Functional identification of microRNA-centered complexes in C. elegans.

microRNAs (miRNAs) are crucial for normal development and physiology. To identify factors that might coordinate with miRNAs to regulate gene expression, we used 2'O-methylated oligonucleotides to precipitate Caenorhabditis elegans let-7, miR-58, and miR-2 miRNAs and the associated proteins. A total of 211 proteins were identified through mass-spectrometry analysis of miRNA co-precipitates, which included previously identified interactors of key miRNA pathway components.

Single nucleotide substitutions effectively block Cas9 and allow for scarless genome editing in Caenorhabditis elegans.

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location.

An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in Caenorhabditis elegans.

The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C.

microRNA strand selection: Unwinding the rules.

microRNAs (miRNAs) play a central role in the regulation of gene expression by targeting specific mRNAs for degradation or translational repression. Each miRNA is post-transcriptionally processed into a duplex comprising two strands. One of the two miRNA strands is selectively loaded into an Argonaute protein to form the miRNA-Induced Silencing Complex (miRISC) in a process referred to as miRNA strand selection.

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans.

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques.

Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal.

MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans microRNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)]. alg-1(anti) mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1.

Mutations in conserved residues of the C. elegans microRNA Argonaute ALG-1 identify separable functions in ALG-1 miRISC loading and target repression.

microRNAs function in diverse developmental and physiological processes by regulating target gene expression at the post-transcriptional level. ALG-1 is one of two Caenorhabditis elegans Argonautes (ALG-1 and ALG-2) that together are essential for microRNA biogenesis and function. Here, we report the identification of novel antimorphic (anti) alleles of ALG-1 as suppressors of lin-28(lf) precocious developmental phenotypes.

Complex network of Wnt signaling regulates neuronal migrations during Caenorhabditis elegans development.

Members of the Wnt family of secreted glycoproteins regulate many developmental processes, including cell migration. We and others have previously shown that the Wnts egl-20, cwn-1, and cwn-2 are required for cell migration and axon guidance. However, the roles in cell migration of all of the Caenorhabditis elegans Wnt genes and their candidate receptors have not been explored fully.

The C. elegans histone deacetylase HDA-1 is required for cell migration and axon pathfinding.

Histone proteins play integral roles in chromatin structure and function. Histones are subject to several types of posttranslational modifications, including acetylation, which can produce transcriptional activation. The converse, histone deacetylation, is mediated by histone deacetylases (HDACs) and often is associated with transcriptional silencing.

The C. elegans Frizzled CFZ-2 is required for cell migration and interacts with multiple Wnt signaling pathways.

Members of the Frizzled family of integral membrane proteins are implicated in many developmental events, including specifying cell fate, orienting cell and planar polarity, and directing cell migration. Frizzleds function as cell surface receptors for secreted Wnt proteins. We report here the isolation of a mutation in cfz-2, a Caenorhabditis elegans Frizzled gene.