Laminin induced local axonal translation of β-actin mRNA is impaired in SMN-deficient motoneurons

Reduced levels of the SMN (survival of motoneuron) protein cause spinal muscular atrophy, the main form of motoneuron disease in children and young adults. In cultured motoneurons, reduced SMN levels lead to disturbed axon growth that correlates with reduced actin mRNA and protein in growth cones, indicating that anterograde transport and local translation of β-actin mRNA are altered in this disease. However, it is not fully understood how local translation of the β-actin mRNA is regulated in SMN-deficient motoneurons. Here, we established a lentiviral GFP-based reporter construct to monitor local translation of β-actin mRNA. Time-lapse imaging of fluorescence recovery after photobleaching (FRAP) in living motoneurons revealed that β-actin is locally translated in the growth cones of embryonic motoneurons. Interestingly, local translation of the β-actin reporter construct was differentially regulated by various Laminin isoforms, indicating that Laminins provide extracellular cues for the regulation of local translation in growth cones. Notably, local translation of β-actin mRNA was deregulated in motoneurons from a mouse model for the most severe form of SMA (Smn ^?/? ;SMN2). Taken together our findings suggest that local translation of β-actin in growth cones of motoneurons is regulated by Laminin signalling and that this signalling is disturbed in SMA.     

Local translation and directional steering in axons

The assembly of functional neural circuits in the developing brain requires neurons to extend axons to the correct targets. This in turn requires the navigating tips of axons to respond appropriately to guidance cues present along the axonal pathway, despite being cellular 'outposts' far from the soma. Work over the past few years has demonstrated a critical role for local translation within the axon in this process in vitro, making axon guidance another process that requires spatially localized translation, among others such as synaptic plasticity, cell migration, and cell polarity. This article reviews recent findings in local axonal translation and discusses how new protein synthesis may function in growth cone guidance, with a comparative view toward models of local translation in other systems.     

A collection of caged compounds for probing roles of local translation in neurobiology

Spatially localized translation plays a vital role in the normal functioning of neuronal systems and is widely believed to be involved in both learning and memory formation. It is of central interest to understand both the phenomenon and molecular mechanisms of local translation using new tools and approaches. Caged compounds can, in principle, be used as tools to investigate local translation since optical activation of bioactive molecules can achieve both spatial and temporal resolution on the micron scale and on the order of seconds or less, respectively. Successful caging of bioactive molecules requires the identification of key functional groups in appropriate molecules and the introduction of a suitable caging moiety. Here we present the design, synthesis and testing of a collection of three caged compounds: anisomycin caged with a diethylaminocoumarin moiety and dimethoxynitrobenzyl caged versions of 4E-BP and rapamycin. Whereas caged anisomycin can be used to control general translation, caged 4E-BP serves as a probe of cap-dependent translation initiation and caged rapamycin serves a probe of the role of mTORC1 in translation initiation. In vitro translation assays demonstrate that these caging strategies, in combination with the aforementioned compounds, are effective for optical control making it likely that such strategies can successfully employed in the study of local translation in living systems.     

Correlation between the effectiveness of translation initiation and secondary structure of mRNA in the hybrid gene cro-lacIZ

A set of plasmids containing short DNA deletions in the N-part of cro-lacIZ coding zone as constructed using Bal31 nuclease. Development of models of mRNA secondary structure was carried out stepwise beginning from the 5'-end by taking into consideration the hairpins first formed during mRNA synthesis. Comparison of the results of mRNA secondary structure determination and protein production analysis demonstrated a correlation between the efficiency of translation initiation and the appearance of a single-stranded region upon disruption of the mRNA local secondary structure in the translation initiation zone generated by ribosomes. These results confirm the suggestion of the central role played by the Shine-Dalgarno sequence in the generation of a single-stranded region. Some plasmids from the set are supposed to determine protein synthesis by a translation reinitiation mechanism in the absence of Shine--Dalgarno interaction. In this case, correlation between reinitiation efficiency and local disruption of the mRNA secondary structure by the terminating ribosome was also observed. The terminating ribosome that forms the single-stranded region near the initiation codon fulfils the major function of the Shine--Dalgarno interaction. In addition, the possible effect of another mRNA secondary structure region on the translation initiation efficiency is discussed.     

Regulation of local mRNA translation

Regulated local mRNA translation is one mechanism cells employ to concentrate proteins in particular locations. However, cells use many different strategies to accomplish this task; for example, some mRNAs are destroyed in regions where they are not wanted, other mRNAs are repressed in areas where their translation would be deleterious, and yet other mRNAs are transported, in a quiescent state, to the sites where their translation is activated. The importance of local translation cannot be overstated, for, depending on the species or cell type, it is required for cell division, establishment of mating type, development and memory formation.     

Molecular biology of axons: "a turning point...".

Previous studies have shown that dendrites and axons contain both mRNAs and the machinery for local protein translation. While a number of studies in recent years have focused on the functional role of protein synthesis in dendrites, relatively less is know about the role of local translation in axons. Campbell and Holt (this issue of Neuron) show that local protein synthesis and degradation are required for proper chemotropic turning responses of isolated retinal growth cones.     

