RNA and sex determination in Caenorhabditis elegans. Post-transcriptional regulation of the sex-determining tra-2 and fem-3 mRNAs in the Caenorhabditis elegans hermaphrodite.

The Caenorhabditis elegans hermaphrodite sequentially produces sperm and oocytes from a single pool of precursors. Therefore, the hermaphrodite's germ line is the site of two major cell fate decisions: a germ cell precursor first undergoes a mitosis/meiosis decision and then a sperm/oocyte decision. While the mitosis/meiosis decision is governed by Notch/GLP-1 signalling, the sperm/oocyte decision relies on post-transcriptional regulation of two key mRNAs, tra-2 and fem-3. This review focuses on factors that are required for the silencing of these mRNAs, which results in the sequential production of sperm and oocytes. Most factors that regulate the expression of tra-2 and fem-3 are homologous to proteins involved in RNA regulation in yeast, mammals or Drosophila, suggesting that at least some of the molecular mechanisms regulating the two worm mRNAs have been conserved throughout evolution.

The hermaphrodite sperm/oocyte switch requires the Caenorhabditis elegans homologs of PRP2 and PRP22.

Sex determination in the hermaphrodite germ line of Caenorhabditis elegans is controlled posttranscriptionally. The switch from spermatogenesis to oogenesis relies on regulation of the fem-3 sex-determining gene via a regulatory element in the fem-3 3' untranslated region. Previous work showed that at least six mog genes are required for repression by the fem-3 3' untranslated region, and that one of those genes, mog-1, encodes a DEAH-box protein. In this paper, we report the cloning of mog-4 and mog-5 and the finding that mog-4 and mog-5 also encode DEAH-box proteins. Our molecular identification of mog-4 and mog-5 relied on genetic mapping and transformation rescue and was confirmed by a missense mutation in each gene. A phylogenetic analysis revealed that the C. elegans MOG-1, MOG-4, and MOG-5 proteins are closely related to the yeast proteins PRP16, PRP2, and PRP22, respectively. In view of their effect on fem-3 regulation and their homology to PRP16, PRP2, and PRP22, we propose that MOG-1, MOG-4, and MOG-5 are required for posttranscriptional regulation, perhaps by modifying the conformation of ribonucleoprotein complexes.

Splicing factor SF1 from Drosophila and Caenorhabditis: presence of an N-terminal RS domain and requirement for viability.

Splicing factor SF1 contributes to the recognition of the 3' splice site by interacting with U2AF65 and binding to the intron branch site during the formation of the early splicing complex E. These interactions and the essential functional domains of SF1 are highly conserved in Saccharomyces cerevisiae. We have isolated cDNAs encoding SF1 from Drosophila (Dm) and Caenorhabditis (Ce). The encoded proteins share the U2AF65 interaction domain, a hnRNP K homology domain, and one or two zinc knuckles required for RNA binding as well as Pro-rich C-terminal sequences with their yeast and mammalian counterparts. In contrast to SF1 in other species, DmSF1 and CeSF1 are characterized by an N-terminal region enriched in Ser, Arg, Lys, and Asp residues with homology to the RS domains of several splicing proteins. These domains mediate protein-protein or protein-RNA interactions, suggesting an additional role for DmSF1 and CeSF1 in pre-mRNA splicing. Human (Hs), fly, and worm SF1 interact equally well with HsU2AF65 or the Drosophila homolog DmU2AF50. Moreover, DmSF1 lacking its N terminus is functional in prespliceosome formation in a HeLa splicing system, emphasizing the conserved nature of interactions at an early step in spliceosome assembly. The Ce-SF1 gene is located in a polycistronic transcription unit downstream of the genes encoding U2AF35 (uaf-2) and a cyclophilin (cyp-13), implying the coordinate transcriptional regulation of these genes. Injection of double-stranded RNA into C. elegans results in embryonic lethality; thus, the SF1 gene is essential not only in yeast but also in at least one metazoan.

The Caenorhabditis elegans sex determination gene mog-1 encodes a member of the DEAH-Box protein family.

In the Caenorhabditis elegans hermaphrodite germ line, the sex-determining gene fem-3 is repressed posttranscriptionally to arrest spermatogenesis and permit oogenesis. This repression requires a cis-acting regulatory element in the fem-3 3' untranslated region; the FBF protein, which binds to this element; and at least six mog genes. In this paper, we report the molecular characterization of mog-1 as well as additional phenotypic characterization of this gene. The mog-1 gene encodes a member of the DEAH-box family. Three mog-1 alleles possess premature stop codons and are likely to be null alleles, and one is a missense mutation and is likely to retain residual activity. mog-1 mRNA is expressed in both germ line and somatic tissues and appears to be ubiquitous. The MOG-1 DEAH-box protein is most closely related to proteins essential for splicing in the yeast Saccharomyces cerevisiae, but splicing appears to occur normally in a mog-1-null mutant. In addition to its involvement in the sperm-oocyte switch and control of fem-3, zygotic mog-1 is required for robust germ line proliferation and for normal growth during development. We suggest that mog-1 plays a broader role in RNA regulation than previously considered.

A conserved RNA-binding protein that regulates sexual fates in the C. elegans hermaphrodite germ line.

The nematode Caenorhabditis elegans has two sexes, males and hermaphrodites. Hermaphrodites Initially produce sperm but switch to producing oocytes. This switch appears to be controlled by the 3' untranslated region of fem-3 messenger RNA. We have now identified a binding factor (FBF) which is a cytoplasmic protein that binds specifically to the regulatory region of fem-3 3'UTR and mediates the sperm/oocyte switch. The RNA-binding domain of FBF consists of a stretch of eight tandem repeats and two short flanking regions. This structural element is conserved in several proteins including Drosophila Pumilio, a regulatory protein that controls pattern formation in the fly by binding to a 3'UTR. We propose that FBF and Pumilio are members of a widespread family of sequence-specific RNA-binding proteins.

Early effect of aldosterone on the rate of synthesis of the epithelial sodium channel alpha subunit in A6 renal cells.

Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. The amiloride-sensitive epithelial sodium channel (ENaC) is a major target for the natriferic action of aldosterone. In this study, the effect of aldosterone on ENaC mRNA abundance and the rate of protein synthesis for each of the three ENaC subunits (alpha, beta and gamma) in the A6 kidney cell line were examined. In cells grown on plastic, aldosterone induced a large and rapid increase in epithelial sodium channel (ENaC) beta and gamma subunit mRNA abundance, but this effect is not translated into the synthesis of the corresponding proteins. In cells grown on a porous substrate, amiloride-sensitive electrogenic sodium transport was expressed and was upregulated by aldosterone (300 nM) as early as 1 h after the addition of the hormone. The alpha, beta, and gamma mRNA abundance was not changed by aldosterone during the first 3 h of stimulation, whereas a fourfold increase over control was observed after 24 h. The rate of synthesis of alpha subunit was significantly increased above control already 60 min after aldosterone addition, whereas beta subunit synthesis increased only 6 h after hormone addition, with no significant change for the gamma subunit. The half-lives of each subunit as assessed by 35S methionine pulse-chase experiments were short (between 40 and 50 min) and were not modified by aldosterone. Taking into account the short half-life of ENaC protein and assuming that the synthesis of the alpha subunit is a limiting factor in the assembly and expression of new channels at the cell surface, it is proposed that the aldosterone regulation of sodium transport might be, in part, mediated by de novo synthesis of the channel protein.

The gamma subunit is a specific component of the Na,K-ATPase and modulates its transport function.

The role of small, hydrophobic peptides that are associated with ion pumps or channels is still poorly understood. By using the Xenopus oocyte as an expression system, we have characterized the structural and functional properties of the gamma peptide which co-purifies with Na,K-ATPase. Immuno-radiolabeling of epitope-tagged gamma subunits in intact oocytes and protease protection assays show that the gamma peptide is a type I membrane protein lacking a signal sequence and exposing the N-terminus to the extracytoplasmic side. Co-expression of the rat or Xenopus gamma subunit with various proteins in the oocyte reveals that it specifically associates only with isozymes of Na,K-ATPase. The gamma peptide does not influence the formation and cell surface expression of functional Na,K-ATPase alpha-beta complexes. On the other hand, the gamma peptide itself needs association with Na,K-ATPase in order to be stably expressed in the oocyte and to be transported efficiently to the plasma membrane. Gamma subunits do not associate with individual alpha or beta subunits but only interact with assembled, transport-competent alpha-beta complexes. Finally, electrophysiological measurements indicate that the gamma peptide modulates the K+ activation of Na,K pumps. These data document for the first time the membrane topology, the specificity of association and a potential functional role for the gamma subunit of Na,K-ATPase.

Novel isoforms of the beta and gamma subunits of the Xenopus epithelial Na channel provide information about the amiloride binding site and extracellular sodium sensing.

We have previously identified three homologous subunits alpha, beta, and gamma of the highly selective amiloride-sensitive Na channel from the Xenopus laevis kidney A6 cell line, which forms a tight epithelium in culture. We report here two novel genes, termed beta2 and gamma2, which share 90 and 92% sequence identity with the previously characterized beta and gamma XENaC, respectively. beta2 and gamma2 transcripts were detected in lung, kidney, and A6 cells grown on porous substrate. The physiological and pharmacological profile of the Na channel expressed after alphabeta2gamma XENaC cRNA injection in Xenopus oocyte did not differ from alphabetagamma XENaC. By contrast, the channel expressed after alphabetagamma2 injection showed: (i) a lower maximal amiloride-sensitive sodium current, (ii) a higher apparent affinity for external sodium and inactivation of the sodium current by high sodium concentrations, and (iii) a lower apparent affinity for amiloride (KI alphabetagamma2; 1.34 microM versus alphabetagamma 0.35 microM). These data indicate that the gamma (and/or gamma2) subunit participates in amiloride binding and the sensing of the extracellular sodium concentration. The close homology between gamma and gamma2 will help to define the domains involved in sensing external sodium and in the structure of this important drug receptor.

Biosynthesis of the side chain of yeast glycosylphosphatidylinositol anchors is operated by novel mannosyltransferases located in the endoplasmic reticulum and the Golgi apparatus.

Glycosylphosphatidylinositol (GPI) anchors of the yeast Saccharomyces cerevisiae have been reported to contain three different types of side chains attached to contain three different types of side chains attached to the alpha 1,2-linked mannose of the conserved protein-ethanolamine-PO4-Man alpha 1,2Man alpha 1,6Man alpha 1,4GlcNH2-inositol glycan core. The possible side chains are Man alpha 1,2- or Man alpha 1,2Man alpha 1,2- or Man alpha 1,3Man alpha 1,2- (Fankhauser, C., Homan, S. W., Thomas Oates, J. E., McConville, M. J., Desponds, C., Conzelmann, A., and Ferguson, M. A. (1993) J. Biol. Chem. 268, 26365-26374). To determine in what subcellular compartment these side chains are made, we metabolically labeled GPI-anchored membrane proteins with myo-[2-3H]inositol in secretion mutants blocked at various stages of the secretory pathway and analyzed the anchor structure of the labeled glycoproteins. When the exit of vesicles from the endoplasmic reticulum or entry into the cis-Golgi were blocked in sec12 or sec18 cells, all anchors contained a side chain consisting of a single alpha 1,2-linked mannose. GPI proteins trapped in the cis-Golgi of sec7 contained Man alpha 1,3Man alpha 1,2- but no Man alpha 1,2Man alpha 1,2-side chains. Mutants blocked at later stages of the secretory pathway made increased amounts of side chains containing two mannoses. Man alpha 1,2Man alpha 1,2- and Man alpha 1,3Man alpha 1,2- side chains were preferentially associated with ceramide- and diacylglycerol-containing GPI anchors, respectively. Mnn1, mnn2, mnn3, mnn5, and mnt1(=kre2), i.e. mutants which lack or down-regulate 1,2- and 1,3- mannosyltransferases used in the elongation of N- and O-glycans in the Golgi, add the fifth mannose to GPI anchors normally. The same conclusion was reached through the analysis of deletion mutants in KTR1, KTR2, KTR3, KTR4, and YUR1 which all are open reading frames with high homology to MNT1. Mutants deficient in the Golgi elongation of N-glycans such as anp1, van1, mnn9 are deficient in the maturation of the N-glycans of GPI-anchored glycoproteins, but process the GPI anchor side chain normally. Data are consistent with the idea that the fourth mannose is added to proteins as part of the anchor precursor glycolipid in the endoplasmic reticulum, whereas the fifth mannose is added by not yet identified alpha 1,3- and alpha 1,2-mannosyltransferases located in the Golgi apparatus.

