Zinc Finger RNA Binding Protein Research Articles

Nanos3 Gene Targeting in Medaka ES Cells

Gene targeting (GT) by homologous recombination offers the best precision for genome editing in mice. nanos3 is a highly conserved gene and encodes a zinc-finger RNA binding protein essential for germ stem cell maintenance in Drosophila, zebrafish and mouse. Here we report nanos3 GT in embryonic stem (ES) cells of the fish medaka as a lower vertebrate model organism. A vector was designed for GT via homologous recombination on the basis of positive-negative selection (PNS). The ES cell line MES1 after gene transfer and PNS produced 56 colonies that were expanded into ES cell sublines. Nine sublines were GT-positive by PCR genotyping, 4 of which were homologous recombinants as revealed by Southern blot. We show that one of the 4, A15, contains a precisely targeted nanos3 allele without any random events, demonstrating the GT feasibility in medaka ES cells. Importantly, A15 retained all features of undifferentiated ES cells, including stable self-renewal, an undifferentiated phenotype, pluripotency gene expression and differentiation during chimeric embryogenesis. These results provide first evidence that the GT procedure and genuine GT on a chromosomal locus such as nanos3 do not compromise pluripotency in ES cells of a lower vertebrate.

NF45 dimerizes with NF90, Zfr and SPNR via a conserved domain that has a nucleotidyltransferase fold

Nuclear factors NF90 and NF45 form a complex involved in a variety of cellular processes and are thought to affect gene expression both at the transcriptional and translational level. In addition, this complex affects the replication of several viruses through direct interactions with viral RNA. NF90 and NF45 dimerize through their common ‘DZF’ domain (domain associated with zinc fingers). NF90 has additional double-stranded RNA-binding domains that likely mediate its association with target RNAs. We present the crystal structure of the NF90/NF45 dimerization complex at 1.9-Å resolution. The DZF domain shows structural similarity to the template-free nucleotidyltransferase family of RNA modifying enzymes. However, both NF90 and NF45 have lost critical catalytic residues during evolution and are therefore not functional enzymes. Residues on NF90 that make up its interface with NF45 are conserved in two related proteins, spermatid perinuclear RNA-binding protein (SPNR) and zinc-finger RNA-binding protein (Zfr). Using a co-immunoprecipitation assay and site-specific mutants, we demonstrate that NF45 is also able to recognize SPNR and Zfr through the same binding interface, revealing that NF45 is able to form a variety of cellular complexes with other DZF-domain proteins.

Xenopus laevis zygote arrest 2 (zar2) encodes a zinc finger RNA-binding protein that binds to the translational control sequence in the maternal Wee1 mRNA and regulates translation

Zygote arrest (Zar) proteins are crucial for early embryonic development, but their molecular mechanism of action is unknown. The Translational Control Sequence (TCS) in the 3′ untranslated region (UTR) of the maternal mRNA, Wee1, mediates translational repression in immature Xenopus oocytes and translational activation in mature oocytes, but the protein that binds to the TCS and mediates translational control is not known. Here we show that Xenopus laevis Zar2 (encoded by zar2) binds to the TCS in maternal Wee1 mRNA and represses translation in immature oocytes. Using yeast 3 hybrid assays and electrophoretic mobility shift assays, Zar2 was shown to bind specifically to the TCS in the Wee1 3′UTR. RNA binding required the presence of Zn2+ and conserved cysteines in the C-terminal domain, suggesting that Zar2 contains a zinc finger. Consistent with regulating maternal mRNAs, Zar2 was present throughout oogenesis, and endogenous Zar2 co-immunoprecipitated endogenous Wee1 mRNA from immature oocytes, demonstrating the physiological significance of the protein–RNA interaction. Interestingly, Zar2 levels decreased during oocyte maturation. Dual luciferase reporter tethered assays showed that Zar2 repressed translation in immature oocytes. Translational repression was relieved during oocyte maturation and this coincided with degradation of Zar2 during maturation. This is the first report of a molecular function of zygote arrest proteins. These data show that Zar2 contains a zinc finger and is a trans-acting factor for the TCS in maternal mRNAs in immature Xenopus oocytes.

