Predicting condensate formation of protein and RNA under various environmental conditions.

BACKGROUND: Liquid-liquid phase separation (LLPS) by biomolecules plays a central role in various biological phenomena and has garnered significant attention. The behavior of LLPS is strongly influenced by the characteristics of RNAs and environmental factors such as pH and temperature, as well as the properties of proteins. Recently, several databases recording LLPS-related biomolecules have been established, and prediction models of LLPS-related phenomena have been explored using these databases. However, a prediction model that concurrently considers proteins, RNAs, and experimental conditions has not been developed due to the limited information available from individual experiments in public databases. RESULTS: To address this challenge, we have constructed a new dataset, RNAPSEC, which serves each experiment as a data point. This dataset was accomplished by manually collecting data from public literature. Utilizing RNAPSEC, we developed two prediction models that consider a protein, RNA, and experimental conditions. The first model can predict the LLPS behavior of a protein and RNA under given experimental conditions. The second model can predict the required conditions for a given protein and RNA to undergo LLPS. CONCLUSIONS: RNAPSEC and these prediction models are expected to accelerate our understanding of the roles of proteins, RNAs, and environmental factors in LLPS.

GSK3ß/axin-1/ß-catenin complex is involved in semaphorin3A signaling.

Semaphorin3A (Sema3A) exerts a wide variety of biological functions by regulating reorganization of actin and tubulin cytoskeletal proteins through signaling pathways including sequential phosphorylation of collapsin response mediator protein 1 (CRMP1) and CRMP2 by cyclin-dependent kinase-5 and glycogen synthase kinase-3ß (GSK3ß). To delineate how GSK3ß mediates Sema3A signaling, we here determined the substrates of GSK3ß involved. Introduction of either GSK3ß mutants, GSK3ß-R96A, L128A, or K85M into chick dorsal root ganglion (DRG) neurons suppressed Sema3A-induced growth cone collapse, thereby suggesting that unprimed as well as primed substrates are involved in Sema3A signaling. Axin-1, a key player in Wnt signaling, is an unprimed substrate of GSK3ß. The phosphorylation of Axin-1 by GSK3ß accelerates the association of Axin-1 with ß-catenin. Immunocytochemical studies revealed that Sema3A induced an increase in the intensity levels of ß-catenin in the DRG growth cones. Axin-1 siRNA knockdown suppressed Sema3A-induced growth cone collapse. The reintroduction of RNAi-resistant Axin-1 (rAxin-1)-wt rescued the responsiveness to Sema3A, while that of nonphosphorylated mutants, rAxin S322A/S326A/S330A and T485A/S490A/S497A, did not. Sema3A also enhanced the colocalization of GSK3ß, Axin-1, and ß-catenin in the growth cones. The increase of ß-catenin in the growth cones was suppressed by the siRNA knockdown of Axin-1. Furthermore, either Axin-1 or ß-catenin RNAi knockdown suppressed the internalization of Sema3A. These results suggest that Sema3A induces the formation of GSK3ß/Axin-1/ß-catenin complex, which regulates signaling cascade of Sema3A via an endocytotic mechanism. This finding should provide clue for understanding of mechanisms of a wide variety of biological functions of Sema3A.

Novel presynaptic assay system revealed that metformin ameliorates exaggerated synaptic release and Munc18-1 accumulation in presynapses of neurons from Fragile X syndrome mouse model.

Fragile X syndrome (FXS) is a developmental disorder characterized by intellectual disability and autistic-like behaviors. These symptoms are supposed to result from dysregulated translation in pre- and postsynapses, resulting in aberrant synaptic plasticity. Although most drug development research on FXS has focused on aberrant postsynaptic functions by excess translation in postsynapses, the effect of drug candidates on FXS in presynaptic release is largely unclear. In this report, we developed a novel assay system using neuron ball culture with beads to induce presynapse formation, allowing for the analysis of presynaptic phenotypes, including presynaptic release. Metformin, which is shown to rescue core phenotypes in FXS mouse model by normalizing dysregulated translation, ameliorated the exaggerated presynaptic release of neurons of FXS model mouse using this assay system. Furthermore, metformin suppressed the excess accumulation of the active zone protein Munc18-1, which is supposed to be locally translated in presynapses. These results suggest that metformin rescues both postsynaptic and presynaptic phenotypes by inhibiting excess translation in FXS neurons.

CRMP5 (collapsin response mediator protein 5) regulates dendritic development and synaptic plasticity in the cerebellar Purkinje cells.

Collapsin response mediator protein 5 (CRMP5) is one of the CRMP members that expresses abundantly in the developing brain. To examine the in vivo function of CRMP5, we generated crmp5-deficient (crmp5(-/-)) mice. Anti-calbindin immunofluorescence studies of crmp5(-/-) mice revealed aberrant dendrite morphology; specifically, a decrease in the size of soma and diameter of primary dendrite of the cerebellar Purkinje cells at postnatal day 21 (P21) and P28, but not at P14. Coincidentally, CRMP5 is detected in Purkinje cells at P21 and P28 from crmp5(+/-) mice. In cerebellar slices of crmp5(-/-) mice, the induction of long-term depression of excitatory synaptic transmission between parallel fibers and Purkinje cells was deficient. Given that brain-derived neurotrophic factor (BDNF) plays major roles in dendritic development, we tried to elucidate the possible roles of CRMP5 in BDNF signaling. The effect of BDNF to induce dendritic branching was markedly attenuated in cultured crmp5(-/-) neurons. Furthermore, CRMP5 was tyrosine phosphorylated when coexpressed with neurotrophic tyrosine kinase receptor type 2 (TrkB), a receptor for BDNF, in HEK293T cells. These findings suggest that CRMP5 is involved in the development, maintenance and synaptic plasticity of Purkinje cells.

