Biomedical subjects

Nancy Standart

Publications and source records attributed to Nancy Standart.

13 recordsLinked to original sources

PubMed2007

How do microRNAs regulate gene expression?

Several thousand human genes, amounting to about one-third of the whole genome, are potential targets for regulation by the several hundred microRNAs (miRNAs) encoded in the genome. The regulation occurs posttranscriptionally and involves the approximately 21-nucleotide miRNA interacting with a target site in the mRNA that generally has imperfect complementarity to the miRNA. The target sites are almost invariably in the 3'-untranslated region of the messenger RNA (mRNA), often in multiple copies. Metazoan miRNAs were previously thought to down-regulate protein expression by inhibiting target mRNA translation at some stage after the translation initiation step, without much effect on mRNA abundance. However, recent studies have questioned these suppositions. With some targets, an increase in the rate of mRNA degradation by the normal decay pathway contributes to the decrease in protein expression. miRNAs can also inhibit translation initiation, specifically the function of the cap-binding initiation factor, eIF4E. Repressed target mRNAs as well as miRNAs themselves accumulate in cytoplasmic foci known as P-bodies, where many enzymes involved in mRNA degradation are concentrated. However, P-bodies may also serve as repositories for the temporary and reversible storage of untranslated mRNA, and reducing the expression (knockdown) of several distinct P-body protein components can alleviate miRNA-mediated repression of gene expression.

Animals↗

PubMed2006

Asymmetrical beta-actin mRNA translation in growth cones mediates attractive turning to netrin-1.

Local protein synthesis regulates the turning of growth cones to guidance cues, yet little is known about which proteins are synthesized or how they contribute to directional steering. Here we show that beta-actin mRNA resides in Xenopus laevis retinal growth cones where it binds to the RNA-binding protein Vg1RBP. Netrin-1 induces the movement of Vg1RBP granules into filopodia, suggesting that it may direct the localization and translation of mRNAs in growth cones. Indeed, a gradient of netrin-1 activates a translation initiation regulator, eIF-4E-binding protein 1 (4EBP), asymmetrically and triggers a polarized increase in beta-actin translation on the near side of the growth cone before growth cone turning. Inhibition of beta-actin translation abolishes both the asymmetric rise in beta-actin and attractive, but not repulsive, turning. Our data suggest that newly synthesized beta-actin, concentrated near sites of signal reception, provides the directional bias for polymerizing actin in the direction of an attractive stimulus.

3' Untranslated Regions↗

PubMed2006

Xenopus ElrB, but not ElrA, binds RNA as an oligomer: possible role of the linker.

We have shown that ElrA and ElrB, Xenopus ELAV homologues, bind the Vg1 mRNA 3'UTR translation element in Xenopus oocytes and implicated ElrB in mediating translational repression during oogenesis. Here we report that, while ElrA and ElrB are 69% identical and both exhibit RNA binding in the nM range, recombinant ElrB, but not ElrA, is able to oligomerise. This oligomerisation is also seen with the endogenous protein. Both RNA binding and oligomerisation require the linker region flanked with two RNA recognition motifs. Our data demonstrate a novel and unique property of ElrB which may be important for its function as a translational regulator.

Animals↗

PubMed2006

The two eIF4A helicases in Trypanosoma brucei are functionally distinct.

Protozoan parasites belonging to the family Trypanosomatidae are characterized by an unusual pathway for the production of mRNAs via polycistronic transcription and trans-splicing of a 5' capped mini-exon which is linked to the 3' cleavage and polyadenylation of the upstream transcript. However, little is known of the mechanism of protein synthesis in these organisms, despite their importance as agents of a number of human diseases. Here we have investigated the role of two Trypanosoma brucei homologues of the translation initiation factor eIF4A (in the light of subsequent experiments these were named as TbEIF4AI and TbEIF4AIII). eIF4A, a DEAD-box RNA helicase, is a subunit of the translation initiation complex eIF4F which binds to the cap structure of eukaryotic mRNA and recruits the small ribosomal subunit. TbEIF4AI is a very abundant predominantly cytoplasmic protein (over 1 x 10(5) molecules/cell) and depletion to approximately 10% of normal levels through RNA interference dramatically reduces protein synthesis one cell cycle following double-stranded RNA induction and stops cell proliferation. In contrast, TbEIF4AIII is a nuclear, moderately expressed protein (approximately 1-2 x 10(4) molecules/cell), and its depletion stops cellular proliferation after approximately four cell cycles. Ectopic expression of a dominant negative mutant of TbEIF4AI, but not of TbEIF4AIII, induced a slow growth phenotype in transfected cells. Overall, our results suggest that only TbEIF4AI is involved in protein synthesis while the properties and sequence of TbEIF4AIII indicate that it may be the orthologue of eIF4AIII, a component of the exon junction complex in mammalian cells.

Amino Acid Sequence↗

PubMed2005

Solution structure and backbone dynamics of the KH-QUA2 region of the Xenopus STAR/GSG quaking protein.

The Quaking protein belongs to the family of STAR/GSG domain RNA-binding proteins and is involved in multiple cell signalling and developmental processes in vertebrates, including the formation of myelin. Heteronuclear NMR methods were used to determine the solution structure of a 134 residue fragment spanning the KH and QUA2 homology regions of the Quaking protein from Xenopus laevis (pXqua) in the absence of RNA. The protein is shown to adopt an extended type I KH domain fold that is connected to a structured alpha-helix in the C-terminal QUA2 region by means of a highly flexible linker. A comparison with the solution structure of the related protein splicing factor 1 (SF1) indicates that most aspects of the RNA-binding interface are conserved in pXqua, although the "variable loop" region that follows the second beta-strand possesses two additional alpha-helices. The structure of pXqua provides an appropriate template for building models of important homologues, such as GLD-1 and Sam68. Measurements of the (15)N relaxation parameters of pXqua confirm that the polypeptide backbone of the QUA2 region is more dynamic than that of the KH portion, and that the C-terminal helix is partially structured in the absence of RNA. By comparison with a random coil reference state, the nascent structure in the QUA2 region is estimated to contribute 15.5kJmol(-1) to the change in conformational free energy that occurs on forming a complex with RNA. Since STAR/GSG proteins may regulate alternative splicing by competing with SF1 in the nucleus for specific branch-point sequences that signal intronic RNA, the formation of secondary structure in the QUA2 region in the unbound state of pXqua has important functional consequences.

Amino Acid Sequence↗

PubMed2005

Translation initiation in Leishmania major: characterisation of multiple eIF4F subunit homologues.

In eukaryotes protein synthesis initiates with the binding of the multimeric translation initiation complex eIF4F - eIF4E, eIF4A and eIF4G - to the monomethylated cap present on the 5' end of mRNAs. eIF4E interacts directly with the cap nucleotide, while eIF4A is a highly conserved RNA helicase and eIF4G acts as a scaffold for the complex with binding sites for both eIF4E and eIF4A. eIF4F binding to the mRNA recruits the small ribosomal subunit to its 5' end. Little is known in detail of protein synthesis in the protozoan parasites belonging to the family Trypanosomatidae. However, the presence of the highly modified cap structure, cap4, and the spliced leader sequence on the 5' ends of all mRNAs suggests possible differences in mRNA recruitment by ribosomes. We identified several potential eIF4F homologues by searching Leishmania major databases: four eIF4Es (LmEIF4E1-4), two eIF4As (LmEIF4A1-2) and five eIF4Gs (LmEIF4G1-5). We report the initial characterisation of LmEIF4E1-3, LmEIF4A1-2 and LmEIF4G3. First, the expression of these proteins in L. major promastigotes was quantitated by Western blotting using isoform specific antibodies. LmEIF4A1 and LmEIF4E3 are very abundant, LmEIF4G3 is moderately abundant and LmEIF4E1/LmEIF4E2/LmEIF4A2 are rare or not detected. In cap-binding assays, only LmEIF4E1 bound to the 7-methyl-GTP-Sepharose resin. Molecular modelling confirmed that LmEIF4E1 has all the structural features of a cap-binding protein. Finally, pull-down assays were used to investigate the potential interaction between the eIF4A (LmEIF4A1/LmEIF4A2) and eIF4G (LmEIF4G1-3) homologues. Only LmEIF4G3, via the HEAT domain, bound specifically both to LmEIF4A1 as well as to human eIF4A. Therefore for each factor, one of the L. major forms seems to fulfil, in part at least, the expected characteristics of a translational initiation factor.

