Li, L. et al. (2019) International Worm Meeting "The role of 5-methylcytosine modification in piRNA regulation."

Li, L., Gu, W.

[International Worm Meeting, 2019]

RNA modifications play important roles in regulating RNA stability and functions. Historically, RNA modification research has been focusing on tRNA and rRNA since these RNAs contains diversified modification detectable using traditional techniques. Recent technical advances have made breakthrough on mRNA modification. However, modifications on miRNA, siRNA and piRNA have not been systematically studied. To study these modifications, we first purified C. elegans small RNAs of ~22 nts long. Then we utilized two methods to identify the modifications on these RNAs: Mass spectrometry and bisulfite treatment followed by high-throughput sequencing. We find these small RNAs do not contain a significant, if any, amount of m6A but piRNA contains much higher 5-methylcytosine(m5C) than siRNA and miRNA. The sequencing result shows that the m5C content likely increases when piRNA biogenesis stops, suggesting m5C may be related to piRNA degradation. Our result suggest RNA modifications may play a role in the stability of small RNA.

Li L et al. (1993) International C. elegans Meeting "Genetic analysis of unc-jd5: a gene affecting DD motoneurons."

Walthall WW, Li L

[International C. elegans Meeting, 1993]

Li L et al. (1995) International C. elegans Meeting "MOSAIC ANALYSIS OF unc-55: A GENE INVOLVED IN SYNAPTIC SPECIFICITY"

Li L, Walthall WW

[International C. elegans Meeting, 1995]

The DD and VD motoneurons (mns) form reciprocal synaptic patterns that result in the formation of a cross inhibitory circuit that is essential for normal movement. Despite different lineal origins the two classes of mns share a number of features related to their structure and function. Mutations in unc-55 alter the synaptic patterns of the VD mns so that it becomes identical to that of the DD mns. Here we performed a mosaic analysis and determined that unc-55 (+) must be expressed in the VD but not the DD mns. In this study, a che-3 mutant was used as a marker because it blocks uptake of FITC by chemosensory neurons, which are lineally related to the D mns. We constructed a duplication strain, unc-55(e1170); che-3(nr2288); hDp69. unc mosaics (n=5) were observed that had lost the duplication in the four phasmid neurons, indicating that the duplication loss had occurred before the AB.p division. In contrast, wild-type mosaic animals (n=17) had lost the duplication in a subset of phasmid neurons, indicating that the duplication loss had occurred after the division of AB.p. These results suggest that a primary target of unc-55 is the descendants of AB.p. Both the DD and VD mn are derived from this lineage. Further analysis showed that 9 of 17 wild-type mosaics had lost the duplication at or after the birth of AB.plp or AB.prp, the lineage from which the DD mns are derived. Thus loss of the duplication in the DD mns had no affect on their synaptic pattern. Therefore, unc-55 must be expressed in the VD mns and is necessary for their synaptic pattern.

Li L et al. (1996) West Coast Worm Meeting "In Vivo Analysis of Differential Expression of tRNATrp Genes in C. elegans"

Honda BM, Li L

[West Coast Worm Meeting, 1996]

In C. elegans the availability of different suptRNA alleles allows us to study expression of individual tRNA gene family members in vivo. Previous in vivo suppression and in vitro transcription results suggested that members of the tRNATrp gene family were differentially expressed. To study sup tRNATrp expression in vivo, an amber site was introduced into a lacZ gene driven by C. elegans heat shock promoters. Transgenic sup7, sup24, sup28 and sup29 strains which represented strong, medium and weak levels of transcription in vitro and suppression in vivo were used. Sup7/+, sup24/+, sup28/+, sup29/sup29 showed good suppression of lacZam in intestine; sup29/sup29 showed stronger expression than sup7/+, sup24/+ and sup28/+. In the pharynx, however, sup24/+, sup28/+ and sup29/sup29 showed weak expression whereas sup7/+ showed much stronger suppression, almost as strong as an N2, wt lacZ. These results suggested that suptRNA genes may be expressed tissue-specifically. Using stable integrated lacZam strains will allow me to further confirm these results, and pursue possible molecular mechanisms. (This research is supported by NSERC Canada.)

