RNA Transport Proteins Research Articles

Analysis of small EV proteomes reveals unique functional protein networks regulated by VAP-A.

Extracellular vesicles (EVs) influence cell phenotypes and functions via protein, nucleic acid, and lipid cargoes. EVs are heterogeneous, due to diverse biogenesis mechanisms that remain poorly understood. Our previous study revealed that the endoplasmic reticulum (ER) membrane contact site (MCS) linker protein vesicle associated protein associated protein A (VAP-A) drives biogenesis of a subset of RNA-enriched EVs. Here, we examine the protein content of VAP-A-regulated EVs. Using label-free proteomics, we identified down- and upregulated proteins in small EVs (SEVs) purified from VAP-A knockdown (KD) colon cancer cells. Gene set enrichment analysis (GSEA) of the data revealed protein classes that are differentially sorted to SEVs dependent on VAP-A. Search Tool for the Retrieval of Reciprocity Genes (STRING) protein-protein interaction network analysis of the RNA-binding protein (RBP) gene set identified several RNA functional machineries that are downregulated in VAP-A KD SEVs, including ribosome, spliceosome, mRNA surveillance, and RNA transport proteins. We also observed downregulation of other functionally interacting protein networks, including cadherin-binding, unfolded protein binding, and ATP-dependent proteins.

Comparative Ubiquitome Analysis Reveals Deubiquitinating Effects Induced by Wolbachia Infection in Drosophila melanogaster.

The endosymbiotic Wolbachia bacteria frequently cause cytoplasmic incompatibility (CI) in their insect hosts, where Wolbachia-infected males cross with uninfected females, leading to no or fewer progenies, indicating a paternal modification by Wolbachia. Recent studies have identified a Wolbachia protein, CidB, containing a DUB (deubiquitylating enzyme) domain, which can be loaded into host sperm nuclei and involved in CI, though the DUB activity is not necessary for CI in Drosophila melanogaster. To investigate whether and how Wolbachia affect protein ubiquitination in testes of male hosts and are thus involved in male fertility, we compared the protein and ubiquitinated protein expressions in D. melanogaster testes with and without Wolbachia. A total of 643 differentially expressed proteins (DEPs) and 309 differentially expressed ubiquitinated proteins (DEUPs) were identified to have at least a 1.5-fold change with a p-value of <0.05. Many DEPs were enriched in metabolic pathway, ribosome, RNA transport, and post-translational protein modification pathways. Many DEUPs were involved in metabolism, ribosome, and proteasome pathways. Notably, 98.1% DEUPs were downregulated in the presence of Wolbachia. Four genes coding for DEUPs in ubiquitin proteasome pathways were knocked down, respectively, in Wolbachia-free fly testes. Among them, Rpn6 and Rpn7 knockdown caused male sterility, with no mature sperm in seminal vesicles. These results reveal deubiquitylating effects induced by Wolbachia infection, suggesting that Wolbachia can widely deubiquitinate proteins that have crucial functions in male fertility of their hosts, but are not involved in CI. Our data provide new insights into the regulatory mechanisms of endosymbiont/host interactions and male fertility.

Survival characteristics and transcriptome profiling reveal the adaptive response of the Brucella melitensis 16M biofilm to osmotic stress

