Yoshina S et al. (2022) MicroPubl Biol "Integration of multicopy extrachromosomal transgenes into defined loci without phenotypes"

Yoshina S, Mitani S

[MicroPubl Biol, 2022]

We show how presumably non-phenotypic loci can be used for integration sites of multi-copy extrachromosomal transgenes, using the CRISPR/Cas9 system. We used four loci, which show no apparent phenotype in our hands, as a model for any other loci with no phenotype.

Yoshina S et al. (2017) MicroPubl Biol "Novel deletion alleles of a C. elegans gene C38D4.9, named as tm4476 and tm4561"

Hori S, Suehiro Y, Yoshina S, Mitani S

[MicroPubl Biol, 2017]

We report tm4476 and tm4561 as novel alleles of C38D4.9 gene that is an ortholog of human METTL5 (methyltransferase like 5)1. Human METTL5 function has not been reported. The alleles were isolated from the comprehensive screening of gene deletions generated by TMP/UV2. In the screening, both the alleles were detected by nested PCR using the following primer sets, 5-CCGCCTATATCATGGCGCTT-3 and 5-GCTGGTAGTTGCCACCGCAT-3 for first round PCR and 5-ACTATCGAAGCCGCGCTTCA-3 and 5-AACCCCAACTCCGTCCCATT-3 for second round PCR. By Sanger sequencing, the 30 bp flanking sequences of the alleles tm4476 and tm4561 were identified as GTATGAGTTGGAGACTGTTTTAGGAGTTGA-(191 bp deletion)-GTACTCTTTAAAAGCGCACATCTTTCTGAA and TAATTGATATCGGATGTGGATGTGGAATGT-(309 bp deletion)-ATTTTCGGCTAAAAATCGTATTTGGGTAAT, respectively (Fig. 1). Based on the information about the splicing isoforms of C38D4.9 (WormBase3 WS259), the second exon of C38D4.9 is deleted in tm4476 and tm4561.

Yoshina S et al. (2012) Mol Biol Cell "Identification of a novel ADAMTS9/GON-1 function for protein transport from the ER to the Golgi."

Sakaki K, Mitani S, Yonezumi-Hayashi A, Inoue H, Gengyo-Ando K, Iino Y, Yoshina S

[Mol Biol Cell, 2012]

A disintegrin-like and metalloprotease with thrombospondin type I motif (ADAMTS9) is a member of the secreted metalloprotease family that is believed to digest extracellular matrix (ECM) proteins outside of cells. Its Caenorhabditis elegans orthologue, GON-1, is involved in ECM degradation and is required for gonad morphogenesis. ADAMTS9 and GON-1 have similar domain structures, and both have a unique C-terminal domain called the "GON domain," whose function remains unknown. Here we show that down-regulation of human ADAMTS9 and C. elegans GON-1 results in the inhibition of protein transport from the endoplasmic reticulum (ER) to the Golgi. This phenotype was rescued by the expression of the GON domain localizing in the ER in human cells and C. elegans. We propose a novel function of ADAMTS9 and GON-1 in the ER that promotes protein transport from the ER to the Golgi. This function is GON-domain dependent but protease activity independent.

Yoshina S et al. (2023) J Physiol Sci "Febuxostat ameliorates muscle degeneration and movement disorder of the dystrophin mutant model in Caenorhabditis elegans."

Mashima R, Mitani S, Maejima Y, Izuhara L, Kamatani N, Yoshina S

[J Physiol Sci, 2023]

Duchenne muscular dystrophy (DMD) is an inherited disorder with mutations in the dystrophin gene characterized by progressive muscle degeneration and weakness. Therapy such as administration of glucocorticoids, exon skipping of mutant genes and introduction of dystrophin mini-genes have been tried, but there is no radical therapy for DMD. In this study, we used C. elegans carrying mutations in the dys-1 gene as a model of DMD to examine the effects of febuxostat (FBX). We applied FBX to dys-1 mutant animals harboring a marker for muscle nuclei and mitochondria, and found that FBX ameliorates the muscle loss. We next used a severer model dys-1; unc-22 double mutant and found the dys-1 mutation causes a weakened muscle contraction. We applied FBX and other compounds to the double mutant animals and assayed the movement. We found that the administration of FBX in combination of uric acid has the best effects on the DMD model.

Yoshina S et al. (2016) Biochem Biophys Rep "Locus-specific integration of extrachromosomal transgenes in C. elegans with the CRISPR/Cas9 system."

Mitani S, Suehiro Y, Kage-Nakadai E, Yoshina S

[Biochem Biophys Rep, 2016]

We established a method to generate integration from extrachromosomal arrays with the CRISPR/Cas9 system. Multi-copy transgenes were integrated into the defined loci of chromosomes by this method, while a multi-copy transgene is integrated into random loci by previous methods, such as UV- and gamma-irradiation. The effects of a combination of sgRNAs, which define the cleavage sites in extrachromosomes and chromosomes, and the copy number of potential cleavable sequences were examined. The relative copy number of cleavable sequences in extrachromosomes affects the frequency of fertile F1 transgenic animals. The expression levels of the reporter gene were almost proportional to the copy numbers of the integrated sequences at the same integration site. The technique is applicable to the transgenic strains abundantly stored and shared among the C. elegans community, particularly when researchers use sgRNAs against common plasmid sequences such as -lactamase.