The highly selective low-conductance epithelial Na channel of Xenopus laevis A6 kidney cells.

In Na-reabsorbing tight epithelia, the rate-limiting step for Na transport is the highly selective low-conductance amiloride-sensitive epithelial Na channel (type 1 ENaC). In rat distal colon, type 1 ENaC is made of three homologous subunits. The aim of this study was to identify the corresponding genes of the renal channel from the kidney-derived A6 cell line of Xenopus laevis. Three homologous subunits were identified and coexpressed in the Xenopus oocyte system. The reconstituted channel had all the characteristics of the native type 1 ENaC described in A6 cells: 1) high selectivity, 2) low single-channel conductance, 3) slow gating kinetics, and 4) high affinity for amiloride. Transcripts for alpha-, beta-, and gamma-subunits of the Xenopus epithelial Na channel (xENaC) were detected in A6 kidney cells, Xenopus kidney, lung, and to a lesser extent in stomach and skin. Each subunit of the xENaC shares approximately 60% overall identity with the corresponding rat homologue (alpha, beta, and gamma rENaC). Our data suggest that the triplication of the ENaC subunits occurred before the divergence between mammalian and amphibian lineages.

Glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae: absence of ceramides from complete precursor glycolipids.

Glycosylphosphatidylinositol (GPI) anchoring of membrane proteins occurs through two distinct steps, namely the assembly of a precursor glycolipid and its subsequent transfer onto newly synthesized proteins. To analyze the structure of the yeast precursor glycolipid we made use of the pmi40 mutant that incorporates very high amounts of [3H]mannose. Two very polar [3H]mannose-labeled glycolipids named CP1 and CP2 qualified as GPI precursor lipids since their carbohydrate head group, Man alpha 1,2(X-->PO4-->6)Man alpha 1,2Man alpha 1,6Man alpha-GlcN-inositol (with X most likely being ethanolamine) comprises the core structure which is common to all GPI anchors described so far. CP1 predominates in cells grown at 24 degrees C whereas CP2 is induced by stress conditions. The apparent structural identity of the head groups suggests that CP1 and CP2 contain different lipid moieties. The lipid moieties of both CP1 and CP2 can be removed by mild alkaline hydrolysis although the protein-bound GPI anchors made by the pmi40 cells under identical labeling conditions contain mild base resistant ceramides. These findings imply that the ceramide moiety found on the majority of yeast GPI anchored proteins is added through a lipid remodeling step that occurs after the addition of the GPI precursor glycolipids to proteins.

Characterization of abnormal free glycophosphatidylinositols accumulating in mutant lymphoma cells of classes B, E, F, and H.

Several mutant lymphoma lines are unable to add glycophosphatidylinositol membrane anchors to proteins. Some of them accumulate abnormal glycolipids which can be labeled by tritiated myo-inositol, mannose, or ethanolamine and which are not present in the corresponding parental cell lines. The [3H]myo-inositol-labeled abnormal lipids were isolated and characterized using hydrofluoric acid dephosphorylation, nitrous acid deamination, acetolysis, and exoglycosidase treatments alone or in combination. This partial characterization suggests that the class F mutant EL-4-f contains 3 abnormal glycolipids containing 3, 2, or 1 mannose residues, the headgroups of which are Man alpha 1,2Man alpha 1,6(X-->)Man alpha-GlcN-acylinositol, Man alpha 1,6(X-->)Man alpha-GlcN-inositol, and (X-->)Man alpha-GlcN-acylinositol where X represents a charged, hydrofluoric acid-sensitive substituent. A fourth, minor abnormal lipid with a Man alpha 1,6(X-->)Man alpha-GlcN-inositol headgroup but a different lipid moiety is also found. The substituent X is likely to consist of phosphoethanolamine since hydrofluoric acid releases [3H]ethanolamine from the [3H]ethanolamine-labeled version of these lipids. Pulse-chase experiments indicate that the abnormal glycophosphatidylinositols of class F mutants are very long-lived. The class B mutant S1A-b has previously been shown to contain an abnormal Man alpha 1,6(phosphoethanolamine-->)Man alpha-GlcN-acylinositol-P-lipid intermediate. Here we show that S1A-b also accumulates a more polar but less abundant lipid which has the identical headgroup structure but lacks the acyl group on the inositol residue. The class E mutant BW5147-e accumulates a hydrophobic glycolipid with the headgroup structure GlcN-acylinositol. All the abnormal glycolipids except those of EL-4-f are heterogeneous with regard to their lipid moiety since base-resistant as well as base-sensitive lipids are present. This suggests that the base-resistant alkylglycerols typical of mammalian anchors can get integrated into anchors at early stages of glycophosphatidylinositol formation.

Structural characterization of free glycolipids which are potential precursors for glycophosphatidylinositol anchors in mouse thymoma cell lines.

Biosynthesis of glycophosphatidylinositol-anchored membrane glycoproteins proceeds through the attachment of a preformed glycolipid onto a C-terminal amino acid rapidly after translation. Here we describe the structural analysis of two very polar glycolipids which can be observed after metabolic labeling of lymphoma cell lines S1A and EL-4 with either tritiated myo-inositol, mannose, or ethanolamine. These lipids are not made by mutant cells deficient in the biosynthesis of glycophosphatidylinositol anchors. The lipids were isolated, and their carbohydrate moiety was characterized using hydrofluoric acid dephosphorylation, nitrous acid deamination, acetolysis, exoglycosidase treatments, and combinations thereof to produce labeled fragments which could be analyzed by paper chromatography. Results are compatible with the structure (X-->)Man alpha 1,2 Man alpha 1,6(Y-->)Man alpha-GlcN-acylinositol, X and Y being hydrofluoric acid-sensitive substituents (most likely phosphoethanolamine). The anchor oligosaccharide of the glycophosphatidylinositol protein anchors of S1A cells was isolated, similarly characterized, and found to contain the identical carbohydrate structure. Pulse-chase experiments indicate that the very polar glycolipids have half-lives which are much longer than the one of phosphatidylinositol. The results suggest that these very polar glycolipids represent supernumerary precursor glycolipids which did not get transferred onto proteins or represent processed forms of such precursors.

Two different types of lipid moieties are present in glycophosphoinositol-anchored membrane proteins of Saccharomyces cerevisiae.

