Spatial code recognition in neuronal RNA targeting: role of RNA-hnRNP A2 interactions

In neurons, regulation of gene expression occurs in part through translational control at the synapse. A fundamental requirement for such local control is the targeted delivery of select neuronal mRNAs and regulatory RNAs to distal dendritic sites. The nature of spatial RNA destination codes, and the mechanism by which they are interpreted for dendritic delivery, remain poorly understood. We find here that in a key dendritic RNA transport pathway (exemplified by BC1 RNA, a dendritic regulatory RNA, and protein kinase M ζ [PKMζ] mRNA, a dendritic mRNA), noncanonical purine•purine nucleotide interactions are functional determinants of RNA targeting motifs. These motifs are specifically recognized by heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2), a trans-acting factor required for dendritic delivery. Binding to hnRNP A2 and ensuing dendritic delivery are effectively competed by RNAs with CGG triplet repeat expansions. CGG repeats, when expanded in the 5′ untranslated region of fragile X mental retardation 1 (FMR1) mRNA, cause fragile X–associated tremor/ataxia syndrome. The data suggest that cellular dysregulation observed in the presence of CGG repeat RNA may result from molecular competition in neuronal RNA transport pathways.     

Multiplexed dendritic targeting of alpha calcium calmodulin-dependent protein kinase II, neurogranin, and activity-regulated cytoskeleton-associated protein RNAs by the A2 pathway

In neurons, many different RNAs are targeted to dendrites where local expression of the encoded proteins mediates synaptic plasticity during learning and memory. It is not known whether each RNA follows a separate trafficking pathway or whether multiple RNAs are targeted to dendrites by the same pathway. Here, we show that RNAs encoding alpha calcium calmodulin-dependent protein kinase II, neurogranin, and activity-regulated cytoskeleton-associated protein are coassembled into the same RNA granules and targeted to dendrites by the same cis/trans-determinants (heterogeneous nuclear ribonucleoprotein [hnRNP] A2 response element and hnRNP A2) that mediate dendritic targeting of myelin basic protein RNA by the A2 pathway in oligodendrocytes. Multiplexed dendritic targeting of different RNAs by the same pathway represents a new organizing principle for coordinating gene expression at the synapse.     

The emerging function of IQD proteins as scaffolds in cellular signaling and trafficking

Calcium (Ca²⁺) signaling modules are essential for adjusting plant growth and performance to environmental constraints. Differential interactions between sensors of Ca²⁺ dynamics and their molecular targets are at the center of the transduction process. Calmodulin (CaM) and CaM-like (CML) proteins are principal Ca²⁺-sensors in plants that govern the activities of numerous downstream proteins with regulatory properties. The families of IQ67-Domain (IQD) proteins are a large class of plant-specific CaM/CML-targets (e.g., 33 members in A. thaliana) which share a unique domain of multiple varied CaM retention motifs in tandem orientation. Genetic studies in Arabidopsis and tomato revealed first roles for IQD proteins related to basal defense response and plant development. Molecular, biochemical and histochemical analysis of Arabidopsis IQD1 demonstrated association with microtubules as well as targeting to the cell nucleus and nucleolus. In vivo binding to CaM and kinesin light chain-related protein-1 (KLCR1) suggests a Ca²⁺-regulated scaffolding function of IQD1 in kinesin motor-dependent transport of multiprotein complexes. Furthermore, because IQD1 interacts in vitro with single-stranded nucleic acids, the prospect arises that IQD1 and other IQD family members facilitate cellular RNA localization as one mechanism to control and fine-tune gene expression and protein sorting.     

A Highlights from MBoC Selection: Dendritic and axonal mechanisms of Ca2+ elevation impair BDNF transport in Aβ oligomer–treated hippocampal neurons

Disruption of fast axonal transport (FAT) and intracellular Ca²⁺ dysregulation are early pathological events in Alzheimer''s disease (AD). Amyloid-β oligomers (AβOs), a causative agent of AD, impair transport of BDNF independent of tau by nonexcitotoxic activation of calcineurin (CaN). Ca²⁺-dependent mechanisms that regulate the onset, severity, and spatiotemporal progression of BDNF transport defects from dendritic and axonal AβO binding sites are unknown. Here we show that BDNF transport defects in dendrites and axons are induced simultaneously but exhibit different rates of decline. The spatiotemporal progression of FAT impairment correlates with Ca²⁺ elevation and CaN activation first in dendrites and subsequently in axons. Although many axonal pathologies have been described in AD, studies have primarily focused only on the dendritic effects of AβOs despite compelling reports of presynaptic AβOs in AD models and patients. Indeed, we observe that dendritic CaN activation converges on Ca²⁺ influx through axonal voltage-gated Ca²⁺ channels to impair FAT. Finally, FAT defects are prevented by dantrolene, a clinical compound that reduces Ca²⁺ release from the ER. This work establishes a novel role for Ca²⁺ dysregulation in BDNF transport disruption and tau-independent Aβ toxicity in early AD.     

