Global profiling and annotation of templated isomiRs dynamics across Caenorhabditis elegans development.

microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through translational repression and mRNA destabilization. During canonical miRNA biogenesis, several miRNA isoforms, or isomiRs, are produced from a single precursor miRNA. Templated isomiRs are generated through Drosha or Dicer cleavage at alternate positions on either the primary or the precursor miRNAs, generating truncated or extended 5' and/or 3' miRNA ends. As changes to the mature miRNA sequence can alter miRNA gene target repertoire, we investigated the extent of templated isomiR prevalence, providing a profiling map for templated isomiRs across stages of C. elegans development. While most miRNA loci did not produce abundant templated isomiRs, a substantial number of miRNA loci produced isomiRs were just as, or more, abundant than their annotated canonical mature miRNAs. 3' end miRNA alterations were more frequent than the seed-altering 5' end extensions or truncations. However, we identified several miRNA loci that produced a considerable amount of isomiRs with 5' end alterations, predicted to target new, distinct sets of genes. Overall, the presented annotation of templated isomiR dynamics across C. elegans developmental stages provides a basis for further studies into miRNA biogenesis and the intriguing potential of functional miRNA diversification through isomiR production.

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. Gene ontology analysis of the identified interactors revealed an enrichment for RNA binding proteins, suggesting that we captured proteins that may be involved in mRNA lifecycle. To determine which miRNA interactors are important for miRNA activity, we used RNAi to deplete putative miRNA co-factors in animals with compromised miRNA activity and looked for alterations of the miRNA mutant phenotypes. Depletion of 25 of 39 tested genes modified the miRNA mutant phenotypes in three sensitized backgrounds. Modulators of miRNA phenotypes ranged from RNA binding proteins RBD-1 and CEY-1 to metabolic factors such as DLST-1 and ECH-5, among others. The observed functional interactions suggest widespread coordination of these proteins with miRNAs to ultimately regulate gene expression. This study provides a foundation for future investigations aimed at deciphering the molecular mechanisms of miRNA-mediated gene regulation.

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. elegans E3 ubiquitin ligases complex [SKR-1/2-CUL-1-F-box (SCF)], targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin derivative/mutant AtTIR1 pair [C. elegans AID version 2 (C.e.AIDv2)] that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand-binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID-targets, the addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expand the utility of the AID system and broaden the number of proteins that can be effectively targeted with it.

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. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize "off-target" sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts postinjection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.