Syncrip/hnRNP Q is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation

Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganization at new synapses, requiring posttranscriptional regulation of localized mRNA a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organizes actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is poorly understood. Here, we show that activity-dependent accumulation of Msp300 in the postsynaptic compartment of the Drosophila larval neuromuscular junction is regulated by the conserved RNA binding protein Syncrip/hnRNP Q. Syncrip (Syp) binds to msp300 transcripts and is essential for plasticity. Single-molecule imaging shows that msp300 is associated with Syp in vivo and forms ribosome-rich granules that contain the translation factor eIF4E. Elevated neural activity alters the dynamics of Syp and the number of msp300:Syp:eIF4E RNP granules at the synapse, suggesting that these particles facilitate translation. These results introduce Syp as an important early acting activity-dependent regulator of a plasticity gene that is strongly associated with human ataxias.

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Syncrip/hnRNPQ is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation

10.1101/585679 ◽

2019 ◽

Cited By ~ 1

Author(s):

Josh Titlow ◽

Francesca Robertson ◽

Aino Järvelin ◽

David Ish-Horowicz ◽

Carlas Smith ◽

...

Keyword(s):

Synaptic Plasticity ◽

Single Molecule ◽

Rna Binding ◽

Synapse Formation ◽

Binding Protein ◽

Rna Binding Protein ◽

Long Distance ◽

Key Factor ◽

Activity Dependent

AbstractMemory and learning involve activity-driven expression of proteins and cytoskeletal reorganisation at new synapses, often requiring post-transcriptional regulation a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organises actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is not yet known. Here, we show that the local translation of msp300 is promoted during activity-dependent plasticity by the conserved RNA binding protein Syncrip/hnRNP Q, which binds to msp300 transcripts and is essential for plasticity. Single molecule imaging shows that Syncrip is associated in vivo with msp300 mRNA in ribosome-rich particles. Elevated neural activity alters the dynamics of Syncrip RNP granules at the synapse, suggesting a change in particle composition or binding that facilitates translation. These results introduce Syncrip as an important early-acting activity-dependent translational regulator of a plasticity gene that is strongly associated with human ataxias.Syncrip regulates synaptic plasticity via msp300Titlow et al. find that Syncrip (hnRNPQ RNA binding protein) acts directly on msp300 to modulate activity-dependent synaptic plasticity. In vivo biophysical experiments reveal activity-dependent changes in RNP complex sizes compatible with an increase in translation at the synapse.

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AUF-1 and YB-1 are critical determinants of β-globin mRNA expression in erythroid cells

Blood ◽

10.1182/blood-2011-10-387316 ◽

2012 ◽

Vol 119 (4) ◽

pp. 1045-1053 ◽

Cited By ~ 9

Author(s):

Sebastiaan van Zalen ◽

Grace R. Jeschke ◽

Elizabeth O. Hexner ◽

J. Eric Russell

Keyword(s):

Posttranscriptional Regulation ◽

Rna Binding ◽

Globin Gene ◽

K562 Cells ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Globin Mrna ◽

Affinity Enrichment

Abstract The normal accumulation of β-globin protein in terminally differentiating erythroid cells is critically dependent on the high stability of its encoding mRNA. The molecular basis for this property, though, is incompletely understood. Factors that regulate β-globin mRNA within the nucleus of early erythroid progenitors are unlikely to account for the constitutively high half-life of β-globin mRNA in the cytoplasm of their anucleate erythroid progeny. We conducted in vitro protein-RNA binding analyses that identified a cytoplasm-restricted β-globin messenger ribonucleoprotein (mRNP) complex in both cultured K562 cells and erythroid-differentiated human CD34+ cells. This novel mRNP targets a specific guanine-rich pentanucleotide in a region of the β-globin 3′untranslated region that has recently been implicated as a determinant of β-globin mRNA stability. Subsequent affinity-enrichment analyses identified AUF-1 and YB-1, 2 cytoplasmic proteins with well-established roles in RNA biology, as trans-acting components of the mRNP. Factor-depletion studies conducted in vivo demonstrated the importance of the mRNP to normal steady-state levels of β-globin mRNA in erythroid precursors. These data define a previously unrecognized mechanism for the posttranscriptional regulation of β-globin mRNA during normal erythropoiesis, providing new therapeutic targets for disorders of β-globin gene expression.

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Characterization and Functional Analysis of Polyadenylation Sites in Fast and Slow Muscles

BioMed Research International ◽

10.1155/2020/2626584 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13

Author(s):

Lulu Deng ◽

Long Li ◽

Cheng Zou ◽

Chengchi Fang ◽

Changchun Li

Keyword(s):

Single Molecule ◽

Posttranscriptional Regulation ◽

Muscle Development ◽

Target Genes ◽

Rna Binding ◽

De Novo ◽

Rna Binding Proteins ◽

Fast And Slow Muscles ◽

Molecular Features ◽

Polyadenylation Sites

Many increasing documents have proved that alternative polyadenylation (APA) events with different polyadenylation sites (PAS) contribute to posttranscriptional regulation. However, little is known about the detailed molecular features of PASs and its role in porcine fast and slow skeletal muscles through microRNAs (miRNAs) and RNA binding proteins (RBPs). In this study, we combined single-molecule real-time sequencing and Illumina RNA-seq datasets to comprehensively analyze polyadenylation in pigs. We identified a total of 10,334 PASs, of which 8734 were characterized by reference genome annotation. 32.86% of PAS-associated genes were determined to have more than one PAS. Further analysis demonstrated that tissue-specific PASs between fast and slow muscles were enriched in skeletal muscle development pathways. In addition, we obtained 1407 target genes regulated by APA events through potential binding 69 miRNAs and 28 RBPs in variable 3′ UTR regions and some are involved in myofiber transformation. Furthermore, the de novo motif search confirmed that the most common usage of canonical motif AAUAAA and three types of PASs may be related to the strength of motifs. In summary, our results provide a useful annotation of PASs for pig transcriptome and suggest that APA may serve as a role in fast and slow muscle development under the regulation of miRNAs and RBPs.

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In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters

Nature Communications ◽

10.1038/ncomms13660 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 15

Author(s):

Adekunle T. Bademosi ◽

Elsa Lauwers ◽

Pranesh Padmanabhan ◽

Lorenzo Odierna ◽

Ye Jin Chai ◽

...

Keyword(s):

Single Molecule ◽

Neurotransmitter Release ◽

Motor Nerve ◽

Living Organism ◽

Neurosecretory Cells ◽

Single Particle Tracking ◽

Snare Complex ◽

Motor Nerve Terminal ◽

Activity Dependent

Abstract Syntaxin1A is organized in nanoclusters that are critical for the docking and priming of secretory vesicles from neurosecretory cells. Whether and how these nanoclusters are affected by neurotransmitter release in nerve terminals from a living organism is unknown. Here we imaged photoconvertible syntaxin1A-mEos2 in the motor nerve terminal of Drosophila larvae by single-particle tracking photoactivation localization microscopy. Opto- and thermo-genetic neuronal stimulation increased syntaxin1A-mEos2 mobility, and reduced the size and molecular density of nanoclusters, suggesting an activity-dependent release of syntaxin1A from the confinement of nanoclusters. Syntaxin1A mobility was increased by mutating its polyphosphoinositide-binding site or preventing SNARE complex assembly via co-expression of tetanus toxin light chain. In contrast, syntaxin1A mobility was reduced by preventing SNARE complex disassembly. Our data demonstrate that polyphosphoinositide favours syntaxin1A trapping, and show that SNARE complex disassembly leads to syntaxin1A dissociation from nanoclusters. Lateral diffusion and trapping of syntaxin1A in nanoclusters therefore dynamically regulate neurotransmitter release.

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Neuron-specific cTag-CLIP reveals cell-specific diversity of functional RNA regulation in the brain

10.1101/244905 ◽

2018 ◽

Cited By ~ 1

Author(s):

Yuhki Saito ◽

Yuan Yuan ◽

Ilana Zucker-Scharff ◽

John J. Fak ◽

Yoko Tajima ◽

...

Keyword(s):

Motor Coordination ◽

Rna Binding ◽

Synapse Formation ◽

Rna Binding Proteins ◽

Cell Types ◽

Knock Out ◽

Knock Out Mice ◽

Cerebellar Purkinje Cells ◽

The Brain

SUMMARYRNA-binding proteins (RBPs) regulate genetic diversity, but the degree to which they do so in individual cell-types in vivo is unknown. We employed NOVA2 cTag-CLIP to generate functional RBP-RNA maps from single neuronal populations in the mouse brain. Combining cell-type specific data from Nova2-cTag and Nova2 conditional knock-out mice revealed differential NOVA2 regulatory actions (e.g. alternative splicing) on the same transcripts in different neurons, including in cerebellar Purkinje cells, where NOVA2 acts as an essential factor for proper motor coordination and synapse formation. This also led to the discovery of a mechanism by which NOVA2 action leads to different outcomes in different cells on the same transcripts: NOVA2 is able to regulate retained introns, which subsequently serve as scaffolds for another trans-acting splicing factor, PTBP2. Our results describe differential roles and mechanisms by which RBPs mediate RNA diversity in different neurons and consequent functional outcomes within the brain.

