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PubMed2001

Lack of mitochondrial anionic phospholipids causes an inhibition of translation of protein components of the electron transport chain. A yeast genetic model system for the study of anionic phospholipid function in mitochondria.

Reduction of mitochondrial cardiolipin (CL) levels has been postulated to compromise directly the function of several essential enzymes and processes of the mitochondria. There is limited genetic evidence for the critical roles with which CL and its precursor phosphatidylglycerol (PG) have been associated. A null allele of the PGS1 gene from Saccharomyces cerevisiae, which encodes the enzyme responsible for the synthesis of the CL precursor PG phosphate, was created in a yeast strain in which PGS1 expression is exogenously regulated by doxycycline. The addition of increasing concentrations of doxycycline to the growth medium causes a proportional decrease to undetectable levels of PGS1 transcript, PG phosphate synthase activity, and PG plus CL. The doubling time of this strain with increasing doxycycline increases to senescence in non-fermentable carbon sources or at high temperatures, conditions that do not support growth of the pgs1Delta strain. Doxycycline addition also causes mitochondrial abnormalities as observed by fluorescence microscopy. Products of four mitochondrial encoded genes (COX1, COX2, COX3, and COB) and one nuclear encoded gene (COX4) associated with the mitochondrial inner membrane are not present when PGS1 expression is fully repressed. No translation of these proteins can be detected in cells lacking the PGS1 gene product, although transcription and splicing appear unaffected. Protein import of other nuclear encoded proteins remains unaffected. The remaining proteins encoded by mitochondrial DNA are expressed and translated normally. Thus, the molecular basis for the lack of mitochondrial function in pgs1Delta cells is the failure to translate gene products essential to the electron transport chain.

Acridine Orange↗
PubMed2006

Cytoplasmic CstF-77 protein belongs to a masking complex with cytoplasmic polyadenylation element-binding protein in Xenopus oocytes.

Regulated mRNA translation is a hallmark of oocytes and early embryos, of which cytoplasmic polyadenylation is a major mechanism. This process involves multiple protein components, including the CPSF (cleavage and polyadenylation specificity factor), which is also required for nuclear polyadenylation. The CstF (cleavage stimulatory factor), with CPSF, is required for the pre-mRNA cleavage before nuclear polyadenylation. However, some evidence suggests that the CstF-77 subunit might have a function independent of nuclear polyadenylation, which could be related to the cell cycle. As such, we addressed the question whether CstF-77 might have a role in cytoplasmic polyadenylation. We investigated the function of the CstF-77 protein in Xenopus oocytes, and show that CstF-77 has indeed a role in the cytoplasm. The Xenopus CstF-77 protein (X77K) localizes mainly to the nucleus, but also in punctuate cytoplasmic foci. We show that X77K resides in a cytoplasmic complex with eIF4E, CPEB (cytoplasmic polyadenylation element-binding protein), CPSF-100 and XGLD2, but is not required for cytoplasmic polyadenylation per se. Impairment of X77K function in ovo leads to an acceleration of the G(2)/M transition, with a premature synthesis of Mos and AuroraA proteins. However, the kinetic of Mos mRNA polyadenylation is not modified. Furthermore, X77K represses mRNA translation in vitro. These results suggest that X77K could be involved in masking of mRNA prior to polyadenylation.

Amino Acid Sequence↗
PubMed1996

GLD-1, a cytoplasmic protein essential for oocyte differentiation, shows stage- and sex-specific expression during Caenorhabditis elegans germline development.

GLD-1, a putative RNA binding protein, is essential for oocyte development in Caenorhabditis elegans. A gld-1 null mutation abolishes hermaphrodite oogenesis and confers a tumorous germline phenotype in which presumptive female germ cells exit the meiotic pathway and return to the mitotic cell cycle. Here we demonstrate that gld-1(null) germ lines express female-specific, but not male-specific, molecular markers, indicating that gld-1 acts downstream of sexual fate specification to regulate oocyte differentiation. Immunolocalization studies identify GLD-1 as a cytoplasmic germline protein that displays differential accumulation during germline development. First, germ cells that are in the mitotic cell cycle contain low levels of GLD-1 that likely reflect a nonessential gld-1 function (negative regulation of proliferation in the mitotic germ line) revealed in previous genetic studies. Second, entry of presumptive oocytes into the meiotic pathway is accompanied by a strong increase in GLD-1 expression/accumulation. GLD-1 levels are high through the pachytene stage but fall to background as germ cells exit pachytene and complete oogenesis. The meiotic prophase accumulation pattern is consistent with GLD-1's essential role in oocyte differentiation, which may be to repress the translation of a subset of maternal RNAs synthesized during early oogenesis until late oogenesis when GLD-1 is absent.

Animals↗
PubMed2001

Identification of target mRNAs for the clock-controlled RNA-binding protein Chlamy 1 from Chlamydomonas reinhardtii.

An endogenous clock regulates the temporal expression of genes/mRNAs that are involved in the circadian output pathway. In the green alga Chlamydomonas reinhardtii, a clock-controlled RNA-binding protein (Chlamy 1) was identified recently, which represents an analog of the circadian trans-acting factor CCTR from the phylogenetically diverse alga Gonyaulax polyedra. In order to identify in C. reinhardtii target mRNAs that can be recognized by Chlamy 1, gel mobility-shift assays and UV-crosslinking experiments were carried out, and revealed that this protein interacts specifically with the 3' untranslated regions of several mRNAs and recognizes them all via a common cis-acting element, composed of at least seven UG repeats. By using competition assays, it was found that the affinity of Chlamy 1 is highest for mRNAs whose products are key components of nitrogen and CO2 metabolism. Since the activities of enzymes involved in nitrogen metabolism vary in a temporal pattern that is opposite in phase to that of Chlamy 1 binding activity, the protein may repress the translation of the cognate mRNAs.

3' Untranslated Regions↗
PubMed1989

Abnormal expression of the RI subunit of cyclic AMP-dependent protein kinase in Y-79 retinoblastoma cells.

The cyclic AMP-dependent protein kinases of Y-79 retinoblastoma cells were analyzed by DEAE Sephacel column chromatography, SDS-polyacrylamide gel electrophoresis, and immunoblotting. Both type I and type II protein kinases (characteristic of fetal and mature retina respectively) were observed. In addition, free RI (type I regulatory subunit) was present in Y-79 cells; three other retinoblastoma cell lines also showed evidence of free RI in immunoblot analysis. Northern blot analysis of poly A+-enriched RNA from Y-79 cells showed a 4.5 kb RI transcript and a 6.0 kb RII transcript. The single 4.5 kb RI message is typical of normal retina, in contrast to other tissues and cell lines in which a 2.0 kb message predominates. Since RI expression appears to be post-transcriptionally inhibited in normal retina, it is suggested that free RI dimers in retinoblastoma is the result of loss of repression of translation of this transcript and that the free RI pool may contribute to the unregulated growth of these cells.

Cell Line↗
PubMed1994

Differential expression in blood stages of the gene coding for the 21-kilodalton surface protein of ookinetes of Plasmodium berghei as detected by RNA in situ hybridisation.

The developmentally regulated transcription of the gene encoding the ookinete surface protein, Pbs21, has been investigated in the rodent malaria parasite, Plasmodium berghei, by RNA in situ hybridisation using fluorescently labelled DNA probes. We used a procedure that will allow the visualisation of cytoplasmic mRNA in the parasite and of high copy DNA repeats in the nucleus. Specific hybridisation to Pbs21 mRNA occurred in the cytoplasm of female gametocytes, zygotes and ookinetes, while asexual blood stages, male gametocytes and gametes showed no fluorescence. Analysis of the transcription of the Pbs21 gene during blood stage development in two tightly synchronised parasite clones using the same methodology revealed that transcription is restricted to sexual stages and is initiated in immature gametocytes at 19 h post invasion (hpi). At this point in development it is not yet possible to discriminate between the morphology of asexual trophozoites and immature gametocytes. At 24 hpi approximately 50% of the gametocytes transcribed the Pbs21 gene and the morphology of these gametocytes was identical and female. The distribution of the mRNA encoding Pbs21 confirmed that post-transcriptional control of expression occurred in the cytoplasm by repression of translation and not through delayed transport of the message to the cytoplasm. The transcription of the Pbs21 gene is the earliest demonstrated event in gametocytogenesis in rodent malaria species to date.

Animals↗
PubMed2004

A microtiter plate assay for assessing the interaction of eukaryotic initiation factor eIF4E with eIF4G and eIF4E binding protein-1.

Modulation of interactions among proteins is an important mechanism for regulating both the subcellular location and the function of proteins. An example of the importance of protein-protein interaction is the reversible association of eukaryotic initiation factor eIF4E with the eIF4E binding proteins 4E-BP1 and eIF4G. When bound to 4E-BP1, eIF4E cannot bind to eIF4G to form the active eIF4F complex, an event that is required for the binding of mRNA to the ribosome. Thus, association of eIF4E with 4E-BP1 represses mRNA translation by preventing the binding of mRNA to the ribosome. Previous studies have measured the amount of 4E-BP1 or eIF4G bound to eIF4E by either affinity chromatography or immunoprecipitation of eIF4E followed by Western blot analysis for quantitation of 4E-BP1 and eIF4G. Both of these techniques have significant limitations. In the present study, we describe a microtiter plate-based assay for quantitation of the amount of 4E-BP1 and eIF4G bound to eIF4E that obviates many of the limitations of the earlier approaches. It also has the advantage that absolute amounts of the individual proteins can be easily estimated. The approach should be applicable to the study of a wide variety of protein-protein interactions.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Regulatory relationship among piwi, pumilio, and bag-of-marbles in Drosophila germline stem cell self-renewal and differentiation.

The transition from a Drosophila ovarian germline stem cell (GSC) to its differentiated daughter cell, the cystoblast, is controlled by both niche signals and intrinsic factors. piwi and pumilio (pum) are essential for GSC self-renewal, whereas bag-of-marbles (bam) is required for cystoblast differentiation. We demonstrate that Piwi and Bam proteins are expressed independently of each other in reciprocal patterns in GSCs and cystoblasts. However, overexpression of either one antagonizes the other in these cells. Furthermore, piwi;bam double mutants phenocopy the bam mutant. This epistasis reflects the niche signaling function of piwi because depleting piwi from niche cells in bam mutant ovaries also phenocopies bam mutants. Thus, bam is epistatic to niche Piwi, but not germline Piwi function. Despite this, bam- ovaries lacking germline Piwi contain approximately 4-fold fewer germ cells than bam- ovaries, consistent with the role of germline Piwi in promoting GSC mitosis by 4-fold. Finally, pum is epistatic to bam, indicating that niche Piwi does not regulate Bam-C through Pum. We propose that niche Piwi maintains GSCs by repressing bam expression in GSCs, which consequently prevents Bam from downregulating Pum/Nos function in repressing the translation of differentiation genes and germline Piwi function in promoting germ cell division.

Animals↗
PubMed2006

Identification of 188 conserved maize microRNAs and their targets.

MicroRNAs (miRNAs) represent a newly identified class of non-protein-coding approximately 20nt small RNAs which play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. After searching a genomic survey sequence database using homologs and secondary structures, we found 188 maize miRNAs belonging to 29 miRNA families. Of the 188 maize miRNA genes, 28 (15%) were found in at least one EST. A total of 115 potential targets were identified for 26 of the miRNA families based on the fact that miRNAs exhibit perfect or nearly perfect complementarity with their target sequences. A majority of the targets are transcription factors which play important roles in maize development, including leaf, shoot, and root development. Additionally, these maize miRNAs are also involved in other cellular processes, such as signal transduction, stress response, sucrose and cellulose synthesis, and ubiquitin protein degradation pathway. Some of the newly identified miRNA targets may be unique to maize.

Base Sequence↗
PubMed2006

Iron chelation in the biological activity of curcumin.

Curcumin is among the more successful chemopreventive compounds investigated in recent years, and is currently in human trials to prevent cancer. The mechanism of action of curcumin is complex and likely multifactorial. We have made the unexpected observation that curcumin strikingly modulates proteins of iron metabolism in cells and in tissues, suggesting that curcumin has properties of an iron chelator. Curcumin increased mRNA levels of ferritin and GSTalpha in cultured liver cells. Unexpectedly, however, although levels of GSTalpha protein increased in parallel with mRNA levels in response to curcumin, levels of ferritin protein declined. Since iron chelators repress ferritin translation, we considered that curcumin may act as an iron chelator. To test this hypothesis, we measured the effect of curcumin on transferrin receptor 1, a protein stabilized under conditions of iron limitation, as well as the ability of curcumin to activate iron regulatory proteins (IRPs). Both transferrin receptor 1 and activated IRP, indicators of iron depletion, increased in response to curcumin. Consistent with the hypothesis that curcumin acts as an iron chelator, mice that were fed diets supplemented with curcumin exhibited a decline in levels of ferritin protein in the liver. These results suggest that iron chelation may be an additional mode of action of curcumin.

Animals↗
PubMed2005

Molecular cloning and functional characterization of two apple S-adenosylmethionine decarboxylase genes and their different expression in fruit development, cell growth and stress responses.

Two full-length S-adenosylmethionine decarboxylase (SAMDC) cDNAs, MdSAMDC1 and MdSAMDC2, were isolated from apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. Both cDNAs encoded tiny and small ORFs in addition to the SAMDC ORFs, and genomic sequences of MdSAMDC1 and MdSAMDC2 contained two or three introns in the 5' upstream regions, respectively. Yeast complementation experiment indicated that two MdSAMDCs encoded functional proteins, and that the tiny and small ORFs possibly repressed their translation efficiency. RNA gel blot analysis showed that MdSAMDC1 were differentially regulated in fruits depending on the developmental stage and in cell suspension during the culture period, but MdSAMDC2 did not. In contrast, MdSAMDC2 was positively induced by cold and salt stresses, but MdSAMDC1 was not. These results suggest that MdSAMDC1 is mainly involved in fruit development and cell growth while MdSAMDC2 in stress responses, compared with their respective counterpart.

Adenosylmethionine Decarboxylase↗
PubMed2004

The C terminus of fragile X mental retardation protein interacts with the multi-domain Ran-binding protein in the microtubule-organising centre.

Absence of the fragile X mental retardation protein (FMRP) causes fragile X syndrome, the most common form of hereditary mental retardation. FMRP is a mainly cytoplasmic protein thought to be involved in repression of translation, through a complex network of protein-protein and protein-RNA interactions. Most of the currently known protein partners of FMRP recognise the conserved N terminus of the protein. No interaction has yet been mapped to the highly charged, poorly conserved C terminus, so far thought to be involved in RNA recognition through an RGG motif. In the present study, we show that a two-hybrid bait containing residues 419-632 of human FMRP fishes out a protein that spans the sequence of the Ran-binding protein in the microtubule-organising centre (RanBPM/RanBP9). Specific interaction of RanBPM with FMRP was confirmed by in vivo and in vitro assays. In brain tissue sections, RanBPM is highly expressed in the neurons of cerebral cortex and the cerebellar purkinje cells, in a pattern similar to that described for FMRP. Sequence analysis shows that RanBPM is a multi-domain protein. The interaction with FMRP was mapped in a newly identified CRA motif present in the RanBPM C terminus. Our results suggest that the functional role of RanBPM binding is modulation of the RNA-binding properties of FMRP.

Amino Acid Motifs↗
PubMed2005

Caenorhabditis elegans DAZ-1 is expressed in proliferating germ cells and directs proper nuclear organization and cytoplasmic core formation during oogenesis.

The deleted in azoospermia (DAZ) family genes encode potential RNA-binding proteins that are expressed exclusively in germ cells in a wide range of metazoans. We have previously shown that mutations in daz-1, the only DAZ family gene in Caenorhabditis elegans, cause pachytene stage arrest of female germ cells but do not affect spermatogenesis. In this study, we report that DAZ-1 protein is most abundantly expressed in proliferating female germ cells, in a manner independent of the GLP-1 signaling pathway. DAZ-1 is dispensable in males but it is expressed also in male mitotic germ cells. Detailed phenotypic analyses with fluorescence microscopy and transmission electron microscopy have revealed that loss of daz-1 function causes multiple abnormalities as early as the onset of meiotic prophase, which include aberrant chromatin structure, small nucleoli, absence of the cytoplasmic core, and precocious cellularization. Although the reduced size of nucleoli is indicative of a low translational activity in these cells, artificial repression of general translation in the germline does not phenocopy the daz-1 mutant. Thus, we propose that DAZ-1 in C. elegans plays essential roles in female premeiotic and early meiotic germ cells, probably via regulating the translational activity of specific target genes required for the progression of oogenesis.

Animals↗
PubMed1999

The structure of threonyl-tRNA synthetase-tRNA(Thr) complex enlightens its repressor activity and reveals an essential zinc ion in the active site.

