Mutation of NEKL-4/NEK10 and TTLL genes opposes loss of the CCPP-1 deglutamylase and prevents neuronal ciliary degeneration

AbstractCiliary microtubules are subject to post-translational modifications that act as a “Tubulin Code” to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. In C. elegans, mutations in ccpp-1, the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in the tubulin tyrosine ligase-like glutamylase gene. We show that mutation in ttll-4, ttll-5, or ttll-11 gene suppressed the hyperglutamylation-induced loss of microtubules and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-4(my31) suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia. C. elegans NEKL-4 does not localize to cilia yet plays a role in regulating axonemal microtubule stability. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifying C. elegans genes important for neuronal and ciliary stability, our work may inform research into human ciliopathies and neurodegenerative diseases.

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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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UNC-2 CaV2 channel localization at presynaptic active zones depends on UNC-10/RIM and SYD-2/Liprin-α in Caenorhabditis elegans

10.1101/2021.01.27.428454 ◽

2021 ◽

Author(s):

Kelly H. Oh ◽

Mia Krout ◽

Janet E. Richmond ◽

Hongkyun Kim

Keyword(s):

Active Zone ◽

Calcium Influx ◽

Genetic Screen ◽

Scaffolding Protein ◽

Tight Coupling ◽

Forward Genetic Screen ◽

Precise Control ◽

C Elegans ◽

Vesicle Exocytosis ◽

Active Zones

AbstractPresynaptic active zone proteins couple calcium influx with synaptic vesicle exocytosis. However, the control of presynaptic calcium channel clustering by active zone proteins is not completely understood. In a C. elegans forward genetic screen, we find that UNC-10/RIM (Rab3-interacting molecule) and SYD-2/Liprin-α regulate presynaptic clustering of UNC-2, the CaV2 channel ortholog. We further quantitatively analyzed live animals using endogenously GFP-tagged UNC-2 and active zone components. Consistent with the interaction between RIM and CaV2 in mammals, the intensity and number of UNC-2 channel clusters at presynaptic terminals were greatly reduced in unc-10 mutant animals. To understand how SYD-2 regulates presynaptic UNC-2 channel clustering, we analyzed presynaptic localization of endogenous SYD-2, UNC-10, RIMB-1/RIM-BP (RIM binding protein), and ELKS-1. Our analysis revealed that while SYD-2 is the most critical for active zone assembly, loss of SYD-2 function does not completely abolish presynaptic localization of UNC-10, RIMB-1, and ELKS-1, suggesting an existence of SYD-2-independent active zone assembly. UNC-2 localization analysis in double and triple mutants of active zone components show that SYD-2 promotes UNC-2 clustering by partially controlling UNC-10 localization, and ELKS-1 and RIMB-1 also contribute to UNC-2 channel clustering. In addition, we find that core active zone proteins are unequal in their abundance. While the abundance of UNC-10 at the active zone is comparable to UNC-2, SYD-2 and ELKS-1 are twice more and RIMB-1 four times more abundant than UNC-2. Together our data show that UNC-10, SYD-2, RIMB-1, and ELKS-1 control presynaptic UNC-2 channel clustering in redundant yet distinct manners.Significance StatementPrecise control of neurotransmission is dependent on the tight coupling of the calcium influx through voltage-gated calcium channels (VGCCs) to the exocytosis machinery at the presynaptic active zones. However, how these VGCCs are tethered to the active zone is incompletely understood. To understand the mechanism of presynaptic VGCC localization, we performed a C. elegans forward genetic screen and quantitatively analyzed endogenous active zones and presynaptic VGCCs. In addition to RIM (Rab3-interacting molecule), our study finds that SYD-2/Liprin-α is critical for presynaptic localization of VGCCs. Yet, the loss of SYD-2, the master active zone scaffolding protein, does not completely abolish the presynaptic localization of the VGCC, showing that the active zone is a resilient structure assembled by redundant mechanisms.

