Zinovyeva AY et al. (2008) Genetics "Complex network of Wnt signaling regulates neuronal migrations during Caenorhabditis elegans development."

Forrester WC, Zinovyeva AY, Yamamoto Y, Sawa H

[Genetics, 2008]

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 C. elegans Wnt genes and their candidate receptors have not been explored fully. We have extended our analysis to include all C. elegans Wnts and six candidate Wnt receptors: four Frizzleds, the sole Ryk family receptor LIN-18 and the Ror receptor tyrosine kinase CAM-1. We show that three of the Wnts, CWN-1, CWN-2, and EGL-20, play major roles in directing cell migrations and that all five Wnts direct specific cell migrations, either by acting redundantly or antagonizing each other''s function. We report that all four Frizzleds function to direct Q descendent cell migrations, but only a subset of the putative Wnt receptors function in directing migrations of other cells. Finally, we find striking differences between the phenotypes of the Wnt quintuple and Frizzled quadruple mutants.

Zinovyeva AY et al. (2006) Dev Biol "The C. elegans histone deacetylase HDA-1 is required for cell migration and axon pathfinding."

Zinovyeva AY, Cloud VJ, Graham SM, Forrester WC

[Dev Biol, 2006]

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. We identified a new mutation, cw2, in the Caenorhabditis elegans hda-1 gene, which encodes a histone deacetylase. Previous studies showed that a mutation in hda-1, e1795, or reduction of hda-1 RNA by RNAi causes defective vulval and gonadal development leading to sterility. The hda-1(cw2) mutation causes defective vulval development and reduced fertility, like hda-1(e1795), albeit with reduced severity. Unlike the previously reported hda-1 mutation, hda-1(cw2) mutants are viable as homozygotes, although many die as embryos or larvae, and are severely uncoordinated. Strikingly, in hda-1(cw2) mutants, axon pathfinding is defective; specific axons often appear to wander randomly or migrate in the wrong direction. In addition, the long range migrations of three neuron types and fasciculation of the ventral nerve cord are defective. Together, our studies define a new role for HDA-1 in nervous system development, and provide the first evidence for HDAC function in regulating neuronal axon guidance.

Zinovyeva AY et al. (2005) Dev Biol "The C. elegans Frizzled CFZ-2 is required for cell migration and interacts with multiple Wnt signaling pathways."

Forrester WC, Zinovyeva AY

[Dev Biol, 2005]

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. Mutation of cfz-2 causes defective cell migration, disorganization of head neurons, and can cause ectopic axon outgrowth. Analysis of mosaic animals shows that CFZ-2 functions cell nonautonomously, but does not rule out an autonomous role. CFZ-2 is expressed primarily in the anterior of embryos and in several cells in the head of adults. Our analysis of interactions between CFZ-2 and other Wnt pathways reveals that three Wnts, CWN-1, CWN-2 and EGL-20, and a Frizzled, MOM-5, function redundantly with one another and with CFZ-2 for specific cell migrations. In contrast, CWN-1, CWN-2, EGL-20, CFZ-2, and MOM-5 antagonize one another for other migrations. Therefore, CFZ-2 functions by collaborating with and/or antagonizing other Wnt signaling pathways to regulate specific cell migrations.

Zinovyeva AY et al. (2015) Proc Natl Acad Sci U S A "Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal."

Zinovyeva AY, Wohlschlegel JA, Ambros VA, Veksler-Lublinsky I, Vashisht AA

[Proc Natl Acad Sci U S A, 2015]

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. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA and protein populations associated with ALG-1(anti) complexes in vivo. We extensively characterized proteins associated with wild-type and mutant ALG-1 and found that the mutant ALG-1(anti) protein fails to interact with numerous miRISC cofactors, including proteins known to be necessary for target repression. In addition, alg-1(anti) mutants dramatically overaccumulated microRNA* (passenger) strands, and immunoprecipitated ALG-1(anti) complexes contained nonstoichiometric yields of mature microRNA and microRNA* strands, with some microRNA* strands present in the ALG-1(anti) Argonaute far in excess of the corresponding mature microRNAs. We show complex and microRNA-specific defects in microRNA strand selection and microRNA* strand disposal. For certain microRNAs (for example mir-58), microRNA guide strand selection by ALG-1(anti) appeared normal, but microRNA* strand release was inefficient. For other microRNAs (such as mir-2), both the microRNA and microRNA* strands were selected as guide by ALG-1(anti), indicating a defect in normal specificity of the strand choice. Our results suggest that wild-type ALG-1 complexes recognize structural features of particular microRNAs in the context of conducting the strand selection and microRNA* ejection steps of miRISC maturation.

Zinovyeva AY et al. (2014) PLoS Genet "Mutations in conserved residues of the C. elegans microRNA Argonaute ALG-1 identify separable functions in ALG-1 miRISC loading and target repression."

Hammell CM, Bouasker S, Ambros V, Simard MJ, Zinovyeva AY

[PLoS Genet, 2014]

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. The alg-1(anti) mutations broadly impair the function of many microRNAs and cause dosage-dependent phenotypes that are more severe than the complete loss of ALG-1. ALG-1(anti) mutant proteins are competent for promoting Dicer cleavage of microRNA precursors and for associating with and stabilizing microRNAs. However, our results suggest that ALG-1(anti) proteins may sequester microRNAs in immature and functionally deficient microRNA Induced Silencing Complexes (miRISCs), and hence compete with ALG-2 for access to functional microRNAs. Immunoprecipitation experiments show that ALG-1(anti) proteins display an increased association with Dicer and a decreased association with AIN-1/GW182. These findings suggest that alg-1(anti) mutations impair the ability of ALG-1 miRISC to execute a transition from Dicer-associated microRNA processing to AIN-1/GW182 associated effector function, and indicate an active role for ALG/Argonaute in mediating this transition.

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.

Li L et al. (2019) PLoS Genet "HRPK-1, a conserved KH-domain protein, modulates microRNA activity during Caenorhabditis elegans development."

Veksler-Lublinsky I, Zinovyeva A, Li L

[PLoS Genet, 2019]

microRNAs (miRNAs) are potent regulators of gene expression that function in diverse developmental and physiological processes. Argonaute proteins loaded with miRNAs form the miRNA Induced Silencing Complexes (miRISCs) that repress gene expression at the post-transcriptional level. miRISCs target genes through partial sequence complementarity between the miRNA and the target mRNA's 3' UTR. In addition to being targeted by miRNAs, these mRNAs are also extensively regulated by RNA-binding proteins (RBPs) through RNA processing, transport, stability, and translation regulation. While the degree to which RBPs and miRISCs interact to regulate gene expression is likely extensive, we have only begun to unravel the mechanisms of this functional cooperation. An RNAi-based screen of putative ALG-1 Argonaute interactors has identified a role for a conserved RNA binding protein, HRPK-1, in modulating miRNA activity during C. elegans development. Here, we report the physical and genetic interaction between HRPK-1 and ALG-1/miRNAs. Specifically, we report the genetic and molecular characterizations of hrpk-1 and its role in C. elegans development and miRNA-mediated target repression. We show that loss of hrpk-1 causes numerous developmental defects and enhances the mutant phenotypes associated with reduction of miRNA activity, including those of lsy-6, mir-35-family, and let-7-family miRNAs. In addition to hrpk-1 genetic interaction with these miRNA families, hrpk-1 is required for efficient regulation of lsy-6 target cog-1. We report that hrpk-1 plays a role in processing of some but not all miRNAs and is not required for ALG-1/AIN-1 miRISC assembly. We suggest that HRPK-1 may functionally interact with miRNAs by both affecting miRNA processing and by enhancing miRNA/miRISC gene regulatory activity and present models for its activity.

Panzade G et al. (2022) RNA Biol "Global profiling and annotation of templated isomiRs dynamics across Caenorhabditis elegans development."

Panzade G, Hebbar S, Li L, Veksler-Lublinsky I, Zinovyeva A

[RNA Biol, 2022]

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.

Zou Y et al. (2013) Science "Developmental decline in neuronal regeneration by the progressive change of two intrinsic timers."

Zinovyeva A, Ambros V, Zou Y, Chuang CF, Chiu H, Chang C

[Science, 2013]

Like mammalian neurons, Caenorhabditis elegans neurons lose axon regeneration ability as they age, but it is not known why. Here, we report that let-7 contributes to a developmental decline in anterior ventral microtubule (AVM) axon regeneration. In older AVM axons, let-7 inhibits regeneration by down-regulating LIN-41, an important AVM axon regeneration-promoting factor. Whereas let-7 inhibits lin-41 expression in older neurons through the lin-41 3' untranslated region, lin-41 inhibits let-7 expression in younger neurons through Argonaute ALG-1. This reciprocal inhibition ensures that axon regeneration is inhibited only in older neurons. These findings show that a let-7-lin-41 regulatory circuit, which was previously shown to control timing of events in mitotic stem cell lineages, is reutilized in postmitotic neurons to control postdifferentiation events.

Duan Y et al. (2024) Proc Natl Acad Sci U S A "Modeling neurodevelopmental disorder-associated human AGO1 mutations in Caenorhabditis elegans Argonaute alg-1."

Piton A, Li L, Duan Y, Ambros V, Panzade GP, Zinovyeva A

[Proc Natl Acad Sci U S A, 2024]

MicroRNAs (miRNA) associate with Argonaute (AGO) proteins and repress gene expression by base pairing to sequences in the 3' untranslated regions of target genes. De novo coding variants in the human AGO genes <i>AGO1</i> and <i>AGO2</i> cause neurodevelopmental disorders (NDD) with intellectual disability, referred to as Argonaute syndromes. Most of the altered amino acids are conserved between the miRNA-associated AGO in <i>Homo sapiens</i> and <i>Caenorhabditis elegans</i>, suggesting that the human mutations could disrupt conserved functions in miRNA biogenesis or activity. We genetically modeled four human <i>AGO1</i> mutations in <i>C. elegans</i> by introducing identical mutations into the <i>C. elegans AGO1</i> homologous gene<i>, alg-1</i>. These <i>alg-1</i> NDD mutations cause phenotypes in <i>C. elegans</i> indicative of disrupted miRNA processing, miRISC (miRNA silencing complex) formation, and/or target repression. We show that the <i>alg-1</i> NDD mutations are antimorphic, causing developmental and molecular phenotypes stronger than those of <i>alg-1</i> null mutants, likely by sequestrating functional miRISC components into non-functional complexes. The <i>alg-1</i> NDD mutations cause allele-specific disruptions in mature miRNA profiles, accompanied by perturbation of downstream gene expression, including altered translational efficiency and/or messenger RNA abundance. The perturbed genes include those with human orthologs whose dysfunction is associated with NDD. These cross-clade genetic studies illuminate fundamental AGO functions and provide insights into the conservation of miRNA-mediated post-transcriptional regulatory mechanisms.