The RabGAP TBC-11 controls Argonaute localization for proper microRNA function in C. elegans

Once loaded onto Argonaute proteins, microRNAs form a silencing complex called miRISC that targets mostly the 3’UTR of mRNAs to silence their translation. How microRNAs are transported to and from their target mRNA remains poorly characterized. While some reports linked intracellular trafficking to microRNA activity, it is still unclear how these pathways coordinate for proper microRNA-mediated gene silencing and turnover. Through a forward genetic screen using Caenorhabditis elegans, we identified the RabGAP tbc-11 as an important factor for the microRNA pathway. We show that TBC-11 acts mainly through the small GTPase RAB-6 and that its regulation is required for microRNA function. The absence of functional TBC-11 increases the pool of microRNA-unloaded Argonaute ALG-1 that is likely associated to endomembranes. Furthermore, in this condition, this pool of Argonaute accumulates in a perinuclear region and forms a high molecular weight complex. Altogether, our data suggest that the alteration of TBC-11 generates a fraction of ALG-1 that cannot bind to target mRNAs, leading to defective gene repression. Our results establish the importance of intracellular trafficking for microRNA function and demonstrate the involvement of a small GTPase and its GAP in proper Argonaute localization in vivo.

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MAK and CCRK kinases regulate kinesin-2 motility in C. elegans neuronal cilia

10.1101/209221 ◽

2017 ◽

Author(s):

Peishan Yi ◽

Chao Xie ◽

Guangshuo Ou

Keyword(s):

Cell Cycle ◽

Sensory Neurons ◽

Intraflagellar Transport ◽

Time Lapse ◽

Genetic Screen ◽

Forward Genetic Screen ◽

C Elegans ◽

Neuronal Cilia ◽

Sensory Cilia

AbstractKinesin-2 motors power the anterograde intraflagellar transport (IFT), a highly ordered process that assembles and maintains cilia. It remains elusive how kinesin-2 motors are regulated in vivo. Here we perform forward genetic screen to isolate suppressors that rescue the ciliary defects in the constitutive active mutation of OSM-3-kinesin (G444E) in C. elegans sensory neurons. We identify the C. elegans DYF-5 and DYF-18, which encode the homologs of mammalian male germ cell-associated kinase (MAK) and cell cycle-related kinase (CCRK). Using time-lapse fluorescence microscopy, we show that DYF-5 and DYF-18 are IFT cargo molecules and are enriched at the distal segments of sensory cilia. Mutations of dyf-5 and dyf-18 generate the elongated cilia and ectopic localization of kinesin-II at the ciliary distal segments. Genetic analyses reveal that dyf-5 and dyf-18 are also important for stabilizing the interaction between IFT particle and OSM-3-kinesin. Our data suggest that DYF-5 and DYF-18 act in the same pathway to promote handover between kinesin-II and OSM-3 in sensory cilia.

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The Amyloid Precursor-like Protein APL-1 Regulates Axon Regeneration

10.1101/305284 ◽

2018 ◽

Cited By ~ 2

Author(s):

Lewie Zeng ◽

Rachid El Bejjani ◽

Marc Hammarlund

Keyword(s):

Motor Neurons ◽

Neuronal Development ◽

Axon Regeneration ◽

Neuronal Response ◽

Small Gtpase ◽

C Elegans ◽

Protein Levels ◽

Mature Neurons ◽

And Function

AbstractMembers of the Amyloid Precursor Protein (APP) family have important functions during neuronal development. However, their physiological functions in the mature nervous system are not fully understood. Here we use the C. elegans GABAergic motor neurons to study the post-developmental function of the APP-like protein APL-1 in vivo. We find that apl-1 has minimum roles in the maintenance of gross neuron morphology and function. However, we show that apl-1 is an inhibitor of axon regeneration, acting on mature neurons to limit regrowth in response to injury. The small GTPase Rab6/RAB-6.2 also inhibits regeneration, and does so in part by maintaining protein levels of APL-1. To inhibit regeneration, APL-1 functions via the E2 domain of its ectodomain; the cytoplasmic tail, transmembrane anchoring, and the E1 domain are not required for this function. Our data defines a novel role for APL-1 in modulating the neuronal response to injury.

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Rap2 and TNIK control Plexin-dependent tiled synaptic innervation in C. elegans

eLife ◽

10.7554/elife.38801 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 4

Author(s):

Xi Chen ◽

Akihiro CE Shibata ◽

Ardalan Hendi ◽

Mizuki Kurashina ◽

Ethan Fortes ◽

...

Keyword(s):

Synapse Formation ◽

Small Gtpase ◽

Negative Regulator ◽

Gtpase Activity ◽

C Elegans ◽

Receptor Interactions ◽

Presynaptic Assembly ◽

Intracellular Mechanisms

During development, neurons form synapses with their fate-determined targets. While we begin to elucidate the mechanisms by which extracellular ligand-receptor interactions enhance synapse specificity by inhibiting synaptogenesis, our knowledge about their intracellular mechanisms remains limited. Here we show that Rap2 GTPase (rap-2) and its effector, TNIK (mig-15), act genetically downstream of Plexin (plx-1) to restrict presynaptic assembly and to form tiled synaptic innervation in C. elegans. Both constitutively GTP- and GDP-forms of rap-2 mutants exhibit synaptic tiling defects as plx-1 mutants, suggesting that cycling of the RAP-2 nucleotide state is critical for synapse inhibition. Consistently, PLX-1 suppresses local RAP-2 activity. Excessive ectopic synapse formation in mig-15 mutants causes a severe synaptic tiling defect. Conversely, overexpression of mig-15 strongly inhibited synapse formation, suggesting that mig-15 is a negative regulator of synapse formation. These results reveal that subcellular regulation of small GTPase activity by Plexin shapes proper synapse patterning in vivo.

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The RhoGAP SPV-1 regulates calcium signaling to control the contractility of theC. elegansspermatheca during embryo transits

10.1101/441386 ◽

2018 ◽

Author(s):

Jeff Bouffard ◽

Alyssa D. Cecchetelli ◽

Coleman Clifford ◽

Kriti Sethi ◽

Ronen Zaidel-Bar ◽

...

Keyword(s):

Calcium Signaling ◽

Signaling Pathways ◽

Calcium Release ◽

Small Gtpase ◽

Negative Regulator ◽

Calcium Signal ◽

C Elegans ◽

Spatial Regulation ◽

Calcium Indicator

AbstractContractility of the non-muscle and smooth muscle cells that comprise biological tubing is regulated by the Rho-ROCK and calcium signaling pathways. Although many molecular details about these signaling pathways are known, less is known about how they are coordinated spatiotemporally in biological tubes. The spermatheca of theC. elegansreproductive system enables study of the signaling pathways regulating actomyosin contractility in live adult animals. The RhoGAP SPV-1 was previously identified as a negative regulator of RHO-1/Rho and spermathecal contractility. Here, we uncover a role for SPV-1 as a key regulator of calcium signaling.spv-1mutants expressing the calcium indicator GCaMP in the spermatheca exhibit premature calcium release, elevated calcium levels, and disrupted spatial regulation of calcium signaling during spermathecal contraction. Although RHO-1 is required for spermathecal contractility, RHO-1 does not play a significant role in regulating calcium. In contrast, activation of CDC-42 recapitulates many aspects ofspv-1mutant calcium signaling. Depletion ofcdc-42by RNAi does not suppress the premature or elevated calcium signal seen inspv-1mutants, suggesting other targets remain to be identified. Our results suggest SPV-1 works through both the Rho-ROCK and calcium signaling pathways to coordinate cellular contractility.Highlight SummaryThroughin vivoimaging of the calcium sensor GCaMP, we show that the RhoGAP SPV-1 is a key regulator of calcium signaling in theC. elegansspermatheca. Our data suggests SPV-1 acts at least partially through the small GTPase CDC-42 to modulate calcium signaling, while also acting on RHO-1 to modulate Rho-ROCK signaling. This places SPV-1 as a central regulator of cellular contractility.