Brucella can inhabit hostile environments, including osmotic stress. How Brucella responds collectively to osmotic stress is largely unexplored, particularly in spatially structured communities such as a biofilm. To gain insight into this growth mode, we set out to characterize the Brucella melitensis 16M biofilm, describe its phenotype, and carry out a comparative transcriptomic analysis between biofilms under osmotic stress and control conditions. We determined that the bacteria challenged with 1.5 M NaCl had a reduced ability to aggregate and form clumps and develop a biofilm; however, the salt stress promoted the release of the outer membrane vesicles from the biofilm. Together with the genotypical response to osmotic stress, we identified 279 differentially expressed genes in B. melitensis 16M grown under osmotic conditions compared with control conditions; 69 genes were upregulated and 210 downregulated. Under osmotic stress, the main changed genes of biofilm were predicted to be involved in flagellar assembly, cell envelope, translation, small RNA regulation, transport and binding proteins, and energy metabolism. In addition, the ABC transporter was enriched in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. We highlight 12 essential ABC transporter genes associated with a bacterial response to osmotic stress at the biofilm stage, including one specific locus, BME_RS12880, mediating betaine accumulation in biofilms to eliminate osmotic stress. The current study results can help researchers gain insights into B. melitensis 16M biofilm adaptation to osmotic stress and provide information for developing intervention strategies to control Brucella.

Stress Granule-Mediated Oxidized RNA Decay in P-Body: Hypothetical Role of ADAR1, Tudor-SN, and STAU1.

Reactive oxygen species (ROS) generated under oxidative stress (OS) cause oxidative damage to RNA. Recent studies have suggested a role for oxidized RNA in several human disorders. Under the conditions of oxidative stress, mRNAs released from polysome dissociation accumulate and initiate stress granule (SG) assembly. SGs are highly enriched in mRNAs, containing inverted repeat (IR) Alus in 3′ UTRs, AU-rich elements, and RNA-binding proteins. SGs and processing bodies (P-bodies) transiently interact through a docking mechanism to allow the exchange of RNA species. However, the types of RNA species exchanged, and the mechanisms and outcomes of exchange are still unknown. Specialized RNA-binding proteins, including adenosine deaminase acting on RNA (ADAR1-p150), with an affinity toward inverted repeat Alus, and Tudor staphylococcal nuclease (Tudor-SN) are specifically recruited to SGs under OS along with an RNA transport protein, Staufen1 (STAU1), but their precise biochemical roles in SGs and SG/P-body docking are uncertain. Here, we critically review relevant literature and propose a hypothetical mechanism for the processing and decay of oxidized-RNA in SGs/P-bodies, as well as the role of ADAR1-p150, Tudor-SN, and STAU1.

A novel paperclip double-stranded RNA structure demonstrates clathrin-independent uptake in the mosquito Aedes aegypti

RNA interference (RNAi) has become a widely used technique of knocking down a gene's expression in insects, but its efficacy in some species is limited by a reduced ability of the cells to take in and disperse the double-stranded RNA (dsRNA) throughout the cytoplasm. While RNA transport proteins such as SID-1 and its orthologues can facilitate dsRNA uptake in some invertebrate species, dsRNA uptake in many insects examined to date appears to be facilitated by clathrin-mediated endocytosis (CME). In this study, we used pharmacological inhibitors and RNAi-mediated knockdown of endocytic genes to provide evidence that CME is the primary means of dsRNA uptake in the mosquito Aedes aegypti. Inhibition of clathrin-mediated endocytosis was sufficient to supress uptake of short (21 nt) interfering RNAs (siRNAs), short (23 nt) hairpin RNAs (shRNAs), and long (>200 nt) dsRNA molecules in Aedes aegypti cultured cells and larvae. In contrast, we observed that short (23 nt) “paperclip” RNAs (pcRNAs), with partially closed ends, efficiently enter cells via a clathrin-independent pathway and effectively facilitate transcript knockdown. This alternative dsRNA structure may prove useful in insects generally considered recalcitrant to RNAi and in insect populations where resistance to RNAi-insecticides may arise through changes in dsRNA uptake mechanisms.

De novo transcriptome assembly of a facultative parasitic nematode Pelodera (syn. Rhabditis) strongyloides

Pelodera strongyloides is a generally free-living gonochoristic facultative nematode. The whole genomic sequence of P. strongyloides remains unknown but 4 small subunit ribosomal RNA (ssrRNA) gene sequences are available. This project launched a de novo transcriptome assembly with 100 bp paired-end RNA-seq reads from normal, starved and wet-plate cultured animals. Trinity assembly tool generated 104,634 transcript contigs with N50 contig being 2195 bp and average contig length at 1103 bp. Transcriptome BLASTX matching results of five nematodes (C. elegans, Strongyloides stercoralis, Necator americanus, Trichuris trichiura, and Pristionchus pacificus) were consistent with their evolutionary relationships. Sixteen genes were identified to be homologous to key elements of the C. elegans RNA interference system, such as Dicer, Argonaute, RNA-dependent RNA polymerase and double strand RNA transport proteins. In starved samples, we observed up-regulation of cuticle related genes and 3 dauer formation genes. Dauer morphology was captured with enlarged phasmid under light microscopy, and dauer and normal larvae counts in clumps had a Pearson's product-moment correlation of 0.805 with P-value = 0.0088. Our results demonstrate that P. strongyloides could be used for studying nematode-related human or pet parasitic diseases. The sequenced assembled transcriptome reported here may be useful to understand the evolution of parasitism in Nematoda.

Identification of gene expression profiles in myocardial infarction: a systematic review and meta-analysis

BackgroundMyocardial infarction (MI) is a multifactorial disease with complex pathogenesis, mainly the result of the interplay of genetic and environmental risk factors. The regulation of thrombosis, inflammation and cholesterol and lipid metabolism are the main factors that have been proposed thus far to be involved in the pathogenesis of MI. Traditional risk-estimation tools depend largely on conventional risk factors but there is a need for identification of novel biochemical and genetic markers. The aim of the study is to identify differentially expressed genes that are consistently associated with the incidence myocardial infarction (MI), which could be potentially incorporated into the traditional cardiovascular diseases risk factors models.MethodsThe biomedical literature and gene expression databases, PubMed and GEO, respectively, were searched following the PRISMA guidelines. The key inclusion criteria were gene expression data derived from case-control studies on MI patients from blood samples. Gene expression datasets regarding the effect of medicinal drugs on MI were excluded. The t-test was applied to gene expression data from case-control studies in MI patients.ResultsA total of 162 articles and 174 gene expression datasets were retrieved. Of those a total of 4 gene expression datasets met the inclusion criteria, which contained data on 31,180 loci in 93 MI patients and 89 healthy individuals. Collectively, 626 differentially expressed genes were detected in MI patients as compared to non-affected individuals at an FDR q-value = 0.01. Of those, 88 genes/gene products were interconnected in an interaction network. Totally, 15 genes were identified as hubs of the network.ConclusionsFunctional enrichment analyses revealed that the DEGs and that they are mainly involved in inflammatory/wound healing, RNA processing/transport mechanisms and a yet not fully characterized pathway implicated in RNA transport and nuclear pore proteins. The overlap between the DEGs identified in this study and the genes identified through genetic-association studies is minimal. These data could be useful in future studies on the molecular mechanisms of MI as well as diagnostic and prognostic markers.

Site Specific Modification of Adeno-Associated Virus Enables Both Fluorescent Imaging of Viral Particles and Characterization of the Capsid Interactome

Adeno-associated viruses (AAVs) are attractive gene therapy vectors due to their low toxicity, high stability, and rare integration into the host genome. Expressing ligands on the viral capsid can re-target AAVs to new cell types, but limited sites have been identified on the capsid that tolerate a peptide insertion. Here, we incorporated a site-specific tetracysteine sequence into the AAV serotype 9 (AAV9) capsid, to permit labelling of viral particles with either a fluorescent dye or biotin. We demonstrate that fluorescently labelled particles are detectable in vitro, and explore the utility of the method in vivo in mice with time-lapse imaging. We exploit the biotinylated viral particles to generate two distinct AAV interactomes, and identify several functional classes of proteins that are highly represented: actin/cytoskeletal protein binding, RNA binding, RNA splicing/processing, chromatin modifying, intracellular trafficking and RNA transport proteins. To examine the biological relevance of the capsid interactome, we modulated the expression of two proteins from the interactomes prior to AAV transduction. Blocking integrin αVβ6 receptor function reduced AAV9 transduction, while reducing histone deacetylase 4 (HDAC4) expression enhanced AAV transduction. Our method demonstrates a strategy for inserting motifs into the AAV capsid without compromising viral titer or infectivity.

