Structure of the Caenorhabditis elegans m6A methyltransferase METT10 that regulates SAM homeostasis.

In Caenorhabditis elegans, the N6-methyladenosine (m6A) modification by METT10, at the 3'-splice sites in S-adenosyl-l-methionine (SAM) synthetase (sams) precursor mRNA (pre-mRNA), inhibits sams pre-mRNA splicing, promotes alternative splicing coupled with nonsense-mediated decay of the pre-mRNAs, and thereby maintains the cellular SAM level. Here, we present structural and functional analyses of C. elegans METT10. The structure of the N-terminal methyltransferase domain of METT10 is homologous to that of human METTL16, which installs the m6A modification in the 3'-UTR hairpins of methionine adenosyltransferase (MAT2A) pre-mRNA and regulates the MAT2A pre-mRNA splicing/stability and SAM homeostasis. Our biochemical analysis suggested that C. elegans METT10 recognizes the specific structural features of RNA surrounding the 3'-splice sites of sams pre-mRNAs, and shares a similar substrate RNA recognition mechanism with human METTL16. C. elegans METT10 also possesses a previously unrecognized functional C-terminal RNA-binding domain, kinase associated 1 (KA-1), which corresponds to the vertebrate-conserved region (VCR) of human METTL16. As in human METTL16, the KA-1 domain of C. elegans METT10 facilitates the m6A modification of the 3'-splice sites of sams pre-mRNAs. These results suggest the well-conserved mechanisms for the m6A modification of substrate RNAs between Homo sapiens and C. elegans, despite their different regulation mechanisms for SAM homeostasis.

Periostin Exon-21 Antibody Neutralization of Triple-Negative Breast Cancer Cell-Derived Periostin Regulates Tumor-Associated Macrophage Polarization and Angiogenesis.

Periostin (Pn) is involved in multiple processes of cancer progression. Previously, we reported that Pn expression is correlated with mesenchymal tumor markers and poor prognosis in triple-negative breast cancer (TNBC). In the TNBC xenograft model, chemotherapy increased expression of a Pn alternative splicing variant (ASV) with exon 21, and administration of the neutralizing antibody against Pn with exon 21 (Pn-21 Ab) overcame chemoresistance with a reduction in the mesenchymal cancer cell fraction. In the present study, the role of Pn ASV with exon 21 in TNBC progression has been addressed. We first established a stable cell line carrying a fluorescence-based splicing reporter. Pn-positive TNBC has higher expression of genes related to tumor-associated macrophage (TAM) recruitment and ECM-receptor interaction than Pn-negative cells. In a xenograft model, only Pn-positive cells initiated tumor formation, and the Pn-21 Ab suppressed tumor cell growth, accompanied by decreased M2 TAM polarization and the number of tumor vessels. These data suggest that cancer cell-derived Pn ASV educates TAMs and regulates angiogenesis, which in turn establishes a microenvironmental niche that is supportive of TNBC.

Loss of Caenorhabditis elegans homologue of human MOB4 compromises life span, health life span and thermotolerance.

Mob1/phocein family proteins are conserved from yeast to mammals. Human has four MOB genes, MOB1, 2, 3 and 4. Human MOB1 protein, which is a component of the Hippo pathway, is involved in the inhibition of yes-associated protein (YAP1) through large tumor suppressor (LATS) kinases and plays a tumor suppressive role. In contrast, MOB4 activates YAP1. Caernorhabditis elegans (C. elegans) also has four MOB genes. Moreover, C. elegans has homologues of YAP1 (Ce_YAP-1) and LATS kinases (WTS-1). Nevertheless, our previous study revealed that the Hippo pathway is not conserved in C. elegans and that heat shock activates Ce_YAP-1. We also reported that Ce_YAP-1 is involved in the regulation of life span, healthy lifespan and thermotolerance. In this study, we raised a question whether and how C. elegans homologue of MOB4 (Ce_MOB-4) is involved in the regulation of Ce_YAP-1. Ce_MOB-4 is ubiquitously expressed in adult worms. This expression pattern is similar to that of Ce_YAP-1. mob-4 loss-of-function mutants show short life span, short health life span and compromise thermotolerance. However, heat shock activates Ce_YAP-1 in mob-4 mutant. In conclusion, the role of MOB4 in the activation of YAP1 is not conserved in C. elegans.

m6 A-mediated alternative splicing coupled with nonsense-mediated mRNA decay regulates SAM synthetase homeostasis.

Alternative splicing of pre-mRNAs can regulate gene expression levels by coupling with nonsense-mediated mRNA decay (NMD). In order to elucidate a repertoire of mRNAs regulated by alternative splicing coupled with NMD (AS-NMD) in an organism, we performed long-read RNA sequencing of poly(A)+ RNAs from an NMD-deficient mutant strain of Caenorhabditis elegans, and obtained full-length sequences for mRNA isoforms from 259 high-confidence AS-NMD genes. Among them are the S-adenosyl-L-methionine (SAM) synthetase (sams) genes sams-3 and sams-4. SAM synthetase activity autoregulates sams gene expression through AS-NMD in a negative feedback loop. We furthermore find that METT-10, the orthologue of human U6 snRNA methyltransferase METTL16, is required for the splicing regulation in␣vivo, and specifically methylates the invariant AG dinucleotide at the distal 3' splice site (3'SS) in␣vitro. Direct RNA sequencing coupled with machine learning confirms m6 A modification of endogenous sams mRNAs. Overall, these results indicate that homeostasis of SAM synthetase in C. elegans is maintained by alternative splicing regulation through m6 A modification at the 3'SS of the sams genes.

UNC119 is a binding partner of tumor suppressor Ras-association domain family 6 and induces apoptosis and cell cycle arrest by MDM2 and p53.

Ras-association domain family 6 (RASSF6) is a tumor suppressor that interacts with MDM2 and stabilizes p53. Caenorhabditis elegans unc-119 encodes a protein that is required for normal development of the nervous system. Humans have 2 unc-119 homologues, UNC119 and UNC119B. We have identified UNC119 as a RASSF6-interacting protein. UNC119 promotes the interaction between RASSF6 and MDM2 and stabilizes p53. Thus, UNC119 induces apoptosis by RASSF6 and p53. UNC119 depletion impairs DNA repair after DNA damage and results in polyploid cell generation. These findings support that UNC119 is a regulator of the RASSF6-MDM2-p53 axis and functions as a tumor suppressor.

Tropomyosin isoforms differentially affect muscle contractility in the head and body regions of the nematode Caenorhabditis elegans.

Tropomyosin, one of the major actin filament-binding proteins, regulates actin-myosin interaction and actin-filament stability. Multicellular organisms express a number of tropomyosin isoforms, but understanding of isoform-specific tropomyosin functions is incomplete. The nematode Caenorhabditis elegans has a single tropomyosin gene, lev-11, which has been reported to express four isoforms by using two separate promoters and alternative splicing. Here, we report a fifth tropomyosin isoform, LEV-11O, which is produced by alternative splicing that includes a newly identified seventh exon, exon 7a. By visualizing specific splicing events in vivo, we find that exon 7a is predominantly selected in a subset of the body wall muscles in the head, while exon 7b, which is the alternative to exon 7a, is utilized in the rest of the body. Point mutations in exon 7a and exon 7b cause resistance to levamisole--induced muscle contraction specifically in the head and the main body, respectively. Overexpression of LEV-11O, but not LEV-11A, in the main body results in weak levamisole resistance. These results demonstrate that specific tropomyosin isoforms are expressed in the head and body regions of the muscles and contribute differentially to the regulation of muscle contractility.

Splicing factors control C. elegans behavioural learning in a single neuron by producing DAF-2c receptor.

Alternative splicing generates protein diversity essential for neuronal properties. However, the precise mechanisms underlying this process and its relevance to physiological and behavioural functions are poorly understood. To address these issues, we focused on a cassette exon of the Caenorhabditis elegans insulin receptor gene daf-2, whose proper variant expression in the taste receptor neuron ASER is critical for taste-avoidance learning. We show that inclusion of daf-2 exon 11.5 is restricted to specific neuron types, including ASER, and is controlled by a combinatorial action of evolutionarily conserved alternative splicing factors, RBFOX, CELF and PTB families of proteins. Mutations of these factors cause a learning defect, and this defect is relieved by DAF-2c (exon 11.5+) isoform expression only in a single neuron ASER. Our results provide evidence that alternative splicing regulation of a single critical gene in a single critical neuron is essential for learning ability in an organism.

Yes-associated protein homolog, YAP-1, is involved in the thermotolerance and aging in the nematode Caenorhabditis elegans.