Yoshina S et al. (2015) PLoS One "Loss of C. elegans GON-1, an ADAMTS9 Homolog, Decreases Secretion Resulting in Altered Lifespan and Dauer Formation."

Yoshina S, Mitani S

[PLoS One, 2015]

ADAMTS9 is a metalloprotease that cleaves components of the extracellular matrix and is also implicated in transport from the endoplasmic reticulum (ER) to the Golgi. It has been reported that an ADAMTS9 gene variant is associated with type 2 diabetes. The underlying pathology of type 2 diabetes is insulin resistance and beta-cell dysfunction. However, the molecular mechanisms underlying ADAMTS9 function in beta cells and peripheral tissues are unknown. We show that loss of C. elegans GON-1, an ADAMTS9 homolog, alters lifespan and dauer formation. GON-1 loss impairs secretion of proteins such as insulin orthologs and TGF-beta, and additionally impacts insulin/IGF-1 signaling in peripheral tissues. The function of the GON domain, but not the protease domain, is essential for normal lifespan and dauer formation in these scenarios. We conclude that the GON domain is critical for ADAMTS9/GON-1 function across species, which should help the understanding of type 2 diabetes in humans.

Vinci CR et al. (2007) J Biol Chem "Recognition of age-damaged (R,S)-adenosyl-L-methionine by two methyltransferases in the yeast Saccharomyces cerevisiae."

Vinci CR, Clarke SG

[J Biol Chem, 2007]

The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.

Iwata, S. et al. (2015) International Worm Meeting "Genetic engineering of new balancers using by CRISPR/Cas9 genome-editing system in Caenorhabditis elegans."

Mitani, S., Iwata, S., Yoshina, S.

[International Worm Meeting, 2015]

Genetic balancers have been used as a tool to efficiently stabilize lethal or sterile mutations as heterozygotes. However, despite the usage of genetic balancers was begun 30 years ago, 20% of the chromosomal regions have not been covered yet. Because, the uncovered regions are mainly comprised of chromosomal regions around pairing centers (PCs) that contributes to the process of homologous pairing and synapsis, it is assumed that generation of the genetic rearrangements such as inversion and translocation are difficult in the PCs. In the present study, we established a methodology for the engineering of the genetic new balancers based on the ability of the CRISPR/Cas9 genome-editing system. We generated these genetic rearrangements generated by homologous recombination (HR) between the targeted regions and homology vectors. We demonstrate that inactivation of non-homologous end-joining (NHEJ) using by a lig-4 mutant helps efficient rearrangements in the targeted regions. Using this approach we isolated new balancers named tmIn1 (IV), tmIn2 (IV) and tmIn3 (IV) that significantly balanced the lethal mutations. The inversion balancer tmIn3 (IV) extensively suppresses recombination in the left arm of chromosome IV from jtr-1 to unc-17, covering about 2.2 Mb. The balancer causes larval arrest in homozygotes but allows animals to be fertile when heterozygous. Further screening and the genetic analysis of chromosome II, we generated a balancer tmIn4 (II). It balances left arm of chromosome II from lin-8 to dpy-2, covering about 3.6 Mb. Homozygous tmIn4 (II) animals are viable that exhibit a dumpy phenotype, whereas heterozygoutes show the wild-type phenotype. The present findings will make it essential for researchers to maintain lethal or sterile mutations on the chromosome II and IV and open the door for development of an efficient system to generate rearrangements at specific sites of interest to model disease mechanisms.

Fanning S et al. (2018) Mol Cell "Lipidomic Analysis of -Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment."

Lou Y, Haque A, Freyzon Y, Farese RV, Terry-Kantor E, Hofbauer HF, Termine D, Welte MA, Barrasa MI, Imberdis T, Noble T, Lindquist S, Clish CB, Jaenisch R, Pincus D, Nuber S, Sandoe J, Kohlwein SD, Kim TE, Ho GPH, Ramalingam N, Walther TC, Baru V, Selkoe D, Srinivasan S, Landgraf D, Soldner F, Dettmer U, Fanning S, Becuwe M, Newby G

[Mol Cell, 2018]

In Parkinson's disease (PD), -synuclein (S) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in S or lipid/fattyacid homeostasis affect each other. Lipidomic profiling of human S-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of S dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased S yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in S-overexpressing rat neurons. In a C.elegans model, SCD knockout prevented S-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on S homeostasis: in human neural cells, excess OA caused S inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for S-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.

Vandecandelaere I et al. (2017) PLoS One "Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus."

Deforce D, Coenye T, Van Nieuwerburgh F, Vandecandelaere I

[PLoS One, 2017]

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved.