ABSTRACT
Hundreds of pathways for degradation converge at ubiquitin recognition by a proteasome. Here, we found that the five known proteasomal ubiquitin receptors in yeast are collectively nonessential for ubiquitin recognition and identified a sixth receptor, Rpn1. A site ( T1: ) in the Rpn1 toroid recognized ubiquitin and ubiquitin-like ( UBL: ) domains of substrate shuttling factors. T1 structures with monoubiquitin or lysine 48 diubiquitin show three neighboring outer helices engaging two ubiquitins. T1 contributes a distinct substrate-binding pathway with preference for lysine 48-linked chains. Proximal to T1 within the Rpn1 toroid is a second UBL-binding site ( T2: ) that assists in ubiquitin chain disassembly, by binding the UBL of deubiquitinating enzyme Ubp6. Thus, a two-site recognition domain intrinsic to the proteasome uses distinct ubiquitin-fold ligands to assemble substrates, shuttling factors, and a deubiquitinating enzyme.
Subject(s)
Proteasome Endopeptidase Complex/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , DNA-Binding Proteins/metabolism , Endopeptidases/metabolism , Metabolic Networks and Pathways , Models, Molecular , Mutation , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/genetics , Saccharomyces cerevisiae Proteins/genetics , Ubiquitin-Specific Proteases/metabolism , UbiquitinationABSTRACT
Mammalian Staufen2 (Stau2) is a member of the double-stranded RNA-binding protein family. Its expression is largely restricted to the brain. It is thought to play a role in the delivery of RNA to dendrites of polarized neurons. To investigate the function of Stau2 in mature neurons, we interfered with Stau2 expression by RNA interference (RNAi). Mature neurons lacking Stau2 displayed a significant reduction in the number of dendritic spines and an increase in filopodia-like structures. The number of PSD95-positive synapses and miniature excitatory postsynaptic currents were markedly reduced in Stau2 down-regulated neurons. Akin effects were caused by overexpression of dominant-negative Stau2. The observed phenotype could be rescued by overexpression of two RNAi cleavage-resistant Stau2 isoforms. In situ hybridization revealed reduced expression levels of beta-actin mRNA and fewer dendritic beta-actin mRNPs in Stau2 down-regulated neurons. Thus, our data suggest an important role for Stau2 in the formation and maintenance of dendritic spines of hippocampal neurons.
Subject(s)
Brain/metabolism , Dendrites , Neurons/cytology , Protein Isoforms/metabolism , RNA-Binding Proteins/metabolism , Actins/genetics , Actins/metabolism , Animals , Cells, Cultured , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , Dendrites/metabolism , Dendrites/ultrastructure , Disks Large Homolog 4 Protein , Down-Regulation , Excitatory Postsynaptic Potentials/physiology , HeLa Cells , Hippocampus/cytology , Humans , In Situ Hybridization , In Situ Hybridization, Fluorescence , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Neurons/physiology , Patch-Clamp Techniques , Protein Isoforms/genetics , RNA Interference , RNA-Binding Proteins/genetics , Rats , Synapses/metabolismABSTRACT
MicroRNAs are small, non-coding RNAs that control the translation of target messenger RNAs, thereby regulating critical aspects of plant and animal development. In the mammalian nervous system, the spatiotemporal control of mRNA translation has an important role in synaptic development and plasticity. Although a number of microRNAs have been isolated from the mammalian brain, neither the specific microRNAs that regulate synapse function nor their target mRNAs have been identified. Here we show that a brain-specific microRNA, miR-134, is localized to the synapto-dendritic compartment of rat hippocampal neurons and negatively regulates the size of dendritic spines--postsynaptic sites of excitatory synaptic transmission. This effect is mediated by miR-134 inhibition of the translation of an mRNA encoding a protein kinase, Limk1, that controls spine development. Exposure of neurons to extracellular stimuli such as brain-derived neurotrophic factor relieves miR-134 inhibition of Limk1 translation and in this way may contribute to synaptic development, maturation and/or plasticity.
