Twitchin kinase inhibits muscle activity

Matsunaga, Yohei; Hwang, Hyundoo; Franke, Barbara; Williams, Rhys M.; Penley, McKenna J.; Qadota, Hiroshi; Yi, Hong; Morran, Levi T.; Lu, Hang; Mayans, Olga; Benian, Guy M.

doi:10.1091/mbc.e16-10-0707

Cited by 18 publications

(21 citation statements)

References 44 publications

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“…Insertions of gfp were performed using a single guide RNA and a double-stranded repair template amplified using PCR (40). Punc-22::unc-22::gfp resulted in GFP fluorescence within the pharynx as reported earlier (41). Additional details are provided in Supplementary Materials and Methods.…”

Section: Methodssupporting

confidence: 82%

Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference

Ravikumar

Devanapally

Jose

2019

Nucleic Acids Research

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Delivery of double-stranded RNA (dsRNA) into animals can silence genes of matching sequence in diverse cell types through mechanisms that have been collectively called RNA interference. In the nematode Caenorhabditis elegans, dsRNA from multiple sources can trigger the amplification of silencing signals. Amplification occurs through the production of small RNAs by two RNA-dependent RNA polymerases (RdRPs) that are thought to be tissue-specific - EGO-1 in the germline and RRF-1 in somatic cells. Here we demonstrate that EGO-1 can compensate for the lack of RRF-1 when dsRNA from neurons is used to silence genes in intestinal cells. However, the lineal origins of cells that can use EGO-1 varies. This variability could be because random sets of cells can either receive different amounts of dsRNA from the same source or use different RdRPs to perform the same function. Variability is masked in wild-type animals, which show extensive silencing by neuronal dsRNA. As a result, cells appear similarly functional despite underlying differences that vary from animal to animal. This functional mosaicism cautions against inferring uniformity of mechanism based on uniformity of outcome. We speculate that functional mosaicism could contribute to escape from targeted therapies and could allow developmental systems to drift over evolutionary time.

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Section: Methodssupporting

confidence: 82%

Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference

Ravikumar

Devanapally

Jose

2019

Nucleic Acids Research

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“…As "regulation of muscle contraction" is one of the most overrepresented biological processes among CEH-60 DamID targets, we speculate that CEH-60 could be involved in contraction of the pharynx, which is indispensable for feeding [61]. Indeed, for 9 out of the 23 genes classified under "muscle structure development", the largest muscle-related biological process in the GO analysis (Fig 1), there is evidence for expression in pharyngeal muscle: unc-89, unc-22, unc-15, emb-9, dyb-1, alp-1, unc-96, mlc-1 and unc-27 [54,[62][63][64][65][66][67][68][69]. These observations indicate that ceh-60's function in the muscle system could be related to the pharynx.…”

Section: Plos Onementioning

confidence: 99%

DamID identifies targets of CEH-60/PBX that are associated with neuron development and muscle structure in Caenorhabditis elegans

et al. 2020

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Transcription factors govern many of the time- and tissue-specific gene expression events in living organisms. CEH-60, a homolog of the TALE transcription factor PBX in vertebrates, was recently characterized as a new regulator of intestinal lipid mobilization in Caenorhabditis elegans. Because CEH-60’s orthologs and paralogs exhibit several other functions, notably in neuron and muscle development, and because ceh-60 expression is not limited to the C. elegans intestine, we sought to identify additional functions of CEH-60 through DNA adenine methyltransferase identification (DamID). DamID identifies protein-genome interaction sites through GATC-specific methylation. We here report 872 putative CEH-60 gene targets in young adult animals, and 587 in L2 larvae, many of which are associated with neuron development or muscle structure. In light of this, we investigate morphology and function of ceh-60 expressing AWC neurons, and contraction of pharyngeal muscles. We find no clear functional consequences of loss of ceh-60 in these assays, suggesting that in AWC neurons and pharyngeal muscle, CEH-60 function is likely more subtle or redundant with other factors.