RNA trafficking signals in human immunodeficiency virus type 1

Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr and tat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelled gag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently as vpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.     

Transport Properties of the Tomato Fruit Tonoplast : III. Temperature Dependence of Calcium Transport

Calcium transport into tomato (Lycopersicon esculentum Mill, cv Castlemart) fruit tonoplast vesicles was studied. Calcium uptake was stimulated approximately 10-fold by MgATP. Two ATP-dependent Ca²⁺ transport activities could be resolved on the basis of sensitivity to nitrate and affinity for Ca²⁺. A low affinity Ca²⁺ uptake system (K_m > 200 micromolar) was inhibited by nitrate and ionophores and is thought to represent a tonoplast localized H⁺/Ca²⁺ antiport. A high affinity Ca²⁺ uptake system (K_m = 6 micromolar) was not inhibited by nitrate, had reduced sensitivity to ionophores, and appeared to be associated with a population of low density endoplasmic reticulum vesicles that contaminated the tonoplast-enriched membrane fraction. Arrhenius plots of the temperature dependence of Ca²⁺ transport in tomato membrane vesicles showed a sharp increase in activation energy at temperatures below 10 to 12°C that was not observed in red beet membrane vesicles. This low temperature effect on tonoplast Ca²⁺/H⁺ antiport activity could only by partially ascribed to an effect of low temperature on H⁺-ATPase activity, ATP-dependent H⁺ transport, passive H⁺ fluxes, or passive Ca²⁺ fluxes. These results suggest that low temperature directly affects Ca²⁺/H⁺ exchange across the tomato fruit tonoplast, resulting in an apparent change in activation energy for the transport reaction. This could result from a direct effect of temperature on the Ca²⁺/H⁺ exchange protein or by an indirect effect of temperature on lipid interactions with the Ca²⁺/H⁺ exchange protein.     

Mutational analysis of a heterogeneous nuclear ribonucleoprotein A2 response element for RNA trafficking

Cytoplasmic transport and localization of mRNA has been reported for a range of oocytes and somatic cells. The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 response element (A2RE) is a 21-nucleotide segment of the myelin basic protein mRNA that is necessary and sufficient for cytoplasmic transport of this message in oligodendrocytes. The predominant A2RE-binding protein in rat brain has previously been identified as hnRNP A2. Here we report that an 11-nucleotide subsegment of the A2RE (A2RE11) was as effective as the full-length A2RE in binding hnRNP A2 and mediating transport of heterologous RNA in oligodendrocytes. Point mutations of the A2RE11 that eliminated binding to hnRNP A2 also markedly reduced the ability of these oligoribonucleotides to support RNA transport. Oligodendrocytes treated with antisense oligonucleotides directed against the translation start site of hnRNP A2 had reduced levels of this protein and disrupted transport of microinjected myelin basic protein RNA. Several A2RE-like sequences from localized neuronal RNAs also bound hnRNP A2 and promoted RNA transport in oligodendrocytes. These data demonstrate the specificity of A2RE recognition by hnRNP A2, provide direct evidence for the involvement of hnRNP A2 in cytoplasmic RNA transport, and suggest that this protein may interact with a wide variety of localized messages that possess A2RE-like sequences.     

SLC25A23 augments mitochondrial Ca2+ uptake,interacts with MCU,and induces oxidative stress–mediated cell death

Emerging findings suggest that two lineages of mitochondrial Ca²⁺ uptake participate during active and resting states: 1) the major eukaryotic membrane potential–dependent mitochondrial Ca²⁺ uniporter and 2) the evolutionarily conserved exchangers and solute carriers, which are also involved in ion transport. Although the influx of Ca²⁺ across the inner mitochondrial membrane maintains metabolic functions and cell death signal transduction, the mechanisms that regulate mitochondrial Ca²⁺ accumulation are unclear. Solute carriers—solute carrier 25A23 (SLC25A23), SLC25A24, and SLC25A25—represent a family of EF-hand–containing mitochondrial proteins that transport Mg-ATP/Pi across the inner membrane. RNA interference–mediated knockdown of SLC25A23 but not SLC25A24 and SLC25A25 decreases mitochondrial Ca²⁺ uptake and reduces cytosolic Ca²⁺ clearance after histamine stimulation. Ectopic expression of SLC25A23 EF-hand–domain mutants exhibits a dominant-negative phenotype of reduced mitochondrial Ca²⁺ uptake. In addition, SLC25A23 interacts with mitochondrial Ca²⁺ uniporter (MCU; CCDC109A) and MICU1 (CBARA1) while also increasing I_MCU. In addition, SLC25A23 knockdown lowers basal mROS accumulation, attenuates oxidant-induced ATP decline, and reduces cell death. Further, reconstitution with short hairpin RNA–insensitive SLC25A23 cDNA restores mitochondrial Ca²⁺ uptake and superoxide production. These findings indicate that SLC25A23 plays an important role in mitochondrial matrix Ca²⁺ influx.     

Rem uncouples excitation–contraction coupling in adult skeletal muscle fibers

In skeletal muscle, excitation–contraction (EC) coupling requires depolarization-induced conformational rearrangements in L-type Ca²⁺ channel (Ca_V1.1) to be communicated to the type 1 ryanodine-sensitive Ca²⁺ release channel (RYR1) of the sarcoplasmic reticulum (SR) via transient protein–protein interactions. Although the molecular mechanism that underlies conformational coupling between Ca_V1.1 and RYR1 has been investigated intensely for more than 25 years, the question of whether such signaling occurs via a direct interaction between the principal, voltage-sensing α_1S subunit of Ca_V1.1 and RYR1 or through an intermediary protein persists. A substantial body of evidence supports the idea that the auxiliary β_1a subunit of Ca_V1.1 is a conduit for this intermolecular communication. However, a direct role for β_1a has been difficult to test because β_1a serves two other functions that are prerequisite for conformational coupling between Ca_V1.1 and RYR1. Specifically, β_1a promotes efficient membrane expression of Ca_V1.1 and facilitates the tetradic ultrastructural arrangement of Ca_V1.1 channels within plasma membrane–SR junctions. In this paper, we demonstrate that overexpression of the RGK protein Rem, an established β subunit–interacting protein, in adult mouse flexor digitorum brevis fibers markedly reduces voltage-induced myoplasmic Ca²⁺ transients without greatly affecting Ca_V1.1 targeting, intramembrane gating charge movement, or releasable SR Ca²⁺ store content. In contrast, a β_1a-binding–deficient Rem triple mutant (R200A/L227A/H229A) has little effect on myoplasmic Ca²⁺ release in response to membrane depolarization. Thus, Rem effectively uncouples the voltage sensors of Ca_V1.1 from RYR1-mediated SR Ca²⁺ release via its ability to interact with β_1a. Our findings reveal Rem-expressing adult muscle as an experimental system that may prove useful in the definition of the precise role of the β_1a subunit in skeletal-type EC coupling.     

The structure and interactions of Ca2+-ATPase

Electron crystallographic studies on membrane crystals of Ca²⁺-ATPase reveal different patterns of ATPase-ATPase interactions depending on enzyme conformation. Physiologically relevant changes in Ca²⁺ concentration and membrane potential affect these interactions. Ca²⁺ induced difference FTIR spectra of Ca²⁺-ATPase triggered by photolysis of caged Ca²⁺ are consistent with changes in secondary structure and carboxylate groups upon Ca²⁺ binding; the changes are reversed during ATP hydrolysis suggesting that a phosphorylated enzyme form of low Ca²⁺ affinity is the dominant intermediate during Ca²⁺ transport. A two-channel model of Ca²⁺ translocation is proposed involving the membrane-spanning helices M2–M5 and M4, M5, M6 and M8 respectively, with separate but interacting Ca²⁺ binding sites.     

Ancient Origins of RGK Protein Function: Modulation of Voltage-Gated Calcium Channels Preceded the Protostome and Deuterostome Split

RGK proteins, Gem, Rad, Rem1, and Rem2, are members of the Ras superfamily of small GTP-binding proteins that interact with Ca²⁺ channel β subunits to modify voltage-gated Ca²⁺ channel function. In addition, RGK proteins affect several cellular processes such as cytoskeletal rearrangement, neuronal dendritic complexity, and synapse formation. To probe the phylogenetic origins of RGK protein–Ca²⁺ channel interactions, we identified potential RGK-like protein homologs in genomes for genetically diverse organisms from both the deuterostome and protostome animal superphyla. RGK-like protein homologs cloned from Danio rerio (zebrafish) and Drosophila melanogaster (fruit flies) expressed in mammalian sympathetic neurons decreased Ca²⁺ current density as reported for expression of mammalian RGK proteins. Sequence alignments from evolutionarily diverse organisms spanning the protostome/deuterostome divide revealed conservation of residues within the RGK G-domain involved in RGK protein – Ca_vβ subunit interaction. In addition, the C-terminal eleven residues were highly conserved and constituted a signature sequence unique to RGK proteins but of unknown function. Taken together, these data suggest that RGK proteins, and the ability to modify Ca²⁺ channel function, arose from an ancestor predating the protostomes split from deuterostomes approximately 550 million years ago.     

Heterogeneous nuclear ribonucleoprotein (hnRNP) E1 binds to hnRNP A2 and inhibits translation of A2 response element mRNAs

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of specific mRNAs. Here, we show by yeast two-hybrid analysis, in vivo and in vitro coimmunoprecipitation, in vitro cross-linking, and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and is recruited to A2RE RNA in an hnRNP A2-dependent manner. hnRNP E1 is colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE RNA. Excess hnRNP E1 added to an in vitro translation system reduces translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP A2-dependent manner. These results are consistent with a model where hnRNP E1 recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of A2RE RNA during granule transport.     

Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons

Pheromones are substances released from animals that, when detected by the vomeronasal organ of other individuals of the same species, affect their physiology and behavior. Pheromone binding to receptors on microvilli on the dendritic knobs of vomeronasal sensory neurons activates a second messenger cascade to produce an increase in intracellular Ca²⁺ concentration. Here, we used whole-cell and inside-out patch-clamp analysis to provide a functional characterization of currents activated by Ca²⁺ in isolated mouse vomeronasal sensory neurons in the absence of intracellular K⁺. In whole-cell recordings, the average current in 1.5 µM Ca²⁺ and symmetrical Cl⁻ was −382 pA at −100 mV. Ion substitution experiments and partial blockade by commonly used Cl⁻ channel blockers indicated that Ca²⁺ activates mainly anionic currents in these neurons. Recordings from inside-out patches from dendritic knobs of mouse vomeronasal sensory neurons confirmed the presence of Ca²⁺-activated Cl⁻ channels in the knobs and/or microvilli. We compared the electrophysiological properties of the native currents with those mediated by heterologously expressed TMEM16A/anoctamin1 or TMEM16B/anoctamin2 Ca²⁺-activated Cl⁻ channels, which are coexpressed in microvilli of mouse vomeronasal sensory neurons, and found a closer resemblance to those of TMEM16A. We used the Cre–loxP system to selectively knock out TMEM16A in cells expressing the olfactory marker protein, which is found in mature vomeronasal sensory neurons. Immunohistochemistry confirmed the specific ablation of TMEM16A in vomeronasal neurons. Ca²⁺-activated currents were abolished in vomeronasal sensory neurons of TMEM16A conditional knockout mice, demonstrating that TMEM16A is an essential component of Ca²⁺-activated Cl⁻ currents in mouse vomeronasal sensory neurons.     

The Actin Nucleator Cobl Is Controlled by Calcium and Calmodulin

Actin nucleation triggers the formation of new actin filaments and has the power to shape cells but requires tight control in order to bring about proper morphologies. The regulation of the members of the novel class of WASP Homology 2 (WH2) domain-based actin nucleators, however, thus far has largely remained elusive. Our study reveals signal cascades and mechanisms regulating Cordon-Bleu (Cobl). Cobl plays some, albeit not fully understood, role in early arborization of neurons and nucleates actin by a mechanism that requires a combination of all three of its actin monomer–binding WH2 domains. Our experiments reveal that Cobl is regulated by Ca²⁺ and multiple, direct associations of the Ca²⁺ sensor Calmodulin (CaM). Overexpression analyses and rescue experiments of Cobl loss-of-function phenotypes with Cobl mutants in primary neurons and in tissue slices demonstrated the importance of CaM binding for Cobl’s functions. Cobl-induced dendritic branch initiation was preceded by Ca²⁺ signals and coincided with local F-actin and CaM accumulations. CaM inhibitor studies showed that Cobl-mediated branching is strictly dependent on CaM activity. Mechanistic studies revealed that Ca²⁺/CaM modulates Cobl’s actin binding properties and furthermore promotes Cobl’s previously identified interactions with the membrane-shaping F-BAR protein syndapin I, which accumulated with Cobl at nascent dendritic protrusion sites. The findings of our study demonstrate a direct regulation of an actin nucleator by Ca²⁺/CaM and reveal that the Ca²⁺/CaM-controlled molecular mechanisms we discovered are crucial for Cobl’s cellular functions. By unveiling the means of Cobl regulation and the mechanisms, by which Ca²⁺/CaM signals directly converge on a cellular effector promoting actin filament formation, our work furthermore sheds light on how local Ca²⁺ signals steer and power branch initiation during early arborization of nerve cells—a key process in neuronal network formation.     

A Highlights from MBoC Selection: Functional analysis of the interface between the tandem C2 domains of synaptotagmin-1

C2 domains are widespread motifs that often serve as Ca²⁺-binding modules; some proteins have more than one copy. An open issue is whether these domains, when duplicated within the same parent protein, interact with one another to regulate function. In the present study, we address the functional significance of interfacial residues between the tandem C2 domains of synaptotagmin (syt)-1, a Ca²⁺ sensor for neuronal exocytosis. Substitution of four residues, YHRD, at the domain interface, disrupted the interaction between the tandem C2 domains, altered the intrinsic affinity of syt-1 for Ca²⁺, and shifted the Ca²⁺ dependency for binding to membranes and driving membrane fusion in vitro. When expressed in syt-1 knockout neurons, the YHRD mutant yielded reductions in synaptic transmission, as compared with the wild-type protein. These results indicate that physical interactions between the tandem C2 domains of syt-1 contribute to excitation–secretion coupling.     

The relationship between vitamin D-stimulated calcium transport and intestinal calcium-binding protein in the chicken

1. The rapid stimulation of intestinal Ca²⁺ transport observed in vitamin D-deficient chicks after receiving 1,25-dihydroxycholecalciferol has necessitated a re-evaluation of the correlation hitherto observed between this stimulation and the induction of calcium-binding protein synthesis. By 1h after a dose of 125ng of 1,25-dihydroxycholecalciferol, Ca²⁺ transport is increased. This is at least 2h before calcium-binding protein can be detected immunologically and 1h before synthesis of the protein begins on polyribosomes, and thus the hormone stimulates Ca²⁺ transport before calcium-binding-protein biosynthesis is induced. 2. The maximum increase in Ca²⁺ transport observed after this dose of 1,25-dihydroxycholecalciferol (attained by 8h) is similar to that observed after 1.25–25μg of cholecalciferol, but the stimulation is only short-lived, in contrast with the effect observed after the vitamin. At later times after the hormone, however, when Ca²⁺ transport has declined to its basal rate, the cellular content of calcium-binding protein remains elevated. 3. Calcium-binding protein is synthesized on free rather than membrane-bound polyribosomes, which implies that it is an intracellular protein. 4. Rachitic chicks require the presence of dietary calcium for maximum stimulation of calcium-binding protein production by cholecalciferol. 5. These results suggest that calcium-binding protein is an intracellular protein, and that its synthesis may be a consequence of the raised intracellular calcium content of the intestinal epithelial cells resulting from 1,25-dihydroxycholecalciferol-stimulated Ca²⁺ transport. We propose that calcium-binding-protein synthesis is necessary for maintaining the stimulated rate of Ca²⁺ transport, which is initiated by other factors.     

Golgi-specific DHHC Zinc Finger Protein GODZ Mediates Membrane Ca2+ Transport

The Golgi-specific zinc finger protein GODZ (palmitoyl acyltransferase/DHHC-3) mediates the palmitoylation and post-translational modification of many protein substrates that regulate membrane-protein interactions. Here, we show that GODZ also mediates Ca²⁺ transport in expressing Xenopus laevis oocytes. Two-electrode voltage-clamp, fluorescence, and ⁴⁵Ca²⁺ isotopic uptake determinations demonstrated voltage- and concentration-dependent, saturable, and substrate-inhibitable Ca²⁺ transport in oocytes expressing GODZ cRNA but not in oocytes injected with water alone. Moreover, we show that GODZ-mediated Ca²⁺ transport is regulated by palmitoylation, as the palmitoyl acyltransferase inhibitor 2-bromopalmitate or alteration of the acyltransferase DHHC motif (GODZ-DHHS) diminished GODZ-mediated Ca²⁺ transport by ∼80%. The GODZ mutation V61R abolished Ca²⁺ transport but did not affect palmitoyl acyltransferase activity. Coexpression of GODZ-V61R with GODZ-DHHS restored GODZ-DHHS-mediated Ca²⁺ uptake to values observed with wild-type GODZ, excluding an endogenous effect of palmitoylation. Coexpression of an independent palmitoyl acyltransferase (HIP14) with the GODZ-DHHS mutant also rescued Ca²⁺ transport. HIP14 did not mediate Ca²⁺ transport when expressed alone. Immunocytochemistry studies showed that GODZ and HIP14 co-localized to the Golgi and the same post-Golgi vesicles, suggesting that heteropalmitoylation might play a physiological role in addition to a biochemical function. We conclude that GODZ encodes a Ca²⁺ transport protein in addition to its ability to palmitoylate protein substrates.