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Impairing one sensory modality enhances another by reprogramming peptidergic circuits inCaenorhabditis elegans

10.1101/2021.03.11.435052 ◽

2021 ◽

Author(s):

Giulio Valperga ◽

Mario de Bono

Keyword(s):

Synaptic Input ◽

Sensory Modality ◽

Genetic Screen ◽

Sensory Loss ◽

Forward Genetic Screen ◽

Sensory Inputs ◽

C Elegans ◽

Transcriptional Reprogramming ◽

Sensory Responses ◽

Necessary And Sufficient

AbstractAnimals that lose one sensory modality often show augmented responses to other sensory inputs. The mechanisms underpinning this cross-modal plasticity are poorly understood. To probe these mechanisms, we perform a forward genetic screen for mutants with enhanced O2perception inC. elegans. Multiple mutants exhibiting increased responsiveness to O2concomitantly show defects in other sensory responses. One mutant,qui-1, defective in a conserved NACHT/WD40 protein, abolishes pheromone-evoked Ca2+responses in the ADL chemosensory neurons. We find that ADL’s responsiveness to pre-synaptic input from O2- sensing neurons is heightened inqui-1and other sensory defective mutants resulting in an enhanced neurosecretion. Expressingqui-1selectively in ADL rescues both thequi-1ADL neurosecretory phenotype and enhanced escape from 21% O2. Profiling of ADL neurons indicates its acquired O2-evoked neurosecretion is the result of a transcriptional reprogramming that up-regulates neuropeptide signalling. We show that the conserved neuropeptide receptor NPR-22 is necessary and sufficient in ADL to enhance its neurosecretion levels. Sensory loss can thus confer cross-modal plasticity by re-wiring peptidergic circuits.

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Caenorhabditis elegans RIG-I Homolog Mediates Antiviral RNA Interference Downstream of Dicer-Dependent Biogenesis of Viral Small Interfering RNAs

mBio ◽

10.1128/mbio.00264-17 ◽

2017 ◽

Vol 8 (2) ◽

Cited By ~ 20

Author(s):

Stephanie R. Coffman ◽

Jinfeng Lu ◽

Xunyang Guo ◽

Jing Zhong ◽

Hongshan Jiang ◽

...

Keyword(s):

Innate Immunity ◽

Rna Interference ◽

Caenorhabditis Elegans ◽

Virus Infection ◽

Rna Virus ◽

Genetic Screen ◽

Small Interfering Rnas ◽

Forward Genetic Screen ◽

C Elegans ◽

Terminal Domains

ABSTRACT Dicer enzymes process virus-specific double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans . Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans . Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1 , three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors. IMPORTANCE The helicase and C-terminal domains of mammalian RLRs sense intracellular viral RNAs to initiate the interferon-regulated innate immunity against RNA virus infection. Both of the domains from human RIG-I can substitute for the corresponding domains of DRH-1 to mediate antiviral RNAi in C. elegans , suggesting an analogous role for DRH-1 as an intracellular dsRNA sensor to initiate antiviral RNAi. Here, we developed a forward genetic screen for the identification of host factors required for antiviral RNAi in C. elegans . Characterization of four distinct drh-1 mutants obtained from the screen revealed that DRH-1 did not function to initiate antiviral RNAi. We show that DRH-1 acted in a downstream step to enhance Dicer-dependent biogenesis of viral siRNAs in C. elegans . As mammals produce Dicer-dependent viral siRNAs to target RNA viruses, our findings suggest a possible role for mammalian RLRs and interferon signaling in the biogenesis of viral siRNAs.

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Antagonistic paralogs control a switch between growth and pathogen resistance in C. elegans

10.1101/357756 ◽

2018 ◽

Author(s):

Kirthi C. Reddy ◽

Tal Dror ◽

Ryan S. Underwood ◽

Guled A. Osman ◽

Christopher A. Desjardins ◽

...