E. coli threonyl-tRNA synthetase (ThrRS) is a class II enzyme that represses the translation of its own mRNA. We report the crystal structure at 2.9 A resolution of the complex between tRNA(Thr) and ThrRS, whose structural features reveal novel strategies for providing specificity in tRNA selection. These include an amino-terminal domain containing a novel protein fold that makes minor groove contacts with the tRNA acceptor stem. The enzyme induces a large deformation of the anticodon loop, resulting in an interaction between two adjacent anticodon bases, which accounts for their prominent role in tRNA identity and translational regulation. A zinc ion found in the active site is implicated in amino acid recognition/discrimination.

Amino Acyl-tRNA Synthetases↗
PubMed2004

RISC assembly defects in the Drosophila RNAi mutant armitage.

The putative RNA helicase, Armitage (Armi), is required to repress oskar translation in Drosophila oocytes; armi mutant females are sterile and armi mutations disrupt anteroposterior and dorsoventral patterning. Here, we show that armi is required for RNAi. armi mutant male germ cells fail to silence Stellate, a gene regulated endogenously by RNAi, and lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes in wild-type and armi mutant ovary lysates suggests that armi mutants support early steps in the RNAi pathway but are defective in the production of active RNA-induced silencing complex (RISC), which mediates target RNA destruction in RNAi. Our results suggest that armi is required for RISC maturation.

Animals↗
PubMed2000

The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo.

The yeast superkiller (SKI) genes were originally identified from mutations allowing increased production of killer toxin encoded by M "killer" virus, a satellite of the dsRNA virus L-A. XRN1 (SKI1) encodes a cytoplasmic 5'-exoribonuclease responsible for the majority of cytoplasmic RNA turnover, whereas SKI2, SKI3, and SKI8 are required for normal 3'-degradation of mRNA and for repression of translation of poly(A) minus RNA. Ski2p is a putative RNA helicase, Ski3p is a tetratricopeptide repeat (TPR) protein, and Ski8p contains five WD-40 (beta-transducin) repeats. An xrn1 mutation in combination with a ski2, ski3, or ski8 mutation is lethal, suggesting redundancy of function. Using functional epitope-tagged Ski2, Ski3, and Ski8 proteins, we show that Ski2p, Ski3p, and Ski8p can be coimmunoprecipitated as an apparent heterotrimeric complex. With epitope-tagged Ski2p, there was a 1:1:1 stoichiometry of the proteins in the complex. Ski2p did not associate with Ski3p in the absence of Ski8p, nor did Ski2p associate with Ski8p in the absence of Ski3p. However, the Ski3p/Ski8p interaction did not require Ski2p. In addition, ski6-2 or ski4-1 mutations or deletion of SKI7 did not affect complex formation. The identification of a complex composed of Ski2p, Ski3p, and Ski8p explains previous results showing phenotypic similarity between mutations in SKI2, SKI3, and SKI8. Indirect immunofluorescence of Ski3p and subcellular fractionation of Ski2p and Ski3p suggest that Ski2p and Ski3p are cytoplasmic. These data support the idea that Ski2p, Ski3p, and Ski8p function in the cytoplasm in a 3'-mRNA degradation pathway.

Antiviral Agents↗
PubMed2003

The N-terminus of the fragile X mental retardation protein contains a novel domain involved in dimerization and RNA binding.

Fragile X syndrome, the most common cause of inherited mental retardation, is caused by the absence of the fragile X mental retardation protein (FMRP). The emerging picture is that FMRP is involved in repression of translation through a complex network of protein-protein and protein-RNA interactions. Very little structural information is, however, available for FMRP that could help to understand its function. In particular, no structural studies are available about the N-terminus of the protein, a highly conserved region which is involved in several molecular interactions. Here, we explore systematically the ability of the FMRP N-terminus to form independently folded units (domains). We produced deletion mutants and tested their fold and functional properties by mutually complementary biophysical and biochemical techniques. On the basis of our data, we conclude that the N-terminus contains a domain, that we named NDF, comprising the first 134 amino acids. Most interestingly, NDF comprises two copies of a newly identified Agenet motif. NDF is thermally stable and has a high content of beta structure. In addition to being able to bind to RNA and to recognize some of the FMRP interacting proteins, NDF forms stable dimers and is able to interact, although weakly, with the full-length protein. Our data provide conclusive evidence that NDF is a novel motif for protein-protein and protein-RNA interactions and contains a previously unidentified dimerization site.

Amino Acid Sequence↗