West KH et al. (2016) Planta Med "Marine Biodiscovery Goes Deeper: Using In Vivo Bioassays Based on Model Organisms to Identify Biomedically Relevant Marine Metabolites."

West KH, Crawford AD

[Planta Med, 2016]

Secondary metabolites from marine organisms are structurally diverse small molecules with high levels of bioactivity, and represent an underutilized resource for modern drug discovery. To facilitate the identification of drug-like marine metabolites, the significant potential of in vivo models of human disease - in particular those suitable for medium-throughput screening and bioassay-guided fractionation - should be explored in future marine biodiscovery efforts. Here, we explore the advantages of Caenorhabditis elegans, Drosophila, and zebrafish bioassays for marine biodiscovery, and review recent progress in using these in vivo models to identify bioactive marine metabolites.

Christian Eckmann et al. (2002) Mid-west Worm Meeting "GLD-3, a KH protein and Bicaudal C homolog, antagonizes FBF and controls germline sex determination"

Marvin Wickens, Brian Kraemer, Christian Eckmann, Judith Kimble

[Mid-west Worm Meeting, 2002]

The FBF RNA binding protein controls germline sex determination in the nematode C. elegans (Zhang et al. 1997). Normally, XX hermaphrodites make sperm and then oocytes, while XO males make sperm continuously. FBF, a 3UTR-binding repressor in the PUF family, is essential for the hermaphrodite switch from sperm to oocytes. Here we report identification of GLD-3, a protein discovered in a two-hybrid screen for FBF interactors. GLD-3s motif architecture places it in the Bicaudal-C family of RNA binding proteins. It possesses five tandemly repeated, imperfect KH repeats. GLD3 is cytoplasmic and associated with P granules during germline development and in the early embryo. The gld-3 gene is required maternally for embryogenesis and germline survival and zygotically for spermatogenesis: gld-3 is critical not only for progression through spermatogenesis, but also for making the correct number of sperm. Genetically, GLD-3 antagonizes FBF: in fbf mutants, sperm are made continuously, whereas in gld-3 mutants, germ cells switch aberrantly from spermatogenesis to oogenesis. To explore the molecular basis of this antagonism, we have tested FBF binding in a three-hybrid system. Without GLD-3, FBF binds to the fem-3 PME regulatory element. When GLD-3 is introduced, FBF binding is markedly reduced. Controls indicate that this interference with FBF binding is specific for FBF and for GLD-3. For example, the binding of other PUF proteins to their targets is unaffected by GLD-3. We conclude that GLD-3 antagonizes FBF function and that a balance between FBF and GLD-3 activities is required to control the sperm/oocyte switch and specify the correct number of sperm.

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.

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.

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.

Braddock DT et al. (2002) Nature "Structure and dynamics of KH domains from FBP bound to single-stranded DNA."

Baber JL, Clore GM, Levens D, Louis JM, Braddock DT

[Nature, 2002]

Gene regulation can be tightly controlled by recognition of DNA deformations that are induced by stress generated during transcription. The KH domains of the FUSE-binding protein (FBP), a regulator of c-myc expression, bind in vivo and in vitro to the single-stranded far-upstream element (FUSE), 1,500 base pairs upstream from the c-myc promoter. FBP bound to FUSE acts through TFIIH at the promoter. Here we report the solution structure of a complex between the KH3 and KH4 domains of FBP and a 29-base single-stranded DNA from FUSE. The KH domains recognize two sites, 9-10 bases in length, separated by 5 bases, with KH4 bound to the 5' site and KH3 to the 3' site. The central portion of each site comprises a tetrad of sequence 5'd-ATTC for KH4 and 5'd-TTTT for KH3. Dynamics measurements show that the two KH domains bind as articulated modules to single-stranded DNA, providing a flexible framework with which to recognize transient, moving targets.

Fuglsang H et al. (1976) Tropenmed Parasitol "Seasonal variation in the concentration of Onchocerca volvulus microfilariae in the skin?"

Fuglsang H, Fisiy C, Ayonge S, Anderson J, de C Marshall TF

[Tropenmed Parasitol, 1976]

Repeated multiple skin snips in 18 persons with onchocerciasis from the Sudan-savanna of West Africa suggested the possibility of a seasonal variation in microfilarial concentrations. This variation may be an evolutionary adaptation of the parasite to the climate conditions that affect the seasonal distribution of the vector, but a migration of the microfilariae in the skin layers caused by the Simulium bites cannot be excluded.

Haskell, D. et al. (2019) International Worm Meeting "KH domain containing RNA binding proteins coordinate with microRNAs to regulate gene expression."

Haskell, D., Zinovyeva , A.

[International Worm Meeting, 2019]

Precisely controlled gene regulation is essential for proper organism development and homeostasis. Small non-coding RNAs called microRNAs (miRNAs) play a critical role in post-transcriptional regulation of gene expression. miRNAs regulate gene expression by binding to partially complementary sites in mRNA targets and recruiting protein factors to form the miRNA induced silencing complex (miRISC). The miRISC represses mRNA translation and/or targets the mRNA for degradation. Several KH domain-containing RNA binding proteins (RBPs) have been implicated in modulating miRNA activity, including hrpk-1 1, vgln-1 2, and gld-1 3. We hypothesized that additional KH domain RNA binding proteins may coordinate with miRNAs to regulate gene expression. To test this hypothesis, we carried out functional assays in several genetically sensitized backgrounds where miRNA activity is partially compromised. Specifically, we used RNAi to knock down the function of 27 KH domain protein coding genes in the following sensitized backgrounds: lsy-6(ot150), mir-48 mir-241(nDf51), mir-35-mir-41(nDf50), let-7(n2853) and alg-1(ma202) to determine whether this class of RBPs coordinates with miRNA activity. We found that RNAi of several KH domain proteins genetically interacted with miRNA reduction-of-function mutations suggesting that these proteins may directly or indirectly coordinate with miRNAs to regulate gene expression. Interestingly, activity of some KH domain proteins was important for all miRNAs assayed (for example, sfa-1and asd-2), while others were more specific to certain miRNAs (for example, pno-1, akap-1, nova-1, and fubl-1). This specificity could reflect the molecular function of a given RBP, its expression pattern, or the mode of interaction with the miRNA pathway. Understanding the molecular mechanisms through which these RNA binding proteins coordinate with the miRNAs would enhance our understanding of the complex process of gene regulation. 1Li et al., 2019. Biorxiv doi: https://doi.org/10.1101/569905 2Zabinksy et., 2017. G3 doi: doi: 10.1534/g3.117.043414 3Akay et., 2013. Open Biol doi: 10.1098/rsob.130151