Genome-Wide Assessment of AU-Rich Elements by the AREScore Algorithm

In mammalian cells, AU-rich elements (AREs) are well known regulatory sequences located in the 3′ untranslated region (UTR) of many short-lived mRNAs. AREs cause mRNAs to be degraded rapidly and thereby suppress gene expression at the posttranscriptional level. Based on the number of AUUUA pentamers, their proximity, and surrounding AU-rich regions, we generated an algorithm termed AREScore that identifies AREs and provides a numerical assessment of their strength. By analyzing the AREScore distribution in the transcriptomes of 14 metazoan species, we provide evidence that AREs were selected for in several vertebrates and Drosophila melanogaster. We then measured mRNA expression levels genome-wide to address the importance of AREs in SL2 cells derived from D. melanogaster hemocytes. Tis11, a zinc finger RNA–binding protein homologous to mammalian tristetraprolin, was found to target ARE–containing reporter mRNAs for rapid degradation in SL2 cells. Drosophila mRNAs whose expression is elevated upon knock down of Tis11 were found to have higher AREScores. Moreover high AREScores correlate with reduced mRNA expression levels on a genome-wide scale. The precise measurement of degradation rates for 26 Drosophila mRNAs revealed that the AREScore is a very good predictor of short-lived mRNAs. Taken together, this study introduces AREScore as a simple tool to identify ARE–containing mRNAs and provides compelling evidence that AREs are widespread regulatory elements in Drosophila.

Defining potentially conserved RNA regulons of homologous zinc-finger RNA-binding proteins

BackgroundGlucose inhibition of gluconeogenic growth suppressor 2 protein (Gis2p) and zinc-finger protein 9 (ZNF9) are conserved yeast and human zinc-finger proteins. The function of yeast Gis2p is unknown, but human ZNF9 has been reported to bind nucleic acids, and mutations in the ZNF9 gene cause the neuromuscular disease myotonic dystrophy type 2. To explore the impact of these proteins on RNA regulation, we undertook a systematic analysis of the RNA targets and of the global implications for gene expression.ResultsHundreds of mRNAs were associated with Gis2p, mainly coding for RNA processing factors, chromatin modifiers and GTPases. Target mRNAs contained stretches of G(A/U)(A/U) trinucleotide repeats located in coding sequences, which are sufficient for binding to both Gis2p and ZNF9, thus implying strong structural conservation. Predicted ZNF9 targets belong to the same functional categories as seen in yeast, indicating functional conservation, which is further supported by complementation of the large cell-size phenotype of gis2 mutants with ZNF9. We further applied a matched-sample proteome-transcriptome analysis suggesting that Gis2p differentially coordinates expression of RNA regulons, primarily by reducing mRNA and protein levels of genes required for ribosome assembly and by selectively up-regulating protein levels of myosins.ConclusionsThis integrated systematic exploration of RNA targets for homologous RNA-binding proteins indicates an unexpectedly high conservation of the RNA-binding properties and of potential targets, thus predicting conserved RNA regulons. We also predict regulation of muscle-specific genes by ZNF9, adding a potential link to the myotonic dystrophy related phenotypes seen in ZNF9 mouse models.

Early Developmental Expression ofMus musculus Zinc Finger RNA-Binding ProteinCompared to Orthologs inCaenorhabditis elegansandDanio rerioand Subcellular Localization ofMus musculusandCaenorhabditis elegansZinc Finger RNA-Binding Protein in 2-CellMus musculusEmbryos

In mouse, knock-out of the Zfr gene encoding the zinc finger RNA-binding protein (ZFR) is associated with early lethality during gastrulation, suggesting that a pool of maternally contributed Zfr mRNA might compensate to allow development. ZFR is an ancient and highly conserved chromosome-associated protein from nematodes to mammals. We characterized expression of the Zfr transcript during early development in Mus musculus, Danio rerio, and Caenorhabditis elegans by quantitative real-time polymerase chain reaction. Mouse Zfr mRNA was detected in all stages tested during mouse preimplantation, with higher levels at the 1-cell stage that includes the maternal contribution of Zfr mRNA. In D. rerio, Zfr mRNA expression was highest in unfertilized eggs and declines throughout development. In C. elegans, Zfr mRNA expression was barely detectable in the fertilized egg and the L1 stage, but increased in the adult organism. Microinjections of green fluorescent protein (GFP)-tagged versions of in vitro-transcribed mouse and C. elegans Zfr mRNAs into early mouse embryos allowed analysis of the intracellular localization of the protein. Mouse ZFR-GFP was localized in the nucleus in 2-cell stage embryos although absent from nucleoli. Deletion studies revealed that this nuclear localization required the C-terminal part of ZFR, as deletion of the C-terminal resulted in the localization to the nuclear membrane. Despite the lack of a conserved nuclear localization signal, the C. elegans ZFR-GFP fusion protein also displayed an intranuclear localization in the 2-cell mouse embryo.

