Dietary-derived vitamin B12 protects Caenorhabditis elegans from thiol-reducing agents.

BACKGROUND: One-carbon metabolism, which includes the folate and methionine cycles, involves the transfer of methyl groups which are then utilised as a part of multiple physiological processes including redox defence. During the methionine cycle, the vitamin B12-dependent enzyme methionine synthetase converts homocysteine to methionine. The enzyme S-adenosylmethionine (SAM) synthetase then uses methionine in the production of the reactive methyl carrier SAM. SAM-binding methyltransferases then utilise SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. RESULTS: We describe a novel SAM methyltransferase, RIPS-1, which was the single gene identified from forward genetic screens in Caenorhabditis elegans looking for resistance to lethal concentrations of the thiol-reducing agent dithiothreitol (DTT). As well as RIPS-1 mutation, we show that in wild-type worms, DTT toxicity can be overcome by modulating vitamin B12 levels, either by using growth media and/or bacterial food that provide higher levels of vitamin B12 or by vitamin B12 supplementation. We show that active methionine synthetase is required for vitamin B12-mediated DTT resistance in wild types but is not required for resistance resulting from RIPS-1 mutation and that susceptibility to DTT is partially suppressed by methionine supplementation. A targeted RNAi modifier screen identified the mitochondrial enzyme methylmalonyl-CoA epimerase as a strong genetic enhancer of DTT resistance in a RIPS-1 mutant. We show that RIPS-1 is expressed in the intestinal and hypodermal tissues of the nematode and that treating with DTT, ß-mercaptoethanol, or hydrogen sulfide induces RIPS-1 expression. We demonstrate that RIPS-1 expression is controlled by the hypoxia-inducible factor pathway and that homologues of RIPS-1 are found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels. CONCLUSIONS: This work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans, defines a new role for this vitamin in countering reductive stress, and identifies RIPS-1 as a novel methyltransferase in the methionine cycle.

Fate specification and tissue-specific cell cycle control of the Caenorhabditis elegans intestine.

Coordination between cell fate specification and cell cycle control in multicellular organisms is essential to regulate cell numbers in tissues and organs during development, and its failure may lead to oncogenesis. In mammalian cells, as part of a general cell cycle checkpoint mechanism, the F-box protein beta-transducin repeat-containing protein (beta-TrCP) and the Skp1/Cul1/F-box complex control the periodic cell cycle fluctuations in abundance of the CDC25A and B phosphatases. Here, we find that the Caenorhabditis elegans beta-TrCP orthologue LIN-23 regulates a progressive decline of CDC-25.1 abundance over several embryonic cell cycles and specifies cell number of one tissue, the embryonic intestine. The negative regulation of CDC-25.1 abundance by LIN-23 may be developmentally controlled because CDC-25.1 accumulates over time within the developing germline, where LIN-23 is also present. Concurrent with the destabilization of CDC-25.1, LIN-23 displays a spatially dynamic behavior in the embryo, periodically entering a nuclear compartment where CDC-25.1 is abundant.

Combined extracellular matrix cross-linking activity of the peroxidase MLT-7 and the dual oxidase BLI-3 is critical for post-embryonic viability in Caenorhabditis elegans.

The nematode cuticle is a protective collagenous extracellular matrix that is modified, cross-linked, and processed by a number of key enzymes. This Ecdysozoan-specific structure is synthesized repeatedly and allows growth and development in a linked degradative and biosynthetic process known as molting. A targeted RNA interference screen using a cuticle collagen marker has been employed to identify components of the cuticle biosynthetic pathway. We have characterized an essential peroxidase, MoLT-7 (MLT-7), that is responsible for proper cuticle molting and re-synthesis. MLT-7 is an active, inhibitable peroxidase that is expressed in the cuticle-synthesizing hypodermis coincident with each larval molt. mlt-7 mutants show a range of body morphology defects, most notably molt, dumpy, and early larval stage arrest phenotypes that can all be complemented with a wild type copy of mlt-7. The cuticles of these mutants lacks di-tyrosine cross-links, becomes permeable to dye and accessible to tyrosine iodination, and have aberrant collagen protein expression patterns. Overexpression of MLT-7 causes mutant phenotypes further supporting its proposed enzymatic role. In combination with BLI-3, an H2O2-generating NADPH dual oxidase, MLT-7 is essential for post-embryonic development. Disruption of mlt-7, and particularly bli-3, via RNA interference also causes dramatic changes to the in vivo cross-linking patterns of the cuticle collagens DPY-13 and COL-12. This points toward a functionally cooperative relationship for these two hypodermally expressed proteins that is essential for collagen cross-linking and proper extracellular matrix formation.