Keyword(s):

Gene Expression ◽

Pathogen Resistance ◽

Genetic Screen ◽

Specific Gene ◽

Defense Gene Expression ◽

Forward Genetic Screen ◽

C Elegans ◽

Evolutionary Features ◽

Species Specific ◽

Mutant Phenotypes

AbstractImmune genes are under intense pressure from pathogens, which cause these genes to diversify over evolutionary time and become species-specific. Through a forward genetic screen we recently described a C. elegans-specific gene called pals-22 to be a repressor of “Intracellular Pathogen Response” or IPR genes. Here we describe pals-25, which, like pals-22, is a species-specific gene of unknown biochemical function. We identified pals-25 in a screen for suppression of pals-22 mutant phenotypes and found that mutations in pals-25 suppress all known phenotypes caused by mutations in pals-22. These phenotypes include increased IPR gene expression, thermotolerance, and immunity against natural pathogens. Mutations in pals-25 also reverse the reduced lifespan and slowed growth of pals-22 mutants. Transcriptome analysis indicates that pals-22 and pals-25 control expression of genes induced not only by natural pathogens of the intestine, but also by natural pathogens of the epidermis. Indeed, in an independent forward genetic screen we identified pals-22 as a repressor and pals-25 as an activator of epidermal defense gene expression. These phenotypic and evolutionary features of pals-22 and pals-25 are strikingly similar to species-specific R gene pairs in plants that control immunity against co-evolved pathogens.

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Genetic, Behavioral and Environmental Determinants of Male Longevity in Caenorhabditis elegans

Genetics ◽

10.1093/genetics/154.4.1597 ◽

2000 ◽

Vol 154 (4) ◽

pp. 1597-1610 ◽

Cited By ~ 1

Author(s):

David Gems ◽

Donald L Riddle

Keyword(s):

Caenorhabditis Elegans ◽

Life Span ◽

Wild Type ◽

Neuronal Function ◽

Nematode Caenorhabditis Elegans ◽

Wild Isolate ◽

Young Males ◽

C Elegans ◽

Single Sex ◽

Solitary Males

Abstract Males of the nematode Caenorhabditis elegans are shorter lived than hermaphrodites when maintained in single-sex groups. We observed that groups of young males form clumps and that solitary males live longer, indicating that male-male interactions reduce life span. By contrast, grouped or isolated hermaphrodites exhibited the same longevity. In one wild isolate of C. elegans, AB2, there was evidence of copulation between males. Nine uncoordinated (unc) mutations were used to block clumping behavior. These mutations had little effect on hermaphrodite life span in most cases, yet many increased male longevity even beyond that of solitary wild-type males. In one case, the neuronal function mutant unc-64(e246), hermaphrodite life span was also increased by up to 60%. The longevity of unc-4(e120), unc-13(e51), and unc-32(e189) males exceeded that of hermaphrodites by 70–120%. This difference appears to reflect a difference in sex-specific life span potential revealed in the absence of male behavior that is detrimental to survival. The greater longevity of males appears not to be affected by daf-2, but is influenced by daf-16. In the absence of male-male interactions, median (but not maximum) male life span was variable. This variability was reduced when dead bacteria were used as food. Maintenance on dead bacteria extended both male and hermaphrodite longevity.

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Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation

Nature Communications ◽

10.1038/s41467-021-24743-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Nathan J. VanDusen ◽

Julianna Y. Lee ◽

Weiliang Gu ◽

Catalina E. Butler ◽

Isha Sethi ◽

...

Keyword(s):

Gene Expression ◽

Genetic Screen ◽

Forward Genetics ◽

Epigenetic Mark ◽

Biological Processes ◽

Dynamic Changes ◽

Forward Genetic Screen ◽

High Resource ◽

Organ Systems

AbstractThe forward genetic screen is a powerful, unbiased method to gain insights into biological processes, yet this approach has infrequently been used in vivo in mammals because of high resource demands. Here, we use in vivo somatic Cas9 mutagenesis to perform an in vivo forward genetic screen in mice to identify regulators of cardiomyocyte (CM) maturation, the coordinated changes in phenotype and gene expression that occur in neonatal CMs. We discover and validate a number of transcriptional regulators of this process. Among these are RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicate that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems.