RACK1 is necessary for the formation of point contacts and regulates axon growth

Receptor for activated C kinase 1 (RACK1) is a multifunctional ribosomal scaffolding protein that can interact with multiple signaling molecules concurrently through its seven WD40 repeats. We recently found that RACK1 is localized to mammalian growth cones, prompting an investigation into its role during neural development. Here, we show for the first time that RACK1 localizes to point contacts within mouse cortical growth cones. Point contacts are adhesion sites that link the actin network within growth cones to the extracellular matrix, and are necessary for appropriate axon guidance. Our experiments show that RACK1 is necessary for point contact formation. Brain‐derived neurotrophic factor (BDNF) stimulates an increase in point contact density, which was eliminated by RACK1 shRNA or overexpression of a nonphosphorylatable mutant form of RACK1. We also found that axonal growth requires both RACK1 expression and phosphorylation. We have previously shown that the local translation of β‐actin mRNA within growth cones is necessary for appropriate axon guidance and is dependent on RACK1. Thus, we examined the location of members of the local translation complex relative to point contacts. Indeed, both β‐actin mRNA and RACK1 colocalize with point contacts, and this colocalization increases following BDNF stimulation. This implies the novel finding that local translation is regulated at point contacts. Taken together, these data suggest that point contacts are a targeted site of local translation within growth cones, and RACK1 is a critical member of the point contact complex and necessary for appropriate neural development. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1038–1056, 2017     

Local translation in neuronal compartments: how local is local?

Efficient neuronal function depends on the continued modulation of the local neuronal proteome. Local protein synthesis plays a central role in tuning the neuronal proteome at specific neuronal regions. Various aspects of translation such as the localization of translational machinery, spatial spread of the newly translated proteins, and their site of action are carried out in specialized neuronal subcompartments to result in a localized functional outcome. In this review, we focus on the various aspects of these local translation compartments such as size, biochemical and organelle composition, structural boundaries, and temporal dynamics. We also discuss the apparent absence of definitive components of translation in these local compartments and the emerging state‐of‐the‐art tools that could help dissecting these conundrums in greater detail in the future.     

Human interferon-gamma mRNA autoregulates its translation through a pseudoknot that activates the interferon-inducible protein kinase PKR

PKR, an interferon (IFN)-inducible protein kinase activated by double-stranded RNA, inhibits translation by phosphorylating the initiation factor eIF2alpha chain. We show that human IFN-gamma mRNA uses local activation of PKR in the cell to control its own translation yield. IFN-gamma mRNA activates PKR through a pseudoknot in its 5' untranslated region. Mutations that impair pseudoknot stability reduce the ability to activate PKR and strongly increase the translation efficiency of IFN-gamma mRNA. Nonphosphorylatable mutant eIF2alpha, knockout of PKR and PKR inhibitors 2-aminopurine, transdominant-negative PKR, or vaccinia E3L correspondingly enhances translation of IFN-gamma mRNA. The potential to form the pseudoknot is phylogenetically conserved. We propose that the RNA pseudoknot acts to adjust translation of IFN-gamma mRNA to the PKR level expressed in the cell.     

Dysregulated Translation in Neurodevelopmental Disorders: An Overview of Autism‐Risk Genes Involved in Translation

Regulated local translation—whereby specific mRNAs are transported and localized in subcellular domains where they are translated in response to regional signals—allows for remote control of gene expression to concentrate proteins in subcellular compartments. Neurons are highly polarized cells with unique features favoring local control for axonal pathfinding and synaptic plasticity, which are key processes involved in constructing functional circuits in the developing brain. Neurodevelopmental disorders are caused by genetic or environmental factors that disturb the nervous system’s development during prenatal and early childhood periods. The growing list of genetic mutations that affect mRNA translation raises the question of whether aberrant translatomes in individuals with neurodevelopmental disorders share common molecular features underlying their stereotypical phenotypes and, vice versa, cause a certain degree of phenotypic heterogeneity. Here, we briefly give an overview of the role of local translation during neuronal development. We take the autism‐risk gene list and discuss the molecules that (perhaps) are involved in mRNA transport and translation. Both exaggerated and suppressed translation caused by mutations in those genes have been identified or suggested. Finally, we discuss some proof‐of‐principle regimens for use in autism mouse models to correct dysregulated translation.     

Disruption of dendritic translation of CaMKIIalpha impairs stabilization of synaptic plasticity and memory consolidation

Local protein translation in dendrites could be a means for delivering synaptic proteins to their sites of action, perhaps in a spatially regulated fashion that could contribute to plasticity. To directly test the functional role of dendritic translation of calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha) in vivo, we mutated the endogenous gene to disrupt the dendritic localization signal in the mRNA. In this mutant mouse, the protein-coding region of CaMKIIalpha is intact, but mRNA is restricted to the soma. Removal of dendritic mRNA produced a dramatic reduction of CaMKIIalpha in postsynaptic densities (PSDs), a reduction in late-phase long-term potentiation (LTP), and impairments in spatial memory, associative fear conditioning, and object recognition memory. These results demonstrate that local translation is important for synaptic delivery of the kinase and that local translation contributes to synaptic and behavioral plasticity.     