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Redox signaling modulates Rho activity and tissue contractility in the C. elegans spermatheca

10.1101/849463 ◽

2019 ◽

Author(s):

Charlotte A. Kelley ◽

Sasha De Henau ◽

Liam Bell ◽

Tobias B. Dansen ◽

Erin J. Cram

Keyword(s):

Hydrogen Peroxide ◽

Active Site ◽

Myoepithelial Cells ◽

Redox Signaling ◽

Small Gtpase ◽

C Elegans ◽

Hydrogen Peroxide Treatment ◽

Fine Tune ◽

Contractile Tissue

AbstractActomyosin based contractility in smooth muscle and non-muscle cells is regulated by signaling through the small GTPase Rho and by calcium-activated pathways. We use the myoepithelial cells of the Caenorhabditis elegans spermatheca to study the mechanisms of coordinated myosin activation in vivo. Here, we demonstrate that redox signaling regulates RHO-1/Rho activity in this contractile tissue. Exogenous hydrogen peroxide treatment decreases spermathecal contractility by inhibiting RHO-1, which is mediated through a conserved cysteine in its active site (C20). Further, we identify a gradient of oxidation across the spermathecal tissue, which is regulated by the cytosolic superoxide dismutase, SOD-1. SOD-1 functions in the Rho pathway to inhibit RHO-1 through oxidation of C20. Our results suggest that SOD-1 functions to regulate the redox environment and to fine-tune Rho activity across the spermatheca.

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Rap2 and TNIK control Plexin-dependent synaptic tiling in C. elegans

10.1101/238352 ◽

2017 ◽

Author(s):

Xi Chen ◽

Akihiro C.E. Shibata ◽

Ardalan Hendi ◽

Mizuki Kurashina ◽

Ethan Fortes ◽

...

Keyword(s):

Synapse Formation ◽

Small Gtpase ◽

Negative Regulator ◽

Gtpase Activity ◽

C Elegans ◽

Receptor Interactions ◽

Presynaptic Assembly ◽

Intracellular Mechanisms

AbstractDuring development, neurons form synapses with their fate-determined targets. While we begin to elucidate the mechanisms by which extracellular ligand-receptor interactions enhance synapse specificity by inhibiting synaptogenesis, our knowledge about their intracellular mechanisms remains limited. Here we show that Rap2 GTPase (rap-2) and its effector, TNIK (mig-15), act downstream of Plexin (plx-1) to restrict presynaptic assembly and to form tiled synaptic innervation in C. elegans. Both constitutively GTP- and GDP-forms of rap-2 mutants exhibit synaptic tiling defects as plx-1 mutants, suggesting that cycling of the RAP-2 nucleotide state is critical for synapse inhibition. Consistently, RAP-2 activity is locally suppressed by PLX-1. Excessive ectopic synapse formation in mig-15 mutants causes a severe synaptic tiling defect. Conversely, overexpression of mig-15 strongly inhibited synapse formation, suggesting that mig-15 is a negative regulator of synapse formation. These results reveal that subcellular regulation of small GTPase activity by Plexin shapes proper synapse patterning in vivo.

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MiR-146a wild-type 3’ sequence identity is dispensable for proper innate immune function in vivo

10.1101/474791 ◽

2018 ◽

Author(s):

Grant Bertolet ◽

Natee Kongchan ◽

Rebekah Miller ◽

Ravi K. Patel ◽

Antrix Jain ◽

...

Keyword(s):

Seed Region ◽

Wild Type ◽

C Elegans ◽

Sequence Identity ◽

Mature Microrna ◽

Type 3 ◽

Microrna Function ◽

Type Sequence ◽

Mammalian System

AbstractThe prevailing model of microRNA function is that the “seed” region (nucleotides 2-8) is typically sufficient to mediate target recognition and repression. However, numerous recent studies have challenged this model, either by demonstrating extensive 3’ pairing between physically defined miRNA-mRNA pairs or by showing in C. elegans that disrupted 3’ pairing can result in impaired function in vivo. To test the importance of miRNA 3’ pairing in a mammalian system in vivo, we engineered a mutant murine mir-146a allele in which the 5’ half of the mature microRNA retains its wild-type sequence, but the 3’ half has been altered to be anti-complementary. Mice homozygous or hemizygous for this mutant allele are phenotypically indistinguishable from wild-type controls and do not recapitulate any of the immunopathology previously described for mir-146a-null mice. Our results indicate that 3’ pairing is dispensable for the established myeloid function of this key mammalian microRNA.Summary Blurb3’ sequence identity is dispensable for the established function of a mammalian miRNA in vivo.

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