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Abstract LB247: A forward genetic screen to identify drivers of neuroendocrine prostate cancer

10.1158/1538-7445.am2021-lb247 ◽

2021 ◽

Author(s):

Francisca Nunes de Almeida ◽

Alessandro Vasciaveo ◽

Min Zou ◽

Matteo Di Bernardo ◽

Andrea Califano ◽

...

Keyword(s):

Prostate Cancer ◽

Genetic Screen ◽

Forward Genetic Screen ◽

Neuroendocrine Prostate Cancer

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Forward genetic screen of human transposase genomic rearrangements

BMC Genomics ◽

10.1186/s12864-016-2877-x ◽

2016 ◽

Vol 17 (1) ◽

Cited By ~ 7

Author(s):

Anton G. Henssen ◽

Eileen Jiang ◽

Jiali Zhuang ◽

Luca Pinello ◽

Nicholas D. Socci ◽

...

Keyword(s):

Genetic Screen ◽

Genomic Rearrangements ◽

Forward Genetic Screen

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Regulation of Yeast-to-Hyphae Transition inYarrowia lipolytica

mSphere ◽

10.1128/msphere.00541-18 ◽

2018 ◽

Vol 3 (6) ◽

Cited By ~ 9

Author(s):

Kyle R. Pomraning ◽

Erin L. Bredeweg ◽

Eduard J. Kerkhoven ◽

Kerrie Barry ◽

Sajeet Haridas ◽

...

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Stress Response ◽

Hyphal Growth ◽

Genetic Screen ◽

Morphological Transition ◽

Histidine Kinases ◽

Content Type ◽

Forward Genetic Screen ◽

Response To Stress

ABSTRACTThe yeastYarrowia lipolyticaundergoes a morphological transition from yeast-to-hyphal growth in response to environmental conditions. A forward genetic screen was used to identify mutants that reliably remain in the yeast phase, which were then assessed by whole-genome sequencing. All thesmoothmutants identified, so named because of their colony morphology, exhibit independent loss of DNA at a repetitive locus made up of interspersed ribosomal DNA and short 10- to 40-mer telomere-like repeats. The loss of repetitive DNA is associated with downregulation of genes with stress response elements (5′-CCCCT-3′) and upregulation of genes with cell cycle box (5′-ACGCG-3′) motifs in their promoter region. The stress response element is bound by the transcription factor Msn2p inSaccharomyces cerevisiae. We confirmed that theY. lipolyticamsn2(Ylmsn2) ortholog is required for hyphal growth and found that overexpression of Ylmsn2enables hyphal growth insmoothstrains. The cell cycle box is bound by the Mbp1p/Swi6p complex inS. cerevisiaeto regulate G1-to-S phase progression. We found that overexpression of either the Ylmbp1or Ylswi6homologs decreased hyphal growth and that deletion of either Ylmbp1or Ylswi6promotes hyphal growth insmoothstrains. A second forward genetic screen for reversion to hyphal growth was performed with thesmooth-33mutant to identify additional genetic factors regulating hyphal growth inY. lipolytica. Thirteen of the mutants sequenced from this screen had coding mutations in five kinases, including the histidine kinases Ylchk1and Ylnik1and kinases of the high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase cascade Ylssk2, Ylpbs2, and Ylhog1. Together, these results demonstrate thatY. lipolyticatransitions to hyphal growth in response to stress through multiple signaling pathways.IMPORTANCEMany yeasts undergo a morphological transition from yeast-to-hyphal growth in response to environmental conditions. We used forward and reverse genetic techniques to identify genes regulating this transition inYarrowia lipolytica. We confirmed that the transcription factor Ylmsn2is required for the transition to hyphal growth and found that signaling by the histidine kinases Ylchk1and Ylnik1as well as the MAP kinases of the HOG pathway (Ylssk2, Ylpbs2, and Ylhog1) regulates the transition to hyphal growth. These results suggest thatY. lipolyticatransitions to hyphal growth in response to stress through multiple kinase pathways. Intriguingly, we found that a repetitive portion of the genome containing telomere-like and rDNA repeats may be involved in the transition to hyphal growth, suggesting a link between this region and the general stress response.

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