Lee KH et al. (2016) Curr Protoc Toxicol "A Simple Light Stimulation of Caenorhabditis elegans."

Aschner M, Lee KH

[Curr Protoc Toxicol, 2016]

Response via noxious stimulus can be an important indicator of sensory neuron function and overall health of an organism. If the stimulation is quick and simple, and the animal can be rescued afterwards, such a method not only allows for assays pertaining to changed sensory ability after various treatments, but also increases the reliability of the statistical relationships that are established. This protocol demonstrates a stimulation assay in Caenorhabditis elegans, using blue light from common laboratory equipment: the fluorescent microscope. The nematode detects blue light using a set of amphid ciliary sensory neurons, and blue light is detrimental to its overall health after a prolonged exposure. However, under brief exposure, blue light stimulation provides a rapid and easy method for quantifying sensory functions and health without harming the animal. 2016 by John Wiley & Sons, Inc.

Lee KH et al. (2003) Mol Cells "Dna2 requirement for normal reproduction of Caenorhabditis elegans is temperature-dependent."

Han JW, Lee KH, Lee TH, Park YJ, Ahnn J, Seo YS, Koo HS, Lee MH

[Mol Cells, 2003]

To determine the function of Dna2 in a multicellular organism, the Caenorhabditis elegans Dna2 expression was probed and deletion mutant phenotypes were analyzed. Dna2 was localized to the nuclei of C. elegans oocytes and early embryos by immunostaining or green fluorescent protein-tagging. A homozygous dna2 deletion mutant showed a reduced brood size and embryonic lethality, and the phenotypes greatly depended on growth temperature and aggravated in the succeeding generation. The mutant embryos also showed delayed cell divisions, which together with temperature-dependence of the mutant phenotypes supported the well-conserved role of Dna2 in DNA replication.

Haskell D et al. (2021) G3 (Bethesda) "KH domain containing RNA-binding proteins coordinate with microRNAs to regulate Caenorhabditis elegans development."

Zinovyeva A, Haskell D

[G3 (Bethesda), 2021]

MicroRNAs (miRNAs) and RNA-binding proteins (RBPs) regulate gene expression at the post-transcriptional level, but the extent to which these key regulators of gene expression coordinate their activities and the precise mechanisms of this coordination are not well understood. RBPs often have recognizable RNA binding domains that correlate with specific protein function. Recently, several RBPs containing K homology (KH) RNA binding domains were shown to work with miRNAs to regulate gene expression, raising the possibility that KH domains may be important for coordinating with miRNA pathways in gene expression regulation. To ascertain whether additional KH domain proteins functionally interact with miRNAs during Caenorhabditis elegans development, we knocked down twenty-four genes encoding KH-domain proteins in several miRNA sensitized genetic backgrounds. Here, we report that a majority of the KH domain-containing genes genetically interact with multiple miRNAs and Argonaute alg-1. Interestingly, two KH domain genes, predicted splicing factors sfa-1 and asd-2, genetically interacted with all of the miRNA mutants tested, whereas other KH domain genes showed genetic interactions only with specific miRNAs. Our domain architecture and phylogenetic relationship analyses of the C. elegans KH domain-containing proteins revealed potential groups that may share both structure and function. Collectively, we show that many C. elegans KH domain RBPs functionally interact with miRNAs, suggesting direct or indirect coordination between these two classes of post-transcriptional gene expression regulators.

Nakel K et al. (2010) RNA "Four KH domains of the C. elegans Bicaudal-C ortholog GLD-3 form a globular structural platform."

Bonneau F, Eckmann CR, Hartung SA, Nakel K, Conti E

[RNA, 2010]

Caenorhabditis elegans GLD-3 is a five K homology (KH) domain-containing protein involved in the translational control of germline-specific mRNAs during embryogenesis. GLD-3 interacts with the cytoplasmic poly(A)-polymerase GLD-2. The two proteins cooperate to recognize target mRNAs and convert them into a polyadenylated, translationally active state. We report the 2.8-A-resolution crystal structure of a proteolytically stable fragment encompassing the KH2, KH3, KH4, and KH5 domains of C. elegans GLD-3. The structure reveals that the four tandem KH domains are organized into a globular structural unit. The domains are involved in extensive side-by-side interactions, similar to those observed in previous structures of dimeric KH domains, as well as head-to-toe interactions. Small-angle X-ray scattering reconstructions show that the N-terminal KH domain (KH1) forms a thumb-like protrusion on the KH2-KH5 unit. Although KH domains are putative RNA-binding modules, the KH region of GLD-3 is unable in isolation to cross-link RNA. Instead, the KH1 domain mediates the direct interaction with the poly(A)-polymerase GLD-2, pointing to a function of the KH region as a protein-protein interaction platform.

Abate JP et al. (2009) Cell Metab "Life is short, if sweet."

Blackwell TK, Abate JP

[Cell Metab, 2009]

Insulin is essential for glucose homeostasis, but reducing its activity delays the aging process in model organisms. In this issue of Cell Metabolism, Lee et al. (2009) show how these effects of insulin signaling intersect when glucose is fed to C. elegans.

Daubner GM et al. (2014) Nucleic Acids Res "Structural and functional implications of the QUA2 domain on RNA recognition by GLD-1."

Ciosk R, Gerhardy S, Allain FH, Tocchini C, Brummer A, Daubner GM, Zavolan M

[Nucleic Acids Res, 2014]

The STAR family comprises ribonucleic acid (RNA)-binding proteins that play key roles in RNA-regulatory processes. RNA recognition is achieved by a KH domain with an additional -helix (QUA2) that seems to extend the RNA-binding surface to six nucleotides for SF1 (Homo sapiens) and seven nucleotides for GLD-1 (Caenorhabditis elegans). To understand the structural basis of this probable difference in specificity, we determined the solution structure of GLD-1 KH-QUA2 with the complete consensus sequence identified in the tra-2 gene. Compared to SF1, the GLD-1 KH-QUA2 interface adopts a different conformation resulting indeed in an additional sequence-specific binding pocket for a uracil at the 5'end. The functional relevance of this binding pocket is emphasized by our bioinformatics analysis showing that GLD-1 binding sites with this 5'end uracil are more predictive for the functional response of the messenger RNAs to gld-1 knockout. We further reveal the importance of the KH-QUA2 interface in vitro and that its alteration in vivo affects the level of translational repression dependent on the sequence of the GLD-1 binding motif. In conclusion, we demonstrate that the QUA2 domain distinguishes GLD-1 from other members of the STAR family and contributes more generally to the modulation of RNA-binding affinity and specificity of KH domain containing proteins.

Shtang S et al. (1999) Am J Med Genet "Search for a Caenorhabditis elegans FMR1 homologue: identification of a new putative RNA-binding protein (PRP-1) that hybridizes to the mouse FMR1 double K homology domain."

Percy ME, Shtang S, Perry MD

[Am J Med Genet, 1999]

A mixed stage lambdagt10 Caenorhabditis elegans cDNA library was screened with a probe derived by polymerase chain reaction from the double K homology (KH) domain of mouse FMR1 cDNA, a region that is highly conserved in the human, mouse, chicken, and frog FMR1 proteins. Four positively hybridizing cDNAs were cloned and characterized by sequencing. The overlapping sequences map to cosmid R119 from C. elegans linkage group (chromosome) I, and encode a novel proline-, polyglutamine-, and RGG box-rich putative RNA-binding protein. While the cDNA has two regions with similarity to the mouse double KH domain probe at the nucleotide level, there is no significant similarity of the amino acid sequence with human FMR1, FXR1 or FXR2, nor with KH amino acid motifs. The R119 protein, therefore, does not represent an FMR1 homologue.

Lee C et al. (2017) Bio Protoc "Single-molecule RNA Fluorescence in situ Hybridization (smFISH) in Caenorhabditis elegans."

Sorensen EB, Lee C, Seidel HS, Lynch TR, Crittenden SL, Kimble J

[Bio Protoc, 2017]

Single-molecule RNA fluorescence <i>in situ</i> hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA ( Raj <i>et al.</i>, 2008 ; Lee <i>et al.</i>, 2016a ). We adapted this technique to visualize RNAs in the <i>C. elegans</i> whole adult worm or its germline, which enabled simultaneous recording of nascent transcripts at active transcription sites and mature mRNAs in the cytoplasm ( Lee <i>et al.</i>, 2013 and 2016b). Here we describe each step of the smFISH procedure, reagents, and microscope settings optimized for <i>C. elegans</i> extruded gonads.