The RNA-binding zinc-finger protein tristetraprolin regulates AU-rich mRNAs involved in breast cancer-related processes

Tristetraprolin (TTP or ZFP36) is a tandem CCCH zinc-finger RNA-binding protein that regulates the stability of certain AU-rich element (ARE) mRNAs. Recent work suggests that TTP is deficient in cancer cells when compared with normal cell types. In this study we found that TTP expression was lower in invasive breast cancer cells (MDAMB231) compared with normal breast cell lines MCF12A and MCF-10. TTP targets were probed using a novel approach by expressing the C124R zinc-finger TTP mutant that functions as dominant negative and increases target mRNA expression. In contrast to wild-type TTP, C124R TTP was able to increase certain ARE-mRNA expressions in serum-stimulated breast cancer cells. Using an ARE-gene microarray, novel targets of TTP regulation were identified, namely, urokinase plasminogen activator (uPA), uPA receptor and matrix metalloproteinase-1, all known to have prominent roles in breast cancer invasion and metastasis. Expression of these targets was upregulated in tumorigenic types, particularly in highly invasive MDAMB231. The mRNA half-lives of these TTP-regulated genes were increased in TTP-knockout embryonic mouse fibroblasts, as assessed using real-time polymerase chain reaction, whereas forced restoration of TTP by transfection led to a reduction in their mRNA levels. RNA immunoprecipitation confirmed an association of TTP, but not C124R, with these target transcripts. Moreover, TTP reduced, whereas the mutant C124R TTP increased, the activity of reporter constructs fused to target ARE. As a result of TTP regulation, invasiveness of MDAMB231 cells was reduced. The data suggest that TTP, in a 3' untranslated region-and ARE-dependent manner, regulates an important subset of cancer-related genes that are involved in cellular growth, invasion and metastasis.

The endogenous peptides of normal human serum extracted from the acetonitrile-insoluble precipitate using modified aqueous buffer with analysis by LC–ESI–Paul ion trap and Qq-TOF

Many peptides of biological or medicinal importance may be derived from proteolytic actions and are found at low concentrations in human blood fluids. Endogenous polypeptides from human serum were precipitated in acetonitrile and the precipitate was then selectively extracted with water modified by organic solvents and collected over C18 resin. Extraction of serum with C18 alone, and the acetonitrile supernatant or ultrafiltration collected over C18, served as controls. The samples were analyzed by SDS-PAGE, or C18 high pressure liquid chromatography with electrospray ionization using a Paul ion trap and Qq-TOF. Spectra were correlated without specifying an enzyme using the X!TANDEM or the Paragon algorithms. Multiple endogenous peptides from plasminogen, coagulation factors, collagens, serum amyloid, receptors, zinc finger/bromo peptide proteins, ryanodine receptor, calmodulin binding activator, erythroid differentiation factor, testes cancer antigen, extracellular matrix protein, myeloid/lymphoid leukemia 2 and many low abundance proteins were correlated by X!TANDEM with protein expect values of approximately E-16 or less. Proteins with binding sites for nucleic acids, phosphoinositides, and other cellular locations were also observed using the Qq-TOF and Paragon algorithm. Proteins with low expectation scores and overlapping peptides sequences were observed. The existence of these proteins in serum has been confirmed by tryptic digestion and LC-ESI-MS/MS. The presence of plasminogen, serum amyloid and zinc finger RNA binding proteins were confirmed by Western blot. There was agreement on the detection of endogenous peptides from low abundance proteins associated with the biology of cancer from the examination of the blood peptides by ion trap and Qq-TOF, tryptic digests of blood proteins, and Western blot.