The conserved SNARE SEC-22 localizes to late endosomes and negatively regulates RNA interference in Caenorhabditis elegans.

Small RNA pathways, including RNA interference (RNAi), play crucial roles in regulation of gene expression. Initially considered to be cytoplasmic, these processes have later been demonstrated to associate with membranes. For example, maturation of late endosomes/multivesicular bodies (MVBs) is required for efficient RNAi, whereas fusion of MVBs to lysosomes appears to reduce silencing efficiency. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane fusion and are thus at the core of membrane trafficking. In spite of this, no SNARE has previously been reported to affect RNAi. Here, we demonstrate that in Caenorhabditis elegans, loss of the conserved SNARE SEC-22 results in enhanced RNAi upon ingestion of double-stranded RNA. Furthermore, SEC-22 overexpression inhibits RNAi in wild-type animals. We find that overexpression of SEC-22 in the target tissue (body wall muscle) strongly suppresses the sec-22(−) enhanced RNAi phenotype, supporting a primary role for SEC-22 in import of RNAi silencing signals or cell autonomous RNAi. A functional mCherry::SEC-22 protein localizes primarily to late endosomes/MVBs and these compartments are enlarged in animals lacking sec-22. SEC-22 interacts with late endosome-associated RNA transport protein SID-5 in a yeast two-hybrid assay and functions in a sid-5-dependent manner. Taken together, our data indicate that SEC-22 reduces RNAi efficiency by affecting late endosome/MVB function, for example, by promoting fusion between late endosomes/MVBs and lysosomes. To our knowledge, this is the first report of a SNARE with a function in small RNA-mediated gene silencing.

MPL W515L expression induces TGFβ secretion and leads to an increase in chemokinesis via phosphorylation of THOC5.

The thrombopoietin receptor (MPL) has been shown to be mutated (MPL W515L) in myelofibrosis and thrombocytosis yet new approaches to treat this disorder are still required. We have previously shown that transcriptome and proteomic effects do not correlate well in oncogene-mediated leukemogenesis. We therefore investigated the effects of MPL W515L using proteomics. The consequences of MPL W515L expression on over 3300 nuclear and 3500 cytoplasmic proteins were assessed using relative quantification mass spectrometry. We demonstrate that MPL W515L expression markedly modulates the CXCL12/CXCR4/CD45 pathway associated with stem and progenitor cell chemotactic movement. We also demonstrated that MPL W515L expressing cells displayed increased chemokinesis which required the MPL W515L-mediated dysregulation of MYC expression via phosphorylation of the RNA transport protein THOC5 on tyrosine 225. In addition MPL W515L expression induced TGFβ secretion which is linked to sphingosine 1-phosphate production and the increased chemokinesis. These studies identify several pathways which offer potential targets for therapeutic intervention in the treatment of MPL W515L-driven malignancy. We validate our approach by showing that CD34+ cells from MPL W515L positive patients display increased chemokinesis and that treatment with a combination of MYC and sphingosine kinase inhibitors leads to the preferential killing of MPL W515L expressing cells.

Staufen-1 Interacts with the Human Endogenous Retrovirus Family HERV-K(HML-2) Rec and Gag Proteins and Increases Virion Production

The human endogenous retrovirus family HERV-K(HML-2) Rec protein is an RNA transport factor that enhances nuclear export of intron-containing retroviral transcripts. Using the yeast two-hybrid approach, we have newly identified human Staufen-1 as a Rec-interacting protein. The interaction was confirmed by coimmunoprecipitation experiments, and the relevant site in Staufen-1 has been mapped to double-stranded RNA binding domain 4 (RBD4). Staufen-1 is in several aspects functionally related to retroviral RNA transport proteins. It binds mRNAs and targets its ribonuclear cargo to polysomes for efficient translation. We observed an accumulation of Staufen-1 in the nucleus of Rec-expressing cells and colocalization in the nucleoli as well as in the cytoplasm. Overexpression of Staufen-1 resulted in a 5-fold enhancement in nuclear export and/or translation of unspliced HERV-K(HML-2) viral RNAs in the presence of Rec and its Rec-responsive element (RcRE) binding site together with a clear increase in virus production. Staufen-1 was previously shown to interact with the Gag protein of HIV-1, promoting Gag oligomerization and RNA encapsidation. We demonstrate here that Staufen-1 also binds to the Gag protein of HERV-K(HML-2). Under stress conditions, Rec colocalizes with Staufen-1 in stress granules in cells that express viral RNA but not in mRNA-decay-related processing bodies. Our results suggest a new role for Staufen-1 as a cellular Rec and HERV-K(HML-2) Gag cofactor.

Mapping the LINE1 ORF1 protein interactome reveals associated inhibitors of human retrotransposition.

LINE1s occupy 17% of the human genome and are its only active autonomous mobile DNA. L1s are also responsible for genomic insertion of processed pseudogenes and >1 million non-autonomous retrotransposons (Alus and SVAs). These elements have significant effects on gene organization and expression. Despite the importance of retrotransposons for genome evolution, much about their biology remains unknown, including cellular factors involved in the complex processes of retrotransposition and forming and transporting L1 ribonucleoprotein particles. By co-immunoprecipitation of tagged L1 constructs and mass spectrometry, we identified proteins associated with the L1 ORF1 protein and its ribonucleoprotein. These include RNA transport proteins, gene expression regulators, post-translational modifiers, helicases and splicing factors. Many cellular proteins co-localize with L1 ORF1 protein in cytoplasmic granules. We also assayed the effects of these proteins on cell culture retrotransposition and found strong inhibiting proteins, including some that control HIV and other retroviruses. These data suggest candidate cofactors that interact with the L1 to modulate its activity and increase our understanding of the means by which the cell coexists with these genomic ‘parasites’.

The SID-1 double-stranded RNA transporter is not selective for dsRNA length

The double-stranded RNA (dsRNA) transport protein SID-1 enables systemic RNA interference (RNAi) in Caenorhabditis elegans, whereby silencing initiated by local exposure to dsRNA spreads throughout the animal and to its progeny. Previously, we showed that providing dsRNA in the growth medium of Drosophila S2 cells that express C. elegans SID-1 efficiently triggers RNAi. In these experiments long dsRNA proved to be significantly more effective than short dsRNA in silencing the target gene. Here, we show that equivalent masses of long or short dsRNA accumulate in these cells, indicating that size-dependent silencing is not due to size-selective transport through SID-1. Furthermore, using pulse-chase dsRNA uptake experiments, we show that short dsRNA accumulates more rapidly than long dsRNA. We found that import rates are dependent on dsRNA concentration, consistent with energy-independent, diffusion-limited transport through the SID-1 channel. Comparison of silencing efficiencies between Drosophila S2 cells heterologously expressing SID-1 and primary-cultured C. elegans cells shows similar dsRNA concentration and size dependencies, suggesting that C. elegans regulatory proteins do not measurably enhance or restrict dsRNA transport through SID-1. Finally, we find that coexpressing mutant SID-1 with wild-type SID-1 in S2 cells interferes with SID-1 function, indicating that SID-1 may function as a multimer.