The mammalian Hippo pathway comprises mammalian Ste20-like kinases (MST1/2) and large tumor suppressor kinases (LATS1/2). LATS1/2, which are activated by MST1/2, phosphorylate a transcriptional co-activator, yes-associated protein (YAP), and induce the recruitment of YAP by 14-3-3 to cytoplasm, so that the TEAD-dependent gene transcriptions are turned off. Although the core components of the Hippo pathway are well conserved in metazoans, it has been discussed that Caenorhabditis elegans lacks YAP ortholog, we found that F13E6.4 gene encodes a protein that shows sequence similarities to YAP in the N-terminal TEAD-binding domain and in the WW domain. We designated this gene as yap-1. YAP-1 is widely expressed in various cells such as epithelial cells, muscles, hypodermal cells, gonadal sheath cells, spermatheca, and hypodermal cells. YAP-1 is distributed in cytoplasm and nuclei. wts-1 (LATS ortholog) and ftt-2 (14-3-3 ortholog) knockdowns cause nuclear accumulation of YAP-1, supporting that the subcellular localization of YAP-1 is regulated in a similar way as that of YAP. Heat shock also causes the nuclear accumulation of YAP-1 but after heat shock, YAP-1 translocates to cytoplasm. Knockdowns of DAF-21 (HSP90 ortholog) and HSF-1block the nuclear export of YAP-1 during this recovery. YAP-1 overexpression is beneficial for thermotolerance, whereas YAP-1 hyperactivity induced by wts-1 and ftt-2 knockdowns is deleterious on thermal response and yap-1 deficiency promotes health aging. In short, YAP-1 partially shares basal characters with mammalian YAP and plays a role in thermal stress response and healthy aging.

Characterization of RSF-1, the Caenorhabditis elegans homolog of the Ras-association domain family protein 1.

Mammals have 10 RASSF proteins, which are characterized by the Ras-association (RA) domain. Among them, RASSF1 to RASSF6 have the Salvador/RASSF/Hippo (SARAH) domain and form the subclass C-terminal RASSF proteins. Drosophila genome has a single C-terminal RASSF, dRASSF. All these RASSF proteins are related to the tumor suppressive Hippo pathway, and are considered to function as tumor suppressors. Caenorhabditis elegans T24F1.3 encodes a protein with the RA and the SARAH domains. The amino acid sequences are 40% and 55% similar to those of RASSF1 in the RA and the SARAH domains, respectively. We have characterized T24F1.3 gene product and named it RSF-1 as RASSF1 homolog. RSF-1 is widely expressed in epithelial cells. About 14% rsf-1 mutants exhibit defects in embryonal morphogenesis and the actin disorganization. The combinatorial synthetic lethal analysis demonstrates that the lethality is enhanced to more than 80% in rsf-1 mutants with the WASP-family verprolin homologous protein complex-related gene depletions and corroborates the implication of RSF-1 in the regulation of actin cytoskeleton. In rsf-1 mutants, the structures of muscle actin are preserved, but the swimming ability is impaired. Although we could not detect the direct physical interaction of LET-60 with RSF-1, rsf-1 mutants suppress the multivulva phenotype of the active let-60 mutants, suggesting that rsf-1 genetically interacts with the Ras signaling.

Dual role of the carboxyl-terminal region of pig liver L-kynurenine 3-monooxygenase: mitochondrial-targeting signal and enzymatic activity.

l-kynurenine 3-monooxygenase (KMO) is an NAD(P)H-dependent flavin monooxygenase that catalyses the hydroxylation of l-kynurenine to 3-hydroxykynurenine, and is localized as an oligomer in the mitochondrial outer membrane. In the human brain, KMO may play an important role in the formation of two neurotoxins, 3-hydroxykynurenine and quinolinic acid, both of which provoke severe neurodegenerative diseases. In mosquitos, it plays a role in the formation both of eye pigment and of an exflagellation-inducing factor (xanthurenic acid). Here, we present evidence that the C-terminal region of pig liver KMO plays a dual role. First, it is required for the enzymatic activity. Second, it functions as a mitochondrial targeting signal as seen in monoamine oxidase B (MAO B) or outer membrane cytochrome b(5). The first role was shown by the comparison of the enzymatic activity of two mutants (C-terminally FLAG-tagged KMO and carboxyl-terminal truncation form, KMOΔC50) with that of the wild-type enzyme expressed in COS-7 cells. The second role was demonstrated with fluorescence microscopy by the comparison of the intracellular localization of the wild-type, three carboxyl-terminal truncated forms (ΔC20, ΔC30 and ΔC50), C-terminally FLAG-tagged wild-type and a mutant KMO, where two arginine residues, Arg461-Arg462, were replaced with Ser residues.