Amino Acid Sequence↗

PubMed2005

The Xenopus ELAV protein ElrB represses Vg1 mRNA translation during oogenesis.

Xenopus laevis Vg1 mRNA undergoes both localization and translational control during oogenesis. We previously characterized a 250-nucleotide AU-rich element, the Vg1 translation element (VTE), in the 3'-untranslated region (UTR) of this mRNA that is responsible for the translational repression. UV-cross-linking and immunoprecipitation experiments, described here, revealed that the known AU-rich element binding proteins, ElrA and ElrB, and TIA-1 and TIAR interact with the VTE. The levels of these proteins during oogenesis are most consistent with a possible role for ElrB in the translational control of Vg1 mRNA, and ElrB, in contrast to TIA-1 and TIAR, is present in large RNP complexes. Immunodepletion of TIA-1 and TIAR from Xenopus translation extract confirmed that these proteins are not involved in the translational repression. Mutagenesis of a potential ElrB binding site destroyed the ability of the VTE to bind ElrB and also abolished translational repression. Moreover, multiple copies of the consensus motif both bind ElrB and support translational control. Therefore, there is a direct correlation between ElrB binding and translational repression by the Vg1 3'-UTR. In agreement with the reporter data, injection of a monoclonal antibody against ElrB into Xenopus oocytes resulted in the production of Vg1 protein, arguing for a role for the ELAV proteins in the translational repression of Vg1 mRNA during early oogenesis.

3' Untranslated Regions↗

PubMed2004

The active form of Xp54 RNA helicase in translational repression is an RNA-mediated oligomer.

Previously, we reported that in clam oocytes, cytoplasmic polyadenylation element-binding protein (CPEB) co-immunoprecipitates with p47, a member of the highly conserved RCK family of RNA helicases which includes Drosophila Me31B and Saccharomyces cerevisiae Dhh1. Xp54, the Xenopus homologue, with helicase activity, is a component of stored mRNP. In tethered function assays in Xenopus oocytes, we showed that MS2-Xp54 represses the translation of non-adenylated firefly luciferase mRNAs and that mutations in two core helicase motifs, DEAD and HRIGR, surprisingly, activated translation. Here we show that wild-type MS2-Xp54 tethered to the reporter mRNA 3'-untranslated region (UTR) represses translation in both oocytes and eggs in an RNA-dependent complex with endogenous Xp54. Injection of mutant helicases or adenylated reporter mRNA abrogates this association. Thus Xp54 oligomerization is a hallmark of translational repression. Xp54 complexes, which also contain CPEB and eIF4E in oocytes, change during meiotic maturation. In eggs, CPEB is degraded and, while eIF4E still interacts with Xp54, this interaction becomes RNA dependent. Supporting evidence for RNA-mediated oligomerization of endogenous Xp54, and RNA-independent association with CPEB and eIF4E in oocytes was obtained by gel filtration. Altogether, our data are consistent with a model in which the active form of the Xp54 RNA helicase is an oligomer in vivo which, when tethered, via either MS2 or CPEB to the 3'UTR, represses mRNA translation, possibly by sequestering eIF4E from the translational machinery.

Animals↗

PubMed2004

Two distinct Staufen isoforms in Xenopus are vegetally localized during oogenesis.

Localization of mRNA is an important way of generating early asymmetries in the developing embryo. In Drosophila, Staufen is intimately involved in the localization of maternally inherited mRNAs critical for cell fate determination in the embryo. We show that double-stranded RNA-binding Staufen proteins are present in the oocytes of a vertebrate, Xenopus, and are localized to the vegetal cytoplasm, a region where important mRNAs including VegT and Vg1 mRNA become localized. We identified two Staufen isoforms named XStau1 and XStau2, where XStau1 was found to be the principal Staufen protein in oocytes, eggs, and embryos, the levels of both proteins peaking during mid-oogenesis. In adults, Xenopus Staufens are principally expressed in ovary and testis. XStau1 was detectable throughout the oocyte cytoplasm by immunofluorescence and was concentrated in the vegetal cortical region from stage II onward. It showed partial codistribution with subcortical endoplasmic reticulum (ER), raising the possibility that Staufen may anchor mRNAs to specific ER-rich domains. We further showed that XStau proteins are transiently phosphorylated by the MAPK pathway during meiotic maturation, a period during which RNAs such as Vg1 RNA are released from their tight localization at the vegetal cortex. These findings provide evidence that Staufen proteins are involved in targeting and/or anchoring of maternal determinants to the vegetal cortex of the oocyte in Xenopus. The Xenopus oocyte should thus provide a valuable system to dissect the role of Staufen proteins in RNA localization and vertebrate development.

