In neurons, dendritic protein synthesis is required for many forms of long-term synaptic plasticity. The population of mRNAs that are localized to dendrites, however, remains sparsely identified. Here, we use deep sequencing to identify the mRNAs resident in the synaptic neuropil in the hippocampus. Analysis of a neuropil data set yielded a list of 8,379 transcripts of which 2,550 are localized in dendrites and/or axons. Using a fluorescent barcode strategy to label individual mRNAs, we show that their relative abundance in the neuropil varies over 3 orders of magnitude. High-resolution in situ hybridization validated the presence of mRNAs in both cultured neurons and hippocampal slices. Among the many mRNAs identified, we observed a large fraction of known synaptic proteins including signaling molecules, scaffolds and receptors. These results reveal a previously unappreciated enormous potential for the local protein synthesis machinery to supply, maintain and modify the dendritic and synaptic proteome.

It is clear that de novo protein synthesis has an important function in synaptic transmission and plasticity. A substantial amount of work has shown that mRNA translation in the hippocampus is spatially controlled and that dendritic protein synthesis is required for different forms of long-term synaptic plasticity. More recently, several studies have highlighted a function for protein degradation by the ubiquitin proteasome system in synaptic plasticity. These observations suggest that changes in synaptic transmission involve extensive regulation of the synaptic proteome. Here, we review experimental data supporting the idea that protein homeostasis is a regulatory motif for synaptic plasticity.

A major challenge in current molecular biology is to understand how sequential steps in gene expression are coupled. Recently, much attention has been focused on the linkage of transcription, processing, and mRNA export. Here we describe the cytoplasmic rearrangement for shuttling mRNA binding proteins in Saccharomyces cerevisiae during translation. While the bulk of Hrp1p, Nab2p, or Mex67p is not associated with polysome containing mRNAs, significant amounts of the serine/arginine (SR)-type shuttling mRNA binding proteins Npl3p, Gbp2p, and Hrb1p remain associated with the mRNA-protein complex during translation. Interestingly, a prolonged association of Npl3p with polysome containing mRNAs results in translational defects, indicating that Npl3p can function as a negative translational regulator. Consistent with this idea, a mutation in NPL3 that slows down translation suppresses growth defects caused by the presence of translation inhibitors or a mutation in eIF5A. Moreover, using sucrose density gradient analysis, we provide evidence that the import receptor Mtr10p, but not the SR protein kinase Sky1p, is involved in the timely regulated release of Npl3p from polysomeassociated mRNAs. Together, these data shed light onto the transformation of an exporting to a translating mRNP.

Premature termination (nonsense) codons trigger rapid mRNA decay by the nonsense-mediated mRNA decay (NMD) pathway. Two conserved proteins essential for NMD, UPF1 and UPF2, are phosphorylated in higher eukaryotes. The phosphorylation and dephosphorylation of UPF1 appear to be crucial for NMD, as blockade of either event in Caenorhabditis elegans and mammals largely prevents NMD. The universality of this phosphorylation/dephosphorylation cycle pathway has been questioned, however, because the well-studied Saccharomyces cerevisiae NMD pathway has not been shown to be regulated by phosphorylation. Here, we used in vitro and in vivo biochemical techniques to show that both S. cerevisiae Upf1p and Upf2p are phosphoproteins. We provide evidence that the phosphorylation of the N-terminal region of Upf2p is crucial for its interaction with Hrp1p, an RNA-binding protein that we previously showed is essential for NMD. We identify specific amino acids in Upf2p's N-terminal domain, including phosphorylated serines, which dictate both its interaction with Hrp1p and its ability to elicit NMD. Our results indicate that phosphorylation of UPF1 and UPF2 is a conserved event in eukaryotes and for the first time provide evidence that Upf2p phosphorylation is crucial for NMD.Cells have evolved many quality control mechanisms to eliminate aberrant proteins and mRNAs that interfere with normal cellular functions. One such mechanism is the nonsense-mediated mRNA decay (NMD) pathway, which eliminates mRNAs that contain premature termination (nonsense) codons within the protein coding region, thereby preventing the synthesis of truncated proteins with dominant-negative and deleterious gainof-function activities (3,7,14,16,24,42,47). The importance of this surveillance mechanism is underscored by its conservation in a wide variety of organisms, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, plants, and mammals (12,13,16,25,27,34,38).Several genes essential for NMD have been identified in yeast, most notably UPF1, UPF2, and UPF3, all of which destabilize nonsense codon-containing mRNAs without affecting the decay rate of most wild-type mRNAs (16,25). The yeast UPF1 gene encodes the protein Upf1p, which has RNA-binding and RNA-dependent ATPase/helicase activities (8, 9, 45). Yeast UPF3 encodes the basic protein Upf3p, which harbors several nuclear localization and nuclear export signals that allow the protein to shuttle between the nucleus and the cytoplasm (39, 40). Yeast UPF2 encodes the adaptor protein Upf2p, which forms a complex with both Upf1p and Upf3p (6, 19). Single or multiple deletions of each of these three UPF genes produce similar effects on mRNA decay, consistent with the notion that the Upf proteins function as a molecular complex in a single pathway (19).The NMD pathway is elicited by recognition of a nonsense codon, but the precise features distinguishing a premature termination codon from a bona fide stop codon remain unknown. In S. cerevisiae, one model for NMD suggests that the de...