Axon guidance genes identified in a large-scale RNAi screen using the RNAi-hypersensitive
            <i>Caenorhabditis elegans</i>
            strain
            <i>nre-1(hd20) lin-15b(hd126</i>
            )

In both metazoan development and metastatic cancer, migrating cells must carry out a detailed, complex program of sensing cues, binding substrates, and moving their cytoskeletons. The linker cell in Caenorhabditis elegans males undergoes a stereotyped migration that guides gonad organogenesis, occurs with precise timing, and requires the nuclear hormone receptor NHR-67. To better understand how this occurs, we performed RNA-seq of individually staged and dissected linker cells, comparing transcriptomes from linker cells of third-stage (L3) larvae, fourth-stage (L4) larvae, and nhr-67-RNAi-treated L4 larvae. We observed expression of 8,000-10,000 genes in the linker cell, 22-25% of which were up-or downregulated 20-fold during development by NHR-67. Of genes that we tested by RNAi, 22% (45 of 204) were required for normal shape and migration, suggesting that many NHR-67-dependent, linker cell-enriched genes play roles in this migration. One unexpected class of genes up-regulated by NHR-67 was tandem pore potassium channels, which are required for normal linker-cell migration. We also found phenotypes for genes with human orthologs but no previously described migratory function. Our results provide an extensive catalog of genes that act in a migrating cell, identify unique molecular functions involved in nematode cell migration, and suggest similar functions in humans.major sperm protein | nematode development | transcriptional profiling | twin pore potassium channels | conserved uncharacterized proteins C ell migration is pervasive in animal development and is also an unwanted part of human cancer. Several different varieties of cell migration, ranging from social protozoa to the human immune system, have been extensively studied (1), but there remains a need for detailed analysis of the different components of migration in a simple cell type that can be exhaustively probed through anatomy, genetics, and genomics. We have undertaken to develop the linker cell (LC) of Caenorhabditis elegans as such a model. The LC is a single cell, attached to the front of a proliferating male gonad, which carries out a stereotypic, long-range migration to generate the shape of the mature gonad (Fig. 1A). During 25 h of the second to fourth larval stages (L2 through L4), the LC moves anteriorly along the ventral body wall, turns dorsally, proceeds posteriorly, switches sides once more to the ventral bodywall, continues posteriorward, reaches the cloaca, and eventually dies (2-4). Throughout this process, the proliferating and lengthening male gonad follows the LC's path to the cloaca. Stage-specific changes occur during the middle to late stages of migration (L3 and L4 larval stages): the LC changes shape from a spheroid to a pointed ellipsoid; it increases its speed of migration; and the netrin receptor gene unc-5 is down-regulated within the LC, which causes it to switch body walls ventrally ( Fig. 1A; ref. 3). These changes between the L3 and L4 stages require the orphan nuclear receptor NHR-67, whose orthologs TAILLESS and TLX...

Section: Discussionsupporting

confidence: 88%

Functional transcriptomics of a migrating cell in Caenorhabditis elegans

Schwarz

Kato

Sternberg

2012

“…The miRNA lsy-6 functions to specify the fate of ASEL, one of two morphologically similar, bilaterally symmetric ASE neurons (ASEL and ASER), which can be monitored by expression of an ASEL-specific Plim-6::gfp reporter (45). Because many neurons in C. elegans are refractory to RNAi (46), we performed RNAi experiments in an nre-1 mutant background, which potentiates RNAi-mediated gene silencing in neurons (47). CK2 RNAi enhances the penetrance of ASEL misspecification in the lsy-6(ot150) hypomorph (48) extent as alg-1 RNAi (Fig.…”

Section: Resultsmentioning

confidence: 99%

Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1

Alessi

Khivansara

Han

et al. 2015

MicroRNAs (miRNAs) play essential, conserved roles in diverse developmental processes through association with the miRNA-induced silencing complex (miRISC). Whereas fundamental insights into the mechanistic framework of miRNA biogenesis and target gene silencing have been established, posttranslational modifications that affect miRISC function are less well understood. Here we report that the conserved serine/threonine kinase, casein kinase II (CK2), promotes miRISC function in Caenorhabditis elegans. CK2 inactivation results in developmental defects that phenocopy loss of miRISC cofactors and enhances the loss of miRNA function in diverse cellular contexts. Whereas CK2 is dispensable for miRNA biogenesis and the stability of miRISC cofactors, it is required for efficient miRISC target mRNA binding and silencing. Importantly, we identify the conserved DEAD-box RNA helicase, CGH-1/DDX6, as a key CK2 substrate within miRISC and demonstrate phosphorylation of a conserved N-terminal serine is required for CGH-1 function in the miRNA pathway.

“…We asked whether knocking down hmgs-1 causes an ASEL specification defect by scoring lim-6 pro ::gfp, an ASEL-specific reporter. To enhance the efficiency of RNAi in neurons, we crossed the lim-6 pro ::gfp reporter into the RNAi-hypersensitive nre-1(hd20) lin15b(hd126) mutant background (28). RNAi was initiated at the L3 stage of lsy-6(ot150); nre-1(hd20) lin-15b(hd126) parental (P 0 ) animals, and lim-6 pro ::gfp was scored in the progeny.…”

Section: Resultsmentioning

confidence: 99%

The mevalonate pathway regulates microRNA activity in Caenorhabditis elegans

Ruvkun¹

2012

The mevalonate pathway is highly conserved and mediates the production of isoprenoids, which feed into biosynthetic pathways for sterols, dolichol, ubiquinone, heme, isopentenyl adenine, and prenylated proteins. We found that in Caenorhabditis elegans , the nonsterol biosynthetic outputs of the mevalonate pathway are required for the activity of microRNAs (miRNAs) in silencing their target mRNAs. Inactivation of genes that mediate multiple steps of the mevalonate pathway causes derepression of several miRNA target genes, with no disruption of the miRNA levels, suggesting a role in miRNA-induced silencing complex activity. Dolichol phosphate, synthesized from the mevalonate pathway, functions as a lipid carrier of the oligosaccharide moiety destined for protein N -linked glycosylation. Inhibition of the dolichol pathway of protein N -glycosylation also causes derepression of miRNA target mRNAs. The proteins that mediate miRNA repression are therefore likely to be regulated by N -glycosylation. Conversely, drugs such as statins, which inhibit the mevalonate pathway, may compromise miRNA repression as well as the more commonly considered cholesterol biosynthesis.