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. (2003) Genome Res "OrthoMCL: identification of ortholog groups for eukaryotic genomes."

Stoeckert CJ, Li L, Roos DS

[Genome Res, 2003]

The identification of orthologous groups is useful for genome annotation, studies on gene/protein evolution, comparative genomics, and the identification of taxonomically restricted sequences. Methods successfully exploited for prokaryotic genome analysis have proved difficult to apply to eukaryotes, however, as larger genomes may contain multiple paralogous genes, and sequence information is often incomplete. OrthoMCL provides a scalable method for constructing orthologous groups across multiple eukaryotic taxa, using a Markov Cluster algorithm to group (putative) orthologs and paralogs. This method performs similarly to the INPARANOID algorithm when applied to two genomes, but can be extended to cluster orthologs from multiple species. OrthoMCL clusters are coherent with groups identified by EGO, but improved recognition of "recent" paralogs permits overlapping EGO groups representing the same gene to be merged. Comparison with previously assigned EC annotations suggests a high degree of reliability, implying utility for automated eukaryotic genome annotation. OrthoMCL has been applied to the proteome data set from seven publicly available genomes (human, fly, worm, yeast, Arabidopsis, the malaria parasite Plasmodium falciparum, and Escherichia coli). A Web interface allows queries based on individual genes or user-defined phylogenetic patterns (http://www.cbil.upenn.edu/gene-family). Analysis of clusters incorporating P. falciparum genes identifies numerous enzymes that were incompletely annotated in first-pass annotation of the parasite genome.

Li L et al. (2024) Nature "The HEAT repeat protein HPO-27 is a lysosome fission factor."

Zhao Q, Feng W, Ma X, Zhang Q, Liu X, Wang X, Liu Y, Hu S, Li M, Yang C, Wang W, Li D, Wu Y, Li L, Yang S, Zhang J, Hu J, Jiang A

[Nature, 2024]

Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing¹. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission^2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function.

Li L et al. (2015) Inorg Chem "A Molecular Chameleon with Fluorescein and Rhodamine Spectroscopic Behaviors."

Li L, Cai YP, Wu J, Wang C, Wong KM, Tse YC

[Inorg Chem, 2015]

A new class of fluorescein/rhodamine hybrids with two spirolactone rings was reported to exhibit dual-output fluorescent behaviors independently. Isolation and characterization for two diastereomers, trans-RhOH and cis-RhOH, have been made and their X-ray crystal structures determined. In a basic environment, the spirolactone ring on the hydroxyl side will be opened to give a fluorescein-like optical output with the lowest absorptions at 485 and 530 nm emission. On the other hand, a rhodamine-like optical output, i.e., 528 nm absorption and 575 nm emission, will be switched on by a H(+) or a Hg(2+) ion, attributed to the spirolactone ring opening on the amino side. In a methanol-buffer system with different pH values, the corresponding pKa values for the hydroxyl and amino groups were determined as 5.7 and 2.3, respectively. Selective Hg(2+)-sensing properties have also been discussed, and log Ks values of about 3.60 and 3.73 were determined. Confocal microscopic images of Caenorhabditis elegans incubated with RhOH were found to show enhanced fluorescent intensity with a Hg(2+) ion, demonstrating the potential application of RhOH for in vivo biological imaging.

Li L et al. (2020) RNA "A convenient strategy to clone small RNA and mRNA for high-throughput sequencing."

Nguyen AP, Dai H, Li L, Gu W

[RNA, 2020]

High-throughput sequencing has become a standard tool for analyzing RNA and DNA. This method usually needs a cDNA/DNA library ligated with specific 5' and 3' linkers. Unlike mRNA, small RNA often contains modifications including 5' cap or triphosphate and 2'-<i>O</i>-methyl, requiring additional processing steps before linker additions during cloning processes; due to low expression levels, it is difficult to clone small RNA with a small amount of total RNA. Here we present a new strategy to clone 5' modified or unmodified small RNA in an all-liquid-based reaction carried out in a single PCR tube with as little as 20 ng total RNA. The 7-h cloning process only needs 1 h of labor. Moreover, this method can also clone mRNA, simplifying the need to prepare two cloning systems for small RNA and mRNA; the barcoded PCR primers are also compatible with non-cDNA cloning applications, including the preparation of genomic libraries. Not only is our method more convenient for cloning modified RNA than available methods, but it is also more sensitive, versatile, and cost-effective. Moreover, the all-liquid-based reaction can be performed in an automated manner.

Li L et al. (2020) J Vis Exp "Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans."

Li L, Zinovyeva AY

[J Vis Exp, 2020]

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. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitateC. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

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. (2015) Cell Biol Int "The effect of the size of fluorescent dextran on its endocytic pathway."

Li L, Wan T, Zhang R, Wan M, Liu B, Cheng R

[Cell Biol Int, 2015]

Fluorescent dextrans are commonly used as macropinocytic probes to study the properties of endocytic cargoes; however, the effect of the size of dextrans on endocytic mechanisms has not been carefully analyzed. By using chemical and siRNA inhibition of individual endocytic pathways, we evaluated the internalization of two commonly used dextrans, Dex10 (dextran 10kDa) and Dex70 (dextran 70kDa), in mammalian HeLa cells and Caenorhabditis elegans coelomocytes. We revealed that Dex70 enters these two cell types predominantly via clathrin- and dynamin-independent and amiloride-sensitive macropinocytosis process; Dex10, on the other hand, enters the two cell types through clathrin-/dynamin-dependent micropinocytosis in addition to macropinocytosis. In addition, although different-sized dextrans follow different endocytic processes, they share common post-endocytic events. Herein, though straightforward, our studies support that the size of nanomaterials could play a paramount role in their inclusion into endocytic vesicles and suggest that care should be taken while selecting endocytic pathway markers. Based on our results, we propose that Dex70 is a better probe for macropinocytosis, whereas Dex10 and smaller molecules are better for probing general fluid-phase endocytosis, which includes macropinocytic and micropinocytic processes.

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.)