Commonalities in the neurobiology between autism and fragile X

There is a close association between autism and fragile X syndrome (FXS) with 30% of males with FXS having autism and 2 to 7% of children with autism having the fragile X mutation. The protein that is missing or deficient in FXS, FMRP, is an RNA binding and transport protein which regulates the translation of many messages important for synaptic plasticity. Typically FMRP inhibits the translation of these messages, such that protein production increases when FMRP is absent. Some of these proteins are known to also cause autism when they are mutated including neuroligin 3 and 4 and the SHANK protein. Therefore, when FMRP is missing there is dysregulation of other proteins that are known to cause autism. FMRP is an important inhibitor of protein production in the metabotropic glutamate receptor 5 pathway (mGluR5) which leads to long term depression (LTD) or the weakening of synaptic connections. Therefore, when FMRP is missing there is enhanced mGluR5 activity leading to enhanced LTD and weak or immature synaptic connections. The use of mGluR5 antagonists to reverse the LTD in the animal models of FXS has led to reversal of the learning, behaviour and dendritic spine abnormalities in these animals. There are now initial studies taking place in humans regarding the use of mGluR5 antagonists to improve behaviour and cognition in FXS. It is likely that these mGluR5 antagonists will also be helpful in a subgroup of patients with non fragile X autism who have similar problems with hyperactivity, hyperarousal and anxiety to those seen in FXS. A second cause of autism is the fragile X premutation but this mechanism of involvement is related to RNA toxicity which perhaps stimulates neuroimmune problems and may mimic other causes of autism. Neurons with the premutation are more vulnerable to environmental toxicity and oxidative stress leading to early cell death.

Localized Regulation of Axonal RanGTPase Controls Retrograde Injury Signaling in Peripheral Nerve

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? Here, we show that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, we find RanGTP in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve.

RNA transport and localized protein synthesis in neurological disorders and neural repair

Neural cells are able to finely tune gene expression through post-transcriptional mechanisms. Localization of mRNAs to subcellular regions has been detected in neurons, oligodendrocytes, and astrocytes providing these domains with a locally renewable source of proteins. Protein synthesis in dendrites has most frequently been associated with synaptic plasticity, while axonally synthesized proteins appear to facilitate pathfinding and injury responses. For oligodendrocytes, mRNAs encoding several proteins for myelin formation are locally generated suggesting that this mechanism assists in myelination. Astrocytic processes have not been well studied but localization of GFAP mRNA has been demonstrated. Both RNA transport and localized translation are regulated processes. RNA transport appears to be highly selective and, at least in part, the destiny of individual mRNAs is determined in the nucleus. RNA-protein and protein-protein interactions determine which mRNAs are targeted to subcellular regions. Several RNA binding proteins that drive mRNA localization have also been shown to repress translation during transport. Activity of the translational machinery is also regulated in distal neural cell processes. Clinically, disruption of mRNA localization and/or localized mRNA translation may contribute to pathophysiology of fragile X mental retardation and spinal muscular atrophy. Axonal injury has been shown to activate localized protein synthesis, providing both a means to initiate regeneration and retrogradely signal injury to the cell body. Decreased capacity to transport mRNAs and translational machinery into distal processes could jeopardize the ability to respond to injury or local stimuli within axons and dendrites.

(318/610): RNA binding and transport proteins staufen and fragile x mental retardation protein are expressed by sensory neurons and localize to peripheral axons of nociceptors

Neuronal proteins have been traditionally viewed as being derived solely from the soma, wherein the preponderance of ribosomal/translational machinery resides. However, accumulating evidence indicates that non-somal sites, such as the dendrite and axon, are capable of a more autonomous role in terms of new protein synthesis. While this mechanism allows for more rapid, on-demand regulation of neuronal structure and function, it remains unclear precisely how the requisite RNA is trafficked into the axon for this purpose.