Li, L. et al. (2017) International Worm Meeting "A conserved RNA binding protein modulates microRNA activity during C. elegans development."

Wang, Y., Zinovyeva, A., Kranick, M., Li, L.

[International Worm Meeting, 2017]

MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression. MicroRNA activity is essential for animal development as alterations in microRNA expression or function have been associated with many developmental pathologies and diseases, making microRNAs of great interest as diagnostic and therapeutic tools. The 21~23 mature single-strand microRNA brings the microRNA Induced Silencing Complex (miRISC) to target mRNAs via imperfect complementary base-paring, resulting in mRNA silencing through translational repression and/or mRNA degradation. However, little is known about how microRNA function itself is regulated by specific developmental and physiological signals. To address how cellular factors affect microRNA-mediated gene repression, we used proteomics approaches and identified a conserved RNA binding protein, HRPK-1, as a novel microRNA interacting factor. Reduction or loss of hrpk-1 function results in a series of developmental phenotypes, including defects in fertility, embryonic development, cell fate, and organ morphogenesis: all phenotypes reminiscent of defects seen in specific microRNA mutants. Knockdown of hrpk-1 by RNAi enhances embryonic lethality of mir-35-41 mutants, cell fate defects of a reduction-of-function allele of lsy-6, and heterochronic phenotypes associated with mir-48 mir-241 mutants, suggesting that HRPK-1 may function as a positive regulator of microRNA activity. To further characterize hrpk-1, we used CRISPR/Cas9 genome editing to generate additional hrpk-1 alleles and to tag hrpk-1 with GFP at its endogenous locus. Interestingly, hrpk-1 mutants show no reduction in mature microRNA production, suggesting that HRPK-1 function may not be required for microRNA biogenesis. microRNA-specific 2'-O-methyl-oligonucleotide pulldown experiments suggest that HRPK-1 co-precipitates with some but not all miRNAs. In addition, absence of HRPK-1 does not negatively affect mature miRISC formation. We will present our current model for HRPK-1 role in microRNA-mediated gene regulation and our progress towards testing that model.

Li, L. et al. (2019) International Worm Meeting "Systematic proteomic identification of miRISC interactors and characterization of their functional requirement identifies multiple RNA binding proteins as modulators of miRNA activity."

Veksler-Lublinsky, I., Li, L., Hebbar, S., Zinovyeva, A.

[International Worm Meeting, 2019]

microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. miRNAs negatively regulate gene expression through the miRNA Induced SilencingComplex (miRISC), which forms when the miRNA-loaded Argonaute associates with members of the GW182 protein family and binds target mRNAs. This targeting via imperfect complementary base-paring results in mRNA silencing through translational repression and/or mRNA degradation. However, little is known about what other molecular factors contribute to this process. Using MudPIT mass spectrometry, we have previously identified proteins that associate with Argonaute ALG-1. To determine which miRISC interactors are important for miRNA activity and/or processing, we carried out functional assays in sensitized genetic backgrounds, including mir-48 mir-241(nDf51), lsy-6(ot150), and others. Several of the identified proteins belong to a class of heterogeneous nuclear ribonucleoproteins (hnRNPs) that are highly conserved and multifunctional. Among those is an hnRNPK homolog and a conserved Argonaute interactor, HRPK-1. We confirmed that HRPK-1 physically co-precipitates with ALG-1 in a reciprocal experiment of HRPK-1 immunoprecipitation followed by ALG-1 detection and show that this interaction is somewhat reduced but not abolished by RNAse treatment. Loss of hrpk-1 results in a series of developmental phenotypes, including defects in fertility, embryonic development, cell fate, and organ morphogenesis, with the antimorphic mutation of hrpk-1 causing more severe phenotypes than hrpk-1 null. Endogenous hrpk-1 is ubiquitously expressed across all tissues and in all stages of development. Importantly, loss of hrpk-1 enhances heterochronic phenotypes of both mir-48 mir-241 and let-7(n2853) mutants, cell fate defects of lsy-6(ot150), and embryonic lethality of mir-35-41 mutants, suggesting that HRPK-1 normally functions as a positive regulator of miRNA activity. In addition to hrkp-1 genetic interaction with multiple miRNA families, hrpk-1 is required for efficient regulation of lsy-6 targetcog-1 in uterine cells. hrpk-1 mutants show modest to no reduction in mature miRNA abundance, suggesting that HRPK-1 function may not be globally required for miRNA biogenesis. Similarly, hrpk-1 activity is dispensable for mature ALG-1/AIN-1 miRISC formation. While we cannot rule out an HRPK-1 role in miRNA processing, we hypothesize that HRPK-1 primarily modulates miRISC activity at the level of target mRNAs and suggest several models for its activity.

Tabuse Y (1997) International C. elegans Meeting "LITHIUM CAUSES DEVELOPMENTAL DISORDER IN C. ELEGANS"

Tabuse Y

[International C. elegans Meeting, 1997]

Lithium (Li) has long been known to have teratogenic effects on the development of many organisms, including sea urchin, Xenopus and Dictyostelium. In Li-treated Xenopus embryos, ventral blastmeres are respecified to develop into dorsal structures, leading to dorsalized embryos lacking ventral mesodermal tissues. In Dictyostelium, Li alters the fate of prespore cells to become prestalk cells instead. Besides teratogenic effects, Li is also known to be a most effective treatment of manic-depressive illness.!@Although several models have been proposed to explain Li action, the molecular mechanism has remained unclear. Recently, GSK (glycogen synthase kinase)-3b was proposed to be a target of Li, suggesting that Li affects the wnt signaling pathway. To understand the mechanism of Li action, I first examined the effect of Li on C. elegans embryogenesis. I inoculated N2 animals at the late L4 stage onto NG plates containing 20 mM LiCl, incubated them at 20 oC. The number of eggs produced by treated animals was reduced to about half of the untreated control. Although cell division seemed to proceed, no embryos hatched on Li plates. Treated-embryos developed to produce gut granules, but did not execute normal morphogenesis at later embryonic stages. To identify genes involved in the action of Li, I have begun to screen for Li-resistant mutants that propagated on Li-containing medium. Several mutants were isolated, and one of them was mapped on the left side of unc-42 on chromosome V. On Li plates, the hatching rate of mutant eggs cross-fertilized by wild-type males was essentially the same as that for self-fertilized mutant eggs. On the contrary, no wild-type eggs cross-fertilized by mutant males hatched on Li-containing plates. So, this mutation appeared to be maternal. Further genetic analyses of the mutants and the observation on the cellular phenotype of Li-treated embryos are underway.

D Royal et al. (2001) International C. elegans Meeting "Towards the Cloning of Gene That Can Mutate to Confer Lithium Resistance In C. elegans"

J White, M Driscoll, E Hsieh, N Sirotin, M A Royal, D Royal, S Kwok, K O'Connell, B Bowerman

[International C. elegans Meeting, 2001]

Little is known about how ions permeate the C. elegans gut and cuticle. How various ions influence development and behavior is also not understood. One ion with considerable impact in human biology is Li + , which modulates bipolar disorder by an unknown mechanism. In particular, the targets of lithium that cause side effects remain to be identified. We are using C. elegans to genetically identify targets of lithium and to elaborate on mechanisms of transport, which may have an impact on human health. Li + has dosage-dependent effects on C. elegans embryos. When L4 animals mature on NGM plates with Li + added to the final concentrations of 10mM-20mM, they produce embryos that are unable to hatch. We demonstrated that this failure to hatch is due to defects in cytokinesis that result in multi-nucleated embryos and symmetrically partitioned cells. Li + also has a dosage-dependent effect on larval development. When adult hermaphrodites are permitted to lay embryos on 10mM-20mM Li + NGM plates, the resulting offspring experience a developmental delay proportional to the concentration of Li + . The offspring become progressively paralyzed as they reach adulthood. We demonstrated earlier that there is a delay in the entry into the S phase of the cell cycle larval stages. To learn more about the biology of Li + sensitivity, we screened for Li + resistant mutants. We developed three screens that took advantage of the embryonic arrest and the larval delay caused by Li + . We isolated one mutant bz71 , in a screen of 44,000 haploid genomes, that is resistant to both the larval and embryonic blocks of 16mM Li + . bz71 is dominant. Using classical mapping techniques, we positioned it on LGIII between unc-32 and dpy-18 . We are currently using SNP strategy to obtain a higher resolution map and we hope to report on the identification and cloning of this locus.