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“…To tag gtbp-1, unc-22 and sur-5 with gfp using genome editing: A single guide RNA was selected <6 base pairs away from the insertion site ( Supplementary Figure S4), and gfp sequences for the homology template was amplified from pTK2 (a derivative of pPD95.75 - Addgene plasmid #1494, a gift from Andrew Fire) using primers with [35][36][37][38][39][40] base pair overhangs matching either side of the cut site. Guide RNAs were amplified using the forward primers P63 for gtbp-1::gfp, P64 for gfp::unc-22, P65 for unc-22::gfp and P66 for sur-5::gfp.…”

Section: Transgenesis and Genome Editingmentioning

confidence: 99%

“…Insertions of gfp were performed using a single guide RNA and a double-stranded repair template amplified using PCR (35). Punc-22::unc-22::gfp resulted in GFP fluorescence within the pharynx as reported earlier (36). Additional details are provided in Supplementary Materials and methods.…”

Section: Introductionmentioning

confidence: 99%

Gene silencing by double-stranded RNA fromC. elegansneurons reveals functional mosaicism of RNA interference

Ravikumar

Devanapally

Jose

2018

Preprint

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Graphical Abstract:Random sets of cells can either receive different amounts of double-stranded RNA from neurons or use different RdRPs -RRF-1 only versus RRF-1 or EGO-1 -to perform the same function. ABSTRACTDelivery of double-stranded RNA (dsRNA) into animals can silence genes of matching sequence in diverse cell types through mechanisms that have been collectively called RNA interference. In the nematode C. elegans, dsRNA from multiple sources can trigger the amplification of silencing signals. Amplification occurs through the production of small RNAs by two RNA-dependent RNA polymerases (RdRPs) that are thought to be tissuespecific -EGO-1 in the germline and RRF-1 in somatic cells. Here we analyze instances of silencing in somatic cells that lack RRF-1. By varying dsRNA sources and target genes, we find that silencing in the absence of RRF-1 is most obvious when dsRNA from neurons is used to silence genes in intestinal cells. This silencing requires EGO-1, but the lineal identity of cells that can use EGO-1 varies. This variability could be because random sets of cells can either receive different amounts of dsRNA from the same source or use different RdRPs to perform the same function. As a result, all cells appear similarly functional despite underlying differences that vary from animal to animal. This functional mosaicism cautions against the use of a few molecules as proxies for predicting the behavior of a cell. 16: P14-P15).Transgenesis: C. elegans was transformed with plasmids and/or PCR products using microinjection (32) to generate extrachromosomal or integrated arrays. pHC337 was used to express an inverted repeat of gfp in neurons (11), which is expected to generate a hairpin RNA (gfp-dsRNA). Generation of the array that expresses unc-22-dsRNA in neurons (qtEx136) was described earlier (12). To rescue silencing defects in rde-1(jam1) and rrf-1(jam3) animals (Supplementary Figure S2), genomic DNA from wild-type animals (N2 gDNA) was used as a template to generate fused promoter/gene products through overlap extension PCR using Expand Long Template polymerase (Roche) and PCR products were purified using QIAquick PCR Purification Kit (Qiagen). The plasmid pHC448for DsRed2 expression in the pharynx or a PCR product, Prgef-1::DsRed2::unc-54 3'UTR, for DsRed2 expression in neurons was used as a co-injection marker (12). Additional details are provided in Supplementary Materials and Methods.Genome editing: Synthetic CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) (IDT) or single guide RNAs (sgRNA) transcribed in vitro were combined with Cas9 protein

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Twitchin kinase inhibits muscle activity

Cited by 18 publications

References 44 publications

Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference

Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference

DamID identifies targets of CEH-60/PBX that are associated with neuron development and muscle structure in Caenorhabditis elegans

Gene silencing by double-stranded RNA fromC. elegansneurons reveals functional mosaicism of RNA interference

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