The RNA binding and transport proteins staufen and fragile X mental retardation protein are expressed by rat primary afferent neurons and localize to peripheral and central axons

Neuronal proteins have been traditionally viewed as being derived solely from the soma; however, accumulating evidence indicates that dendritic and axonal sites are capable of a more autonomous role in terms of new protein synthesis. Such extra-somal translation allows for more rapid, on-demand regulation of neuronal structure and function than would otherwise be possible. While mechanisms of dendritic RNA transport have been elucidated, it remains unclear how RNA is trafficked into the axon for this purpose. Primary afferent neurons of the dorsal root (DRG) and trigeminal (TG) ganglia have among the longest axons in the neuraxis and such axonal protein synthesis would be advantageous, given the greater time involved for protein trafficking to occur via axonal transport. Therefore, we hypothesized that these primary sensory neurons might express proteins involved in RNA transport. Rat DRG and TG neurons expressed staufen (stau) 1 and 2 (detected at the mRNA level) and stau2 and fragile x mental retardation protein (FMRP; detected at the protein level). Stau2 mRNA was also detected in human TG neurons. Stau2 and FMRP protein were localized to the sciatic nerve and dorsal roots by immunohistochemistry and to dorsal roots by Western blot. Stau2 and FMRP immunoreactivities colocalized with transient receptor potential channel type 1 immunoreactivity in sensory axons of the sciatic nerve and dorsal root, suggesting that these proteins are being transported into the peripheral and central terminals of nociceptive sensory axons. Based on these findings, we propose that stau2 and FMRP proteins are attractive candidates to subserve RNA transport in sensory neurons, linking somal transcriptional events to axonal translation.

Chemical Clamping Allows for Efficient Phosphorylation of the RNA Carrier Protein Npl3

Protein kinases phosphorylate the appropriate protein substrate by recognizing residues both proximal and distal to the site of phosphorylation. Although these distal contacts may provide excellent binding affinities (low Km values) through stabilization of the enzyme-substrate complex, these contacts could reduce catalytic turnover (decrease kcat) through slow phosphoprotein release. To investigate how protein kinases can overcome this problem and maintain both high substrate affinities and high turnover rates, the phosphorylation of the yeast RNA transport protein Npl3 by its natural protein kinase, Sky1p, was evaluated. Sky1p bound and phosphorylated Npl3 with a Km that was 2 orders of magnitude lower than a short peptide mimic representing the phosphorylation site and only proximal determinants. Surprisingly, this extraordinary difference is not the result of high affinity Npl3 binding. Rather, Npl3 achieves a low Km through a rapid and favorable phosphoryl transfer step. This step serves as a chemical clamp that locks the protein substrate in the active site without unduly stabilizing the product phosphoprotein and slowing its release. The chemical clamping mechanism offers an efficient means whereby a protein kinase can simultaneously achieve both high turnover and good substrate binding properties.

RNA transport and local protein synthesis in the dendritic compartment.

The shape and composition of the postsynaptic membrane depend on synaptic activity and change throughout the lifetime of the synapse (for references, see Albright et al. 2000). The mechanisms underlying the establishment of the receptor mosaic at the postsynaptic membrane (synthesis, processing, targeting and insertion of receptors) are still poorly understood. It is currently assumed that receptors, like other transmembrane proteins, are targeted to the plasma membrane via the somatic secretory pathway. Hypotheses concerning receptor-targeting mechanisms in the central neuron derive from two different systems: the neuromuscular junction (for references, see Sanes and Lichtman 1999) and the epithelial cell as a paradigm for neuron polarity (Dotti and Simons 1990). Recently, sorting signals have been identified for the metabotropic glutamate receptors (mGluRs, Stowell and Craig 1999). However, the machinery allowing a vectorial transport of neurotransmitter receptors from the cell soma to the postsynaptic differentiation has not been identified yet. The most challenging question is: how can thousands of signals, which notify minute changes occurring at each postsynaptic differentiation and arise from a large number of synapses, be treated somatically and subsequently induce a synaptic change, some synapses being located on dendrites hundreds of microns away?