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Depolarizing GABA Transmission Restrains Activity-Dependent Glutamatergic Synapse Formation in the Developing Hippocampal Circuit

10.1101/742148 ◽

2019 ◽

Author(s):

Christopher K. Salmon ◽

Horia Pribiag ◽

W. Todd Farmer ◽

Scott Cameron ◽

Emma V. Jones ◽

...

Keyword(s):

Neural Activity ◽

Pyramidal Neurons ◽

Synapse Formation ◽

Neural Circuit ◽

Reversal Potential ◽

Glutamatergic Synapse ◽

Inhibitory Neurotransmitter ◽

Key Factor ◽

Circuit Development ◽

Activity Dependent

ABSTRACTGABA is the main inhibitory neurotransmitter in the mature brain but has the paradoxical property of depolarizing neurons during early development. Depolarization provided by GABAA transmission during this early phase regulates neural stem cell proliferation, neural migration, neurite outgrowth, synapse formation, and circuit refinement, making GABA a key factor in neural circuit development. Importantly, depending on the context, depolarizing GABAA transmission can either drive neural activity, or inhibit it through shunting inhibition. The varying roles of depolarizing GABAA transmission during development, and its ability to both drive and inhibit neural activity, makes it a difficult developmental cue to study. This is particularly true in the later stages of development, when the majority of synapses form and GABAA transmission switches from depolarizing to hyperpolarizing. Here we addressed the importance of depolarizing but inhibitory (or shunting) GABAA transmission in glutamatergic synapse formation in hippocampal CA1 pyramidal neurons. We first showed that the developmental depolarizing-to-hyperpolarizing switch in GABAA transmission is recapitulated in organotypic hippocampal slice cultures. Based on the expression profile of K+-Cl- co-transporter 2 (KCC2) and changes in the GABA reversal potential, we pinpointed the timing of the switch from depolarizing to hyperpolarizing GABAA transmission in CA1 neurons. We found that blocking depolarizing but shunting GABAA transmission increased excitatory synapse number and strength, indicating that depolarizing GABAA transmission can restrain glutamatergic synapse formation. The increase in glutamatergic synapses was activity-dependent, but independent of BDNF signalling. Importantly, the elevated number of synapses was stable for more than a week after GABAA inhibitors were washed out. Together these findings point to the ability of immature GABAergic transmission to restrain glutamatergic synapse formation and suggest an unexpected role for depolarizing GABAA transmission in shaping excitatory connectivity during neural circuit development.

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Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF

Journal of Bacteriology ◽

10.1128/jb.00343-16 ◽

2016 ◽

Vol 198 (18) ◽

pp. 2458-2469 ◽

Cited By ~ 14

Author(s):

Kayley H. Schulmeyer ◽

Manisha R. Diaz ◽

Thomas B. Bair ◽

Wes Sanders ◽

Cindy J. Gode ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Binding Sites ◽

Posttranscriptional Regulation ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Consensus Sequence ◽

Stem Loop ◽

Content Type

ABSTRACTCsrA family RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the posttranscriptional level.Pseudomonas aeruginosahas a canonical member of the CsrA family (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for RsmA and RsmF. The initial target library consisted of 62-nucleotide (nt) RNA transcripts with central cores randomized at 15 sequential positions. Most targets selected by RsmA and RsmF were the expected size and shared a common consensus sequence (CANGGAYG) that was positioned in a hexaloop region of the stem-loop structure. RsmA and RsmF also selected for longer targets (≥96 nt) that were likely generated by rare PCR errors. Most of the long targets contained two consensus-binding sites. Representative short (single consensus site) and long (two consensus sites) targets were tested for RsmA and RsmF binding. Whereas RsmA bound the short targets with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long targets. Mutation of either consensus GGA site in the long targets reduced or eliminated RsmF binding, suggesting a requirement for two tandem binding sites. Conversely, RsmA bound long targets containing only a single GGA site with unaltered affinity. The RsmF requirement for two binding sites was confirmed withtssA1, anin vivoregulatory target of RsmA and RsmF. Our findings suggest that RsmF binding requires two GGA-containing sites, while RsmA binding requirements are less stringent.IMPORTANCEThe CsrA family of RNA-binding proteins is widely conserved in bacteria and plays important roles in the posttranscriptional regulation of protein synthesis.P. aeruginosahas two CsrA proteins, RsmA and RsmF. Although RsmA and RsmF share a few RNA targets, RsmF is unable to bind to other targets recognized by RsmA. The goal of the present study was to better understand the basis for differential binding by RsmF. Our data indicate that RsmF binding requires target RNAs with two consensus-binding sites, while RsmA recognizes targets with just a single binding site. This information should prove useful to future efforts to define the RsmF regulon and its contribution toP. aeruginosaphysiology and virulence.

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In vitro reconstitution of an mRNA-transport complex reveals mechanisms of assembly and motor activation

The Journal of Cell Biology ◽

10.1083/jcb.201302095 ◽

2013 ◽

Vol 203 (6) ◽

pp. 971-984 ◽

Cited By ~ 19

Author(s):

Roland G. Heym ◽

Dennis Zimmermann ◽

Franziska T. Edelmann ◽

Lars Israel ◽

Zeynep Ökten ◽

...

Keyword(s):

Nuclear Export ◽

Messenger Rna ◽

Rna Binding ◽

Long Distance ◽

Long Distance Transport ◽

In Vitro Reconstitution ◽

Localization Elements ◽

Transport Complex

The assembly and composition of ribonucleic acid (RNA)–transporting particles for asymmetric messenger RNA (mRNA) localization is not well understood. During mitosis of budding yeast, the Swi5p-dependent HO expression (SHE) complex transports a set of mRNAs into the daughter cell. We recombinantly reconstituted the core SHE complex and assessed its properties. The cytoplasmic precomplex contains only one motor and is unable to support continuous transport. However, a defined interaction with a second, RNA-bound precomplex after its nuclear export dimerizes the motor and activates processive RNA transport. The run length observed in vitro is compatible with long-distance transport in vivo. Surprisingly, SHE complexes that either contain or lack RNA cargo show similar motility properties, demonstrating that the RNA-binding protein and not its cargo activates motility. We further show that SHE complexes have a defined size but multimerize into variable particles upon binding of RNAs with multiple localization elements. Based on these findings, we provide an estimate of number, size, and composition of such multimeric SHE particles in the cell.

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The Arginine-Rich Motif of Bamboo mosaic virus Satellite RNA-Encoded P20 Mediates Self-Interaction, Intracellular Targeting, and Cell-to-Cell Movement

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-19-0758 ◽

2006 ◽

Vol 19 (7) ◽

pp. 758-767 ◽

Cited By ~ 24

Author(s):

Paramasivan Vijaya Palani ◽

Venkatraman Kasiviswanathan ◽

Jeff Chien-Fu Chen ◽

Wei Chen ◽

Yau-Heiu Hsu ◽

...

Keyword(s):

Mosaic Virus ◽

Rna Binding ◽

Cell Movement ◽

The Self ◽

Satellite Rna ◽

Long Distance ◽

Intracellular Targeting ◽

Arginine Rich Motif

Satellite RNA of Bamboo mosaic virus (satBaMV) has a single open reading frame for a nonstructural, RNA-binding protein, P20, which facilitates the long-distance movement of satBaMV in Nicotiana benthamiana. Here, we elucidate various biological properties of P20 and the involvement of a single domain in its activities. P20 displayed a strong self-interaction in vitro and in vivo, and cross-linking assays demonstrated its oligomerization. Domain mapping, using the bacterial two-hybrid system, indicated that the self-interacting domain overlaps the RNA-binding domain in the N-terminal arginine-rich motif (ARM) of P20. The deletion of the ARM abolished the self-interaction of P20 in vitro and in vivo and impaired its intracellular targeting and efficient cell-to-cell movement in N. benthamiana leaves. Moreover, RNA and protein accumulation of the ARM deletion mutant of satBaMV was significantly reduced in leaves systemically coinfected with Bamboo mosaic potexvirus and satBaMV. This is the first report of the involvement of ARM in various biological activities of a satellite RNA-encoded protein during infection of its host.

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