Single molecule-sensitive probes for imaging RNA in live cells.

To visualize native or non-engineered RNA in live cells with single-molecule sensitivity, we developed multiply labeled tetravalent RNA imaging probes (MTRIPs). When delivered with streptolysin O into living human epithelial cancer cells and primary chicken fibroblasts, MTRIPs allowed the accurate imaging of native mRNAs and a non-engineered viral RNA, of RNA co-localization with known RNA-binding proteins, and of RNA dynamics and interactions with stress granules.

Extracellular stimuli specifically regulate localized levels of individual neuronal mRNAs.

Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein-beta-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous beta-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs.

Phosphorylation of zipcode binding protein 1 is required for brain-derived neurotrophic factor signaling of local beta-actin synthesis and growth cone turning.

The localization of specific mRNAs and their local translation in growth cones of developing axons has been shown to play an important mechanism to regulate growth cone turning responses to attractive or repulsive cues. However, the mechanism whereby local translation and growth cone turning may be controlled by specific mRNA-binding proteins is unknown. Here we demonstrate that brain-derived neurotrophic factor (BDNF) signals the Src-dependent phosphorylation of the beta-actin mRNA zipcode binding protein 1 (ZBP1), which is necessary for beta-actin synthesis and growth cone turning. We raised a phospho-specific ZBP1 antibody to Tyr396, which is a Src phosphorylation site, and immunofluorescence revealed BDNF-induced phosphorylation of ZBP1 within growth cones. The BDNF-induced increase in fluorescent signal of a green fluorescent protein translation reporter with the 3' untranslated region of beta-actin was attenuated with the Src family kinase-specific inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine]. Furthermore, a nonphosphorylatable mutant, ZBP1 Y396F, suppressed the BDNF-induced and protein synthesis-dependent increase in beta-actin localization in growth cones. Last, the ZBP1 Y396F mutant blocked BDNF-induced attractive growth cone turning. These results indicate that phosphorylation of ZBP1 at Tyr396 within growth cones has a critical role to regulate local protein synthesis and growth cone turning. Our findings provide new insight into how the regulated phosphorylation of mRNA-binding proteins influences local translation underlying growth cone motility and axon guidance.

Semaphorin3A signaling mediated by Fyn-dependent tyrosine phosphorylation of collapsin response mediator protein 2 at tyrosine 32.

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr(32) residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr(32) of CRMP showed that Tyr(32)-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr(32) to Phe(32)) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr(32) is involved in Sema3A signaling.

An essential role for beta-actin mRNA localization and translation in Ca2+-dependent growth cone guidance.

Axon pathfinding requires directional responses of growth cones to extracellular cues, which have been shown to involve local synthesis of protein. The identity and functions of the locally produced proteins remain, however, unclear. Here we report that Ca(2+)-dependent bidirectional turning of Xenopus laevis growth cones requires localized distribution and translation of beta-actin messenger RNA. Both beta-actin mRNA and its zipcode-binding protein, ZBP1, are localized at the growth cone and become asymmetrically distributed upon local exposure to brain-derived neurotrophic factor (BDNF). Inhibition of protein synthesis or antisense interference with beta-actin mRNA-ZBP1 binding abolishes both Ca(2+)-mediated attraction and repulsion. In addition, attraction involves a local increase in beta-actin, whereas repulsion is accompanied by a local decrease in beta-actin; thus, both produce a synthesis- and ZBP1 binding-dependent beta-actin asymmetry but with opposite polarities. Together with a similar asymmetry in Src activity during bidirectional responses, our findings indicate that Ca(2+)-dependent spatial regulation of beta-actin synthesis through Src contributes to the directional motility of growth cones during guidance.

Local Translation in Growth Cones and Presynapses, Two Axonal Compartments for Local Neuronal Functions.

During neural development, growth cones, very motile compartments of tips of axons, lead axonal extension to the correct targets. Subsequently, presynapses, another axonal compartment with vigorous trafficking of synaptic vesicles, emerge to form functional synapses with postsynapses. In response to extracellular stimuli, the immediate supply of proteins by local translation within these two axonal compartments far from cell bodies confers high motility of growth cones and active vesicle trafficking in presynapses. Although local translation in growth cones and presynapses occurs at a very low level compared with cell bodies and even dendrites, recent progress in omics and visualization techniques with subcellular fractionation of these compartments has revealed the actual situation of local translation within these two axonal compartments. Here, the increasing evidence for local protein synthesis in growth cones and presynapses for axonal and synaptic functions has been reviewed. Furthermore, the mechanisms regulating local translation in these two compartments and pathophysiological conditions caused by dysregulated local translation are highlighted.