Numerous glycoproteins of Saccharomyces cerevisiae are anchored in the lipid bilayer by a glycophosphatidylinositol (GPI) anchor. Mild alkaline hydrolysis reveals that the lipid components of these anchors are heterogeneous in that both base-sensitive and base-resistant lipid moieties can be found on most proteins. The relative abundance of base-resistant lipid moieties is different for different proteins. Strong alkaline or acid hydrolysis of the mild base-resistant lipid component liberates C18-phytosphingosine indicating the presence of a ceramide. Two lines of evidence suggest that proteins are first attached to a base-sensitive GPI anchor, the lipid moiety of which subsequently gets exchanged for a base-resistant ceramide: (i) an early glycolipid intermediate of GPI biosynthesis only contains base-sensitive lipid moieties; (ii) after a pulse with [3H]myo-inositol the relative abundance of base-sensitive GPI anchors decreases significantly during chase. This decrease does not take place if GPI-anchored proteins are retained in the ER.

Characterization of glycophospholipid intermediate in the biosynthesis of glycophosphatidylinositol anchors accumulating in the Thy-1-negative lymphoma line SIA-b.

Several mammalian mutant cell lines are deficient in the biosynthesis of glycophosphatidylinositol anchors for membrane proteins. When metabolically labeled with [3H]myo-inositol or [3H]mannose, two out of five mutant lines (SIA-b and EL4-f) accumulated abnormal lipids which remained undetectable in the corresponding parental cell lines. The most abundant glycolipid of SIA-b cells (named lipid X) was isolated and partially characterized using hydrofluoric acid, nitrous acid deamination, acetolysis, and exoglycosidase treatments alone or in combination. The partial structure for the carbohydrate moiety of lipid X is Man alpha-(X----)Man alpha-GlcN-inositol, X being a charged, HF-sensitive substituent (possibly phosphoethanolamine). Lipid X is largely resistant to phosphatidylinositol-specific phospholipase C treatment but can be rendered sensitive to the enzyme by treatment with methanolic NH3, which suggests the presence of an acyl chain on the inositol moiety. The lipid moieties of lipid X are heterogenous in that about 50% of headgroups remain bound to a lipid moiety after mild alkaline hydrolysis. Similarly, about 50% of the lipid moieties of Thy-1, a glycophosphatidylinositol-anchored surface glycoprotein, isolated from SIA, the parent of SIA-b cells or from EL4 lymphoma cells, are resistant to mild alkaline hydrolysis. Altogether the data suggest that the SIA-b mutant line lacks an enzyme acting late in the anchor glycolipid biosynthesis pathway.

Biosynthesis of glycophosphoinositol anchors in Saccharomyces cerevisiae.

Numerous membrane glycoproteins of Saccharomyces cerevisiae are posttranslationally modified by the addition of a glycophosphatidylinositol (GPI). These proteins can be detected most easily by metabolic labelling of yeast cells with 3H-myoinositol or 3H-palmitate. This report summarizes what little is known about the identity, biosynthesis and cellular localization of GPI-modified glycoproteins in Saccharomyces cerevisiae as well as what could be learned from the system with respect to the biosynthesis of GPI's in general.

Biosynthesis of mannosylinositolphosphoceramide in Saccharomyces cerevisiae is dependent on genes controlling the flow of secretory vesicles from the endoplasmic reticulum to the Golgi.

Saccharomyces cerevisiae contains several abundant phosphoinositol-containing sphingolipids, namely inositolphosphoceramides (IPCs), mannosyl-inositolphosphoceramide (MIPC), which is substituted on the headgroup with an additional mannose, and M(IP)2C, a ceramide substituted with one mannose and two phosphoinositol groups. Using well-defined temperature-sensitive secretion mutants we demonstrate that the biosynthesis of MIPC, M(IP)2C, and a subclass if IPCs is dependent on genes that are required for the vesicular transport of proteins from the ER to the Golgi. Synthesis of these lipids in intact cells is dependent on metabolic energy. A likely but tentative interpretation of the data is that the biosynthesis of these sphingolipids is restricted to the Golgi apparatus, and that one or more substrates for the biosynthesis of these sphingolipids (phosphatidylinositol, IPCs, or MIPC) are delivered to the Golgi apparatus by an obligatory vesicular transport step. Alternative models to explain the data are also discussed.