Hunter CP et al. (1991) International C. elegans Meeting "NON-AUTONOMY OF HER-1 FUNCTION IMPLICATES CELL INTERACTIONS IN C. ELEGANS SEX DETERMINATION."

Hunter CP, Wood WB

[International C. elegans Meeting, 1991]

Activity of the her-l gene is required for normal male development in C. elegans. Loss-of-function her-l mutations cause feminization of XO animals (normally male) to produce fertile hermaphrodites, while gain-of-function her-l mutations cause masculinization of XX animals ( normally hermaphrodite) to produce pseudo-males. Genetic analysis indicated that her-l functions early in the hierarchy of regulatory interactions among the major sex determination genes (1). We have analyzed her-l genetic mosaics to determine which cells must express her-l for wild-type male development. If her-l functions cell- autonomously, then in XO animals her-l (+) cells will follow male fates, and her-1(-) cells will follow hermaphrodite fates. If her-l or a her-l regulated activity functions non-autonomously, then the her-l genotype of a cell may not determine its sexual phenotype. The phenotypes of XO her-l genetic mosaics are variable, but clearly show that both her-1(-) and her-l (+) cells can follow either hermaphrodite or male fates. We conclude that her-l is neither necessary nor sufficient to cell-autonomously determine male sexual phenotypes and, therefore, that her-l or a her-l regulated activity must be able to function non-autonomously. The observed patterns of non-autonomous effects suggest that many or all cells express and respond to her-l activity. These findings implicate cell interactions in C. elegans sex determination. Earlier genetic analysis indicated that her-l might directly control tra-2 activity (1). Molecular characterization of the gene products of her-l and tra-2 (see abstracts by M. Perry et al. and P. Kuwabara et al) is consistent with a model in which a secreted ligand encoded by her-l interacts with a cell-surface receptor encoded by tra-2. We will discuss the possible role of such an interaction in coordinating the sex determination decision.

Ellis RE et al. (1991) International C. elegans Meeting "PROGRESS IN CLONING fog- 1"

Kimble JE, Ellis RE

[International C. elegans Meeting, 1991]

The fog-l gene plays an important role in the decision of germ cells to differentiate as either spermatocytes or oocytes (Doniach 1986, Barton and Kimble 1990). In fog-l mutants, all germ cells develop into oocytes. This is true for both males and hermaphrodites. However, these fog-l mutations do not appear to affect other aspects of sexual development. Furthermore, the fog-l mutant phenotype is not suppressed by mutations in any of the other genes known to control sex- determination. Thus fog-l might function to initiate spermatogenesis in response to other genes that regulate general sexdetermination. There are 42 fog-l mutations. These mutations arise at a frequency typical for knockout mutations in other C. elegans genes, and four of these fog-l alleles were isolated from a screen capable of recovering deletions. Thus it is likely that these mutations cause a loss of fog- l function. In order to study the mechanisms by which fog-l functions, and to determine how fog-l expression is regulated by the genes that control sex-determination, we are now cloning this gene. The fog-l gene is located on the left arm of LGI, between sup-ll and unc-ll, a region spanned by a large contig of about one megabase in size. By mapping DNA polymorphisms with respect to fog-l, we have narrowed down the region in which fog-l must be located, and are now injecting YAC DNA that covers this region in an attempt to rescue fog-l mutants.