Chapter 7. Nonsense-mediated mRNA decay in Caenorhabditis elegans.

The nonsense-mediated mRNA decay (NMD) pathway is a surveillance mechanism that targets the degradation of mRNAs harboring premature termination codons (PTCs). Two key aspects of NMD are the definition of a PTC codon and the identification of the molecular machinery dedicated to this mechanism. This chapter describes the development of transgenic reporters as well as the use of genome-wide RNAi and genetic screens to identify novel components of the NMD pathway in the nematode Caenorhabditis elegans.

The cuticle.

The nematode cuticle is an extremely flexible and resilient exoskeleton that permits locomotion via attachment to muscle, confers environmental protection and allows growth by molting. It is synthesised five times, once in the embryo and subsequently at the end of each larval stage prior to molting. It is a highly structured extra-cellular matrix (ECM), composed predominantly of cross-linked collagens, additional insoluble proteins termed cuticlins, associated glycoproteins and lipids. The cuticle collagens are encoded by a large gene family that are subject to strict patterns of temporal regulation. Cuticle collagen biosynthesis involves numerous co- and post-translational modification, processing, secretion and cross-linking steps that in turn are catalysed by specific enzymes and chaperones. Mutations in individual collagen genes and their biosynthetic pathway components can result in a range of defects from abnormal morphology (dumpy and blister) to embryonic and larval death, confirming an essential role for this structure and highlighting its potential as an ECM experimental model system.

Mechanistic insights and identification of two novel factors in the C. elegans NMD pathway.

The nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons (PTCs). Seven genes (smg-1-7, for suppressor with morphological effect on genitalia) that are essential for NMD were originally identified in the nematode Caenorhabditis elegans, and orthologs of these genes have been found in several species. Whereas in humans NMD is linked to splicing, PTC definition occurs independently of exon boundaries in Drosophila. Here, we have conducted an analysis of the cis-acting sequences and trans-acting factors that are required for NMD in C. elegans. We show that a PTC codon is defined independently of introns in C. elegans and, consequently, components of the exon junction complex (EJC) are dispensable for NMD. We also show a distance-dependent effect, whereby PTCs that are closer to the 3' end of the mRNA are less sensitive to NMD. We also provide evidence for the existence of previously unidentified components of the NMD pathway that, unlike known smg genes, are essential for viability in C. elegans. A genome-wide RNA interference (RNAi) screen resulted in the identification of two such novel NMD genes, which are essential for proper embryonic development, and as such represent a new class of essential NMD genes in C. elegans that we have termed smgl (for smg lethal). We show that the encoded proteins are conserved throughout evolution and are required for NMD in C. elegans and also in human cells.

Loss of SEC-23 in Caenorhabditis elegans causes defects in oogenesis, morphogenesis, and extracellular matrix secretion.

SEC-23 is a component of coat protein complex II (COPII)-coated vesicles involved in the endoplasmic reticulum-to-Golgi transport pathway of eukaryotes. During postembryonic life, Caenorhabditis elegans is surrounded by a collagenous exoskeleton termed the cuticle. From a screen for mutants defective in cuticle secretion, we identified and characterized a sec-23 mutant of C. elegans. By sequence homology, C. elegans has only the single sec-23 gene described herein. In addition to the cuticle secretion defect, mutants fail to complete embryonic morphogenesis. However, they progress through the earlier stages of embryogenesis, including gastrulation, and achieve substantial morphogenesis before death. We demonstrated a maternal component of SEC-23 function sufficient for progression through the earlier stages of embryogenesis and explaining the limited phenotype of the zygotic mutant. By RNA-mediated interference, we investigated the effects of perturbing COPII function during various postembryonic stages. During larval stages, major defects in cuticle synthesis and molting were observed. In the adult hermaphrodite, reduction of SEC-23 function by RNA-mediated interference caused a rapid onset of sterility, with defects in oogenesis including early maturation of the germline nuclei, probably a result of the observed loss of the GLP-1 receptor from the membrane surfaces adjacent to the developing germline nuclei.

An evolutionarily conserved role for SRm160 in 3'-end processing that functions independently of exon junction complex formation.

SRm160 (the SR-related nuclear matrix protein of 160 kDa) functions as a splicing coactivator and 3'-end cleavage-stimulatory factor. It is also a component of the splicing-dependent exon-junction complex (EJC), which has been implicated in coupling of pre-mRNA splicing with mRNA turnover and mRNA export. We have investigated whether the association of SRm160 with the EJC is important for efficient 3'-end cleavage. The EJC components RNPS1, REF, UAP56, and Y14 interact with SRm160. However, when these factors were tethered to transcripts, only SRm160 and RNPS1 stimulated 3'-end cleavage. Whereas SRm160 stimulated cleavage to a similar extent in the presence or absence of an active intron, stimulation of 3'-end cleavage by tethered RNPS1 is dependent on an active intron. Assembly of an EJC adjacent to the cleavage and polyadenylation signal in vitro did not significantly affect cleavage efficiency. These results suggest that SRm160 stimulates cleavage independently of its association with EJC components and that the cleavage-stimulatory activity of RNPS1 may be an indirect consequence of its ability to stimulate splicing. Using RNA interference (RNAi) in Caenorhabditis elegans, we determined whether interactions between SRm160 and the cleavage machinery are important in a whole organism context. Simultaneous RNAi of SRm160 and the cleavage factor CstF-50 (Cleavage stimulation factor 50-kDa subunit) resulted in late embryonic developmental arrest. In contrast, RNAi of CstF-50 in combination with RNPS1 or REFs did not result in an apparent phenotype. Our combined results provide evidence for an evolutionarily conserved interaction between SRm160 and the 3'-end cleavage machinery that functions independently of EJC formation.

M142.2 (cut-6), a novel Caenorhabditis elegans matrix gene important for dauer body shape.

The cuticle of the nematode Caenorhabditis elegans is a collagenous extracellular matrix which forms the exoskeleton and defines the shape of the worm. We have characterized the C. elegans gene M142.2, and we show that this is a developmentally regulated gene important for cuticle structure. Transgenic worms expressing M142.2 promoter fused to green fluorescent protein showed that M142.2 is expressed in late embryos and L2d predauers, in the hypodermal cells which synthesize the cuticle. The same temporal pattern was seen by RT-PCR using RNA purified from specific developmental stages. A recombinant fragment of M142.2 was expressed in Escherichia coli and used to raise an antiserum. Immunohistochemistry using the antiserum localized M142.2 to the periphery of the alae of L1 and dauers, forming two longitudinal ribbons over the hypodermal cells. Loss-of-function of M142.2 by RNAi resulted in a novel phenotype: dumpy dauers which lacked alae. M142.2 therefore plays a major role in the assembly of the alae and the morphology of the dauer cuticle; because of its similarity to the other cut genes of the cuticle, we have named the gene cut-6.

The Ref/Aly proteins are dispensable for mRNA export and development in Caenorhabditis elegans.

The mRNA export pathway is highly conserved throughout evolution. We have used RNA interference (RNAi) to functionally characterize bona fide RNA export factors and components of the exon-exon junction complex (EJC) in Caenorhabditis elegans. RNAi of CeNXT1/p15, the binding partner of CeNXF1/TAP, caused early embryonic lethality, demonstrating an essential function of this gene during C. elegans development. Moreover, depletion of this protein resulted in nuclear accumulation of poly(A)(+) RNAs, supporting a direct role of NXT1/p15 in mRNA export in C. elegans. Previously, we have shown that RNAi of CeSRm160, a protein of the EJC complex, resulted in wild-type phenotype; in the present study, we demonstrate that RNAi of CeY14, another component of this complex, results in embryonic lethality. In contrast, depletion of the EJC component CeRNPS1 results in no discernible phenotype. Proteins of the REF/Aly family act as adaptor proteins mediating the recruitment of the mRNA export factor, NXF1/TAP, to mRNAs. The C. elegans genome encodes three members of the REF/Aly family. RNAi of individual Ref genes, or codepletion of two Ref genes in different combinations, resulted in wild-type phenotype. Simultaneous suppression of all three Ref genes did not compromise viability or progression through developmental stages in the affected progeny, and only caused a minor defect in larval mobility. Furthermore, no defects in mRNA export were observed upon simultaneous depletion of all three REF proteins. These results suggest the existence of multiple adaptor proteins that mediate mRNA export in C. elegans.

Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix.

A ubiquitous feature of collagens is protein interaction, the trimerization of monomers to form a triple helix followed by higher order interactions during the formation of the mature extracellular matrix. The Caenorhabditis elegans cuticle is a complex extracellular matrix consisting predominantly of cuticle collagens, which are encoded by a family of approximately 154 genes. We identify two discrete interacting sets of collagens and show that they form functionally distinct matrix substructures. We show that mutation in or RNA-mediated interference of a gene encoding a collagen belonging to one interacting set affects the assembly of other members of that set, but not those belonging to the other set. During cuticle synthesis, the collagen genes are expressed in a distinct temporal series, which we hypothesize exists to facilitate partner finding and the formation of appropriate interactions between encoded collagens. Consistent with this hypothesis, we find for the two identified interacting sets that the individual members of each set are temporally coexpressed, whereas the two sets are expressed approximately 2 h apart during matrix synthesis.

Caenorhabditis elegans exoskeleton collagen COL-19: an adult-specific marker for collagen modification and assembly, and the analysis of organismal morphology.

The integral role that collagens play in the morphogenesis of the nematode exoskeleton or cuticle makes them a useful marker in the examination of the collagen synthesizing machinery. In this study, a green fluorescent protein-collagen fusion has been constructed by using the Caenorhabditis elegans adult-specific, hypodermally synthesized collagen COL-19. In wild-type nematodes, this collagen marker localized to the circumferential annular rings and the lateral trilaminar alae of the cuticle. Crosses carried out between a COL-19::GFP integrated strain and several morphologically mutant strains, including blister, dumpy, long, small, squat, and roller revealed significant COL-19 disruption that was predominantly strain-specific and provided a structural basis for the associated phenotypes. Disruption was most notable in the cuticle overlying the lateral seam cell syncytium, and confirmed the presence of two distinct forms of hypodermis, namely the circumferentially contracting lateral seam cells and the laterally contracting ventral-dorsal hypodermis. The effect of a single aberrant collagen being sufficient to mediate widespread collagen disruption was exemplified by the collagen mutant strain dpy-5 and its disrupted COL-19::GFP and DPY-7 collagen expression patterns. Through the disrupted pattern of COL-19 and DPY-7 in a thioredoxin mutant, dpy-11, and through RNA interference of a dual oxidase enzyme and a vesicular transport protein, we also show the efficacy of the COL-19::GFP strain as a marker for aberrant cuticle collagen synthesis and, thus, for the identification of factors involved in the construction of collagenous extracellular matrices.

Oncogenic potential of a C.elegans cdc25 gene is demonstrated by a gain-of-function allele.

In multicellular organisms, developmental programmes must integrate with central cell cycle regulation to co-ordinate developmental decisions with cell proliferation. Hyperplasia caused by deregulated proliferation without significant change to other aspects of developmental behaviour is a probable step towards full oncogenesis in many malignancies. CDC25 phosphatase promotes progression through the eukaryotic cell cycle by dephosphorylation of cyclin-dependent kinase and, in humans, different cdc25 family members have been implicated as potential oncogenes. Demonstrating the direct oncogenic potential of a cdc25 gene, we identify a gain-of-function mutant allele of the Caenorhabditis elegans gene cdc-25.1 that causes a deregulated proliferation of intestinal cells resulting in hyperplasia, while other aspects of intestinal cell function are retained. Using RNA-mediated interference, we demonstrate modulation of the oncogenic behaviour of this mutant, and show that a reduction of the wild-type cdc-25.1 activity can cause a failure of proliferation of intestinal and other cell types. That gain and loss of CDC-25.1 activity has opposite effects on cellular proliferation indicates its critical role in controlling C.elegans cell number.