Amino Acid Sequence↗

PubMed2003

Identification of a C-terminal poly(A)-binding protein (PABP)-PABP interaction domain: role in cooperative binding to poly (A) and efficient cap distal translational repression.

The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.

Amino Acid Sequence↗

PubMed2003

Role of cdc2 kinase phosphorylation and conserved N-terminal proteolysis motifs in cytoplasmic polyadenylation-element-binding protein (CPEB) complex dissociation and degradation.

Cytoplasmic polyadenylation-element-binding protein (CPEB) is a well-characterized and important regulator of translation of maternal mRNA in early development in organisms ranging from worms, flies and clams to frogs and mice. Previous studies provided evidence that clam and Xenopus CPEB are hyperphosphorylated at germinal vesicle breakdown (GVBD) by cdc2 kinase, and degraded shortly after. To examine the conserved features of CPEB that mediate its modification during meiotic maturation, we microinjected mRNA encoding wild-type and mutated clam CPEB into Xenopus oocytes that were subsequently allowed to mature with progesterone. We observed that (i) ectopically expressed clam CPEB is phosphorylated at GVBD and subsequently degraded, mirroring the fate of the endogenous Xenopus CPEB protein, (ii) mutation of nine Ser/Thr Pro-directed kinase sites prevents phosphorylation and degradation and (iii) deletion of the PEST box, and to a lesser extent of the putative cyclin destruction box, generates a stable and phosphorylated version of CPEB. We conclude that phosphorylation of both consensus and non-consensus sites by cdc2 kinase targets clam CPEB for PEST-mediated destruction. We also show that phosphorylation of CPEB mediates its dissociation from ribonucleoprotein complexes, prior to degradation. Our findings reinforce results obtained in Xenopus, and have implications for CPEB from other invertebrates including Drosophila, Caenorhabditis elegans and Aplysia, which lack PEST boxes.

Amino Acid Motifs↗

PubMed2002

The KH domains of Xenopus Vg1RBP mediate RNA binding and self-association.

Xenopus Vg1 mRNA is localized to the vegetal cortex during oogenesis in a process involving microtubules and microfilaments and proteins that specifically recognize the vegetal localization element (VLE) within the 3' untranslated region. One of the best characterized VLE-binding proteins is Vg1RBP or Vera. Primary sequence analysis of Vg1RBP and its homologs suggests that most of its open reading frame is occupied by RNA-binding modules, including two RRMs and four KH domains, arranged as three pairs of didomains. In the first detailed domain analysis of Vg1RBP, we show that the interaction of Vg1RBP with the VLE requires both KH didomains, but not the RRM didomain, and moreover that the KH didomains contribute cooperatively to RNA binding. In the full-length protein, individual KH domains display significant redundancy, and their relative importance appears to vary with the RNA target. We also demonstrate that the KH34 didomain mediates Vg1RBP self-association, which is stabilized by RNA, and occurs in vivo as well as in vitro. Altogether, our findings highlight the importance of multiple KH domains in mediating RNA-protein and protein-protein interactions in the formation of a stable complex of Vg1RBP and Vg1 mRNA.

3' Untranslated Regions↗

PubMedDate not supplied

RNA-binding proteins in early development.

RNA-binding proteins play a major part in the control of gene expression during early development. At this stage, the majority of regulation occurs at the levels of translation and RNA localization. These processes are, in general, mediated by RNA-binding proteins interacting with specific sequence motifs in the 3'-untranslated regions of their target RNAs. Although initial work concentrated on the analysis of these sequences and their trans-acting factors, we are now beginning to gain an understanding of the mechanisms by which some of these proteins function. In this review, we will describe a number of different families of RNA-binding proteins, grouping them together on the basis of common regulatory strategies, and emphasizing the recurrent themes that occur, both across different species and as a response to different biological problems.

Animals↗