Protein functional features are reflected in the patterns of mRNA translation speed

Background

The degeneracy of the genetic code makes it possible for the same amino acid string to be coded by different messenger RNA (mRNA) sequences. These “synonymous mRNAs” may differ largely in a number of aspects related to their overall translational efficiency, such as secondary structure content and availability of the encoded transfer RNAs (tRNAs). Consequently, they may render different yields of the translated polypeptides. These mRNA features related to translation efficiency are also playing a role locally, resulting in a non-uniform translation speed along the mRNA, which has been previously related to some protein structural features and also used to explain some dramatic effects of “silent” single-nucleotide-polymorphisms (SNPs). In this work we perform the first large scale analysis of the relationship between three experimental proxies of mRNA local translation efficiency and the local features of the corresponding encoded proteins.

Results

We found that a number of protein functional and structural features are reflected in the patterns of ribosome occupancy, secondary structure and tRNA availability along the mRNA. One or more of these proxies of translation speed have distinctive patterns around the mRNA regions coding for certain protein local features. In some cases the three patterns follow a similar trend. We also show specific examples where these patterns of translation speed point to the protein’s important structural and functional features.

Conclusions

This support the idea that the genome not only codes the protein functional features as sequences of amino acids, but also as subtle patterns of mRNA properties which, probably through local effects on the translation speed, have some consequence on the final polypeptide. These results open the possibility of predicting a protein’s functional regions based on a single genomic sequence, and have implications for heterologous protein expression and fine-tuning protein function.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1734-7) contains supplementary material, which is available to authorized users.     

How the Sequence of a Gene Specifies Structural Symmetry in Proteins

Internal symmetry is commonly observed in the majority of fundamental protein folds. Meanwhile, sufficient evidence suggests that nascent polypeptide chains of proteins have the potential to start the co-translational folding process and this process allows mRNA to contain additional information on protein structure. In this paper, we study the relationship between gene sequences and protein structures from the viewpoint of symmetry to explore how gene sequences code for structural symmetry in proteins. We found that, for a set of two-fold symmetric proteins from left-handed beta-helix fold, intragenic symmetry always exists in their corresponding gene sequences. Meanwhile, codon usage bias and local mRNA structure might be involved in modulating translation speed for the formation of structural symmetry: a major decrease of local codon usage bias in the middle of the codon sequence can be identified as a common feature; and major or consecutive decreases in local mRNA folding energy near the boundaries of the symmetric substructures can also be observed. The results suggest that gene duplication and fusion may be an evolutionarily conserved process for this protein fold. In addition, the usage of rare codons and the formation of higher order of secondary structure near the boundaries of symmetric substructures might have coevolved as conserved mechanisms to slow down translation elongation and to facilitate effective folding of symmetric substructures. These findings provide valuable insights into our understanding of the mechanisms of translation and its evolution, as well as the design of proteins via symmetric modules.     

Glutamate Stimulates Local Protein Synthesis in the Axons of Rat Cortical Neurons by Activating α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors and Metabotropic Glutamate Receptors

Glutamate is the principal excitatory neurotransmitter in the mammalian CNS. By analyzing the metabolic incorporation of azidohomoalanine, a methionine analogue, in newly synthesized proteins, we find that glutamate treatments up-regulate protein translation not only in intact rat cortical neurons in culture but also in the axons emitting from cortical neurons before making synapses with target cells. The process by which glutamate stimulates local translation in axons begins with the binding of glutamate to the ionotropic AMPA receptors and metabotropic glutamate receptor 1 and members of group 2 metabotropic glutamate receptors on the plasma membrane. Subsequently, the activated mammalian target of rapamycin (mTOR) signaling pathway and the rise in Ca²⁺, resulting from Ca²⁺ influxes through calcium-permeable AMPA receptors, voltage-gated Ca²⁺ channels, and transient receptor potential canonical channels, in axons stimulate the local translation machinery. For comparison, the enhancement effects of brain-derived neurotrophic factor (BDNF) on the local protein synthesis in cortical axons were also studied. The results indicate that Ca²⁺ influxes via transient receptor potential canonical channels and activated the mTOR pathway in axons also mediate BDNF stimulation to local protein synthesis. However, glutamate- and BDNF-induced enhancements of translation in axons exhibit different kinetics. Moreover, Ca²⁺ and mTOR signaling appear to play roles carrying different weights, respectively, in transducing glutamate- and BDNF-induced enhancements of axonal translation. Thus, our results indicate that exposure to transient increases of glutamate and more lasting increases of BDNF would stimulate local protein synthesis in migrating axons en route to their targets in the developing brain.