Van Buskirk, Cheryl (2017) International Worm Meeting "A forward genetic screen for mutants defective in recovery sleep."

Van Buskirk, Cheryl

[International Worm Meeting, 2017]

In response to damaging conditions such as noxious heat or UV exposure, C. elegans enters a period of behavioral quiescence known as stress-induced sleep or recovery sleep (RS), during which sensory responses are dampened and feeding and movement cease1. Recovery sleep is mediated by activation of EGF receptors on the peptidergic ALA interneuron and subsequent release of a collection of neuropeptides1-3. At this time it is not known how cellular damage leads to the initiation of EGF signaling, and gaps remain in our understanding of signal transduction events within ALA as well as in the target tissues affected by ALA peptides. In order to uncover genes required for recovery sleep we have initiated an EMS screen for sleepless F2 animals, using the pore-forming toxin Cry5B as our damage-inducing agent. Progeny of sleepless candidates are tested for responses to other known sleep-inducing stressors, such as heat and UV light. Mutants that are found to be generally defective in recovery sleep are kept for SNP mapping, complementation as needed, and eventual whole-genome sequencing. We will present our detailed methods, some obstacles that have been overcome in our mapping of sleep mutants, and information on candidate identity if available. We also describe how this project has been implemented within the context of an undergraduate laboratory course called BIOL447 FIRE: Full Immersion Research Experience. 1. Hill AJ, Mansfield R, Lopez JMNG, Raizen DM, Van Buskirk C. 2014. Cellular Stress Induces a Protective Sleep-like State in C. elegans. Curr. Biol. 24, 2399-2405. 2. Nelson MD, Lee KH, Churgin MA, Hill AJ, Van Buskirk C, Fang-Yen C, Raizen DM. 2014. FMRFamide-like FLP-13 neuropeptides promote quiescence following heat stress in Caenorhabditis elegans. Curr. Biol. 24:2406-2410. 3. Nath RD, Chow ES, Wang H, Schwarz EM, Sternberg PW. 2016. C. elegans stress- induced sleep emerges from the collective action of multiple neuropeptides. Curr. Bio.l 26:2446-2455.

T Shin et al. (1999) International C. elegans Meeting "Zinc-finger and KH domain Proteins in C. elegans Embryogenesis"

CC Mello, T Shin

[International C. elegans Meeting, 1999]

In the early embryo, the expression of maternally encoded proteins is controlled both spatially and temporally. Two groups of maternal proteins have been implicated in this process. The first consists of Cys3-His zinc-finger proteins including PIE-1, MEX-1, MEX-5/AH6.5 and POS-1. The second class consists of KH domain proteins including ALP-1, MEX-3 and GLD-1. Mutations in most of these genes cause embryonic cell fate transformation, and in some cases, are correlated with clear misexpression of maternal proteins. Both Cys3-His zinc-fingers and KH domains are conserved in a wide variety of organisms and in some cases are implicated in direct RNA binding, raising the possibility that these two families of proteins directly regulate maternal mRNA translation. We identified ALP-1 as a two-hybrid interacter of PIE-1. This interaction requires a part of ALP-1 KH domain and a C-terminal region in PIE-1 outside of its zinc-fingers. We also found that while ALP-1 binds only to PIE-1 among all zinc-finger proteins tested, other KH domain and zinc-finger proteins can interact with multiple partners. For example, GLD-1 can bind strongly to POS-1 and weakly to PIE-1, and POS-1 can interact with MEX-3. These data suggest that zinc-finger/KH domain proteins may function together as a complex and/or regulate each other's activity. Consistent with this view, gld-1(RNAi) produces a no-gut defect very similar to that caused by pos-1 mutations. Furthermore, alp-1 interacts genetically with pos-1 and gld-1 , completely suppressing the no-gut phenotype in both. Thus, the two protein families appear to regulate each other in a complex manner. How do zinc-finger/KH domain proteins function in controlling maternal mRNA translation? Do they directly recognize RNA? What are the biochemical bases for their apparently complex interactions both physically and genetically? Finding answers for these questions will require in vivo biochemical analyses. Toward this end, we have generated transgenic animals that stably express functional HA-tagged PIE-1 protein. We are currently examining in vivo protein-protein interaction between HA-PIE-1 and other members of the two protein families. These studies should shed light on biochemical and physiological roles for the evolutionarily conserved zinc-finger/KH motif proteins.