domain causes AGO2 protein instability, but only T1149 affects RNAi activity. Mass spec analysis shows that several proteasome components co-purify with both wildtype and mutant AGO2, and knockdown of two proteasome pathway components results in AGO2 protein accumulation. Finally, AGO2 protein levels increase after treatment with the proteasome inhibitor MG132. Our results indicate that the ubiquitin-proteasome pathway is involved in AGO2 protein turnover. Shep regulates neuronal remodeling by controlling expression of its chromatin target genes Nervous systems are actively remodeled during the developmental transition from juvenile to adult in a wide range of organisms. This remodeling process often involves pruning of existing connections, regrowth of adult-specific connections and maturation of physiological capabilities. Dysregulation of neuronal remodeling leads to improper connections that are associated with neurological diseases such as schizophrenia and autism. However, our understanding of molecular mechanisms of neuronal remodeling remains largely undefined. Drosophila has been an excellent model to study neuronal remodeling due to its stereotypical and dramatic re-organization of the nervous system during metamorphosis. We have previously identified an RNA-binding protein Shep, which promotes neuronal outgrowth during metamorphic remodeling. Loss of shep leads to locomotor deficits, abnormal sexual behaviors, and morphological defects of synapses and neurons specifically at the adult stage. Shep physically interacts with gypsy chromatin insulator proteins and inhibits their activities specifically in the nervous system. To better understand the mechanisms of shep regulation of metamorphic remodeling, we performed RNA-seq analysis on FACS-sorted larval and pharate adult neurons. Consistent with stage-specific phenotypes, we observed strong effects on the transcriptome specifically in pharate adult neurons. Our results elucidate shep-dependent regulation of synaptic signaling factors and gene expression regulators that control neuronal remodeling during metamorphosis. Bioinformatic analysis further indicated that a significant proportion of these affected genes are chromatin targets of Shep as well as gypsy insulator proteins. Chromatin conformation capture assays revealed shep regulation of promoter-enhancer interactions at the chromatin of the shep-regulated genes, and consequently, we observed shep-dependent inhibition of transcription in our assays monitoring nascent RNA levels. Finally, we employed behavioral and cellular assays to test two candidate shep targets and found that manipulation of their expression suppresses shep-dependent phenotypes, thus validating their biological relevance with respect to shep regulation of neuronal remodeling during metamorphosis. Taken together, our findings provide new insights into gene expression profiles during neuronal remodeling. Furthermore, we identify Shep as a key regulatory factor to control wiring of a differentiated nervous system toward full maturation and function.
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