Targeted disruption of Zfp36l2, encoding a CCCH tandem zinc finger RNA-binding protein, results in defective hematopoiesis

AbstractMembers of the tristetraprolin family of tandem CCCH finger proteins can bind to AU-rich elements in the 3′-untranslated region of mRNAs, leading to their deadenylation and subsequent degradation. Partial deficiency of 1 of the 4 mouse tristetraprolin family members, Zfp36l2, resulted in complete female infertility because of early embryo death. We have now generated mice completely deficient in the ZFP36L2 protein. Homozygous Zfp36l2 knockout (KO) mice died within approximately 2 weeks of birth, apparently from intestinal or other hemorrhage. Analysis of peripheral blood from KO mice showed a decrease in red and white cells, hemoglobin, hematocrit, and platelets. Yolk sacs from embryonic day 11.5 (E11.5) Zfp36l2 KO mice and fetal livers from E14.5 KO mice gave rise to markedly reduced numbers of definitive multilineage and lineage-committed hematopoietic progenitors. Competitive reconstitution experiments demonstrated that Zfp36l2 KO fetal liver hematopoietic stem cells were unable to adequately reconstitute the hematopoietic system of lethally irradiated recipients. These data establish Zfp36l2 as a critical modulator of definitive hematopoiesis and suggest a novel regulatory pathway involving control of mRNA stability in the life cycle of hematopoietic stem and progenitor cells.

Targeted Disruption of Zfp36l2, Encoding a CCCH Tandem Zinc Finger RNA-Binding Protein, Results in Defective Hematopoiesis.

Members of the tristetraprolin (TTP) family of tandem CCCH finger proteins can bind to AU-rich elements in the 3′-untranslated region of mRNAs, leading to their deadenylation and subsequent degradation. In previous work, we disrupted the first exon of one of the four mouse TTP family members, Zfp36l2 (Tis11d, Brf2, Erf2), resulting in the production of decreased levels of a truncated protein lacking the first 29 amino acids. These mice exhibited complete female infertility, with embryos not progressing past the two-cell stage. In order to establish a true null phenotype for this gene, we have generated mice completely lacking the second exon, encoding the RNA-binding tandem zinc finger domain, resulting in a true knockout (KO) with complete lack of mRNA and protein. Surprisingly, these mice exhibited a completely unexpected phenotype involving the development of the hematopoietic system; they appeared otherwise anatomically normal. Homozygous Zfp36l2 KO mice on a mixed C57Bl/6 – 129SvEv background were born with normal Mendelian frequency but generally died about two weeks after birth, apparently from intestinal or other hemorrhage. Analysis of peripheral blood from KO mice at two weeks of age showed significant decreases in red cells (2.3 fold), white cells (2.1 fold), and platelets (11 fold). Flow cytometric analysis of spleen cells demonstrated significant decreases in myeloid cells (Gr-1+; Gr-1+/Mac-1+; 4 fold) and megakaryocytes (CD41+; 14 fold), as well as in c-Kit+ hematopoietic progenitors, without changes in lymphoid cell populations. Bone sections from rare surviving adult mice exhibited hematopoietic cell depletion. In addition, analysis of bone marrow revealed nine-fold decreases in lin-/Sca-1+/c-Kit+ cells; colony forming assays revealed that no hematopoietic progenitors grew from the KO bone marrow, compared to an average of 54 colonies per mouse from the wild-type (WT) mice. We therefore analyzed the development of the hematopoietic system. To do this, we cultured fetal liver cells in semisolid media that supported the proliferation and differentiation of multipotential and lineage-committed hematopoietic progenitors. There were significant decreases in the numbers of erythroid (BFU-E, 10-fold), granulocyte- macrophage (CFU-GM, 32-fold), granulocyte macrophage/macrophage (CFU-GM/M, 14-fold), and multipotential (CFU-GEMM, 14-fold) progenitor cells obtained from Zfp36l2 KO fetal liver cells at embryonic day (E) 14.5 as compared to WT cells from littermates. There were also statistically significant decreases in these progenitors in heterozygous mice compared to WT, suggesting a gene dosage effect. Similar studies of yolk sacs from E11.5 mice revealed significant decreases in myeloid (CFU-GM, 2-fold) and multipotential (CFU-GEMM, 1.7-fold) progenitors in the KO yolk sacs. Primitive hematopoiesis was unaffected, as assessed by in vitro colony forming assays with E8-8.25 yolk sac cells. Competitive reconstitution experiments demonstrated that Zfp36l2 KO fetal cells from E14.5 mice were markedly defective in reconstituting the hematopoietic system of lethally irradiated recipients after 1, 2 and 6 months of follow-up; engraftment rates for these dates were, respectively, 45% (WT) vs. 8% (KO); 65% vs. 3%; and 86% vs. 1%. These studies demonstrated that the development of the definitive hematopoietic system was severely adversely affected in the Zfp36l2 KO mice. This led to the hypothesis that elimination of the RNA destabilizing protein ZFP36L2 leads to the accumulation of one or more transcripts that are toxic to hematopoiesis. To explore this hypothesis, microarray analyses were performed on RNA samples from fetal liver at E14.5. We identified 239 significantly elevated transcripts in the KO samples, including several possible candidates for direct binding targets for ZFP36L2. In addition, 175 transcripts were significantly down-regulated in the KO fetal livers, in many cases in pathways of hematopoiesis or platelet development and function. These results are currently being validated by a variety of functional biochemical approaches, and are being supplemented by analogous microarray assays using transcripts from E11.5 yolk sacs. These data establish Zfp36l2 as a critical modulator of definitive hematopoiesis, and suggest a novel regulatory pathway involving control of mRNA stability in the life cycle of hematopoietic stem and progenitor cells.

The p53 target protein Wig-1 binds hnRNP A2/B1 and RNA Helicase A via RNA

The p53-induced Wig-1 gene encodes a double stranded RNA-binding zinc finger protein. We generated Saos-2 osteosarcoma cells expressing tetracycline-inducible Flag-tagged human Wig-1. Induction of Wig-1 expression by doxycycline inhibited cell growth in a long-term assay but did not cause any changes in cell cycle distribution nor increased fraction of apoptotic cells. Using co-immunoprecipitation and mass spectrometry, we identified two Wig-1-binding proteins, hnRNP A2/B1 and RNA Helicase A, both of which are involved in RNA processing. The binding was dependent on the presence of RNA. Our results establish a link between the p53 tumor suppressor and RNA processing via hnRNPA2/B1 and RNA Helicase A. Structured summary MINT- 6542926, MINT- 6542899: WIG1 (uniprotkb: Q9HA38) physically interacts ( MI:0218) with hnRNP A2/B1 (uniprotkb: P22626) by anti bait coimmunoprecipitation ( MI:0006) MINT- 6542945: RHA (uniprotkb: Q08211) physically interacts ( MI:0218) with hnRNP A2/B1 (uniprotkb: P22626) by anti bait coimmunoprecipitation ( MI:0006) MINT- 6542918, MINT- 6542891: WIG1 (uniprotkb: Q9HA38) physically interacts ( MI:0218) with RHA (uniprotkb: Q08211) by anti bait coimmunoprecipitation ( MI:0006) MINT- 6542867: WIG1 (uniprotkb: Q9HA38) physically interacts ( MI:0218) with RHA (uniprotkb: Q08211) by anti tag coimmunoprecipitation ( MI:0007) MINT- 6542879: WIG1 (uniprotkb: Q9HA38) physically interacts ( MI:0218) with hnRNP A2/B1(uniprotkb: P22626) by anti tag coimmunoprecipitation ( MI:0007)

Fine mapping and positional candidate studies on chromosome 5p13 identify multiple asthma susceptibility loci

Genome-wide linkage scans to identify asthma susceptibility loci have revealed many linked regions, including a broad region on chromosome 5p. To identify a 5p-linked asthma or bronchial hyperresponsiveness (BHR) locus. We performed fine mapping and positional candidate studies of this region in the Hutterites and an outbred case-control sample from Germany by genotyping 89 single nucleotide polymorphisms (SNPs) in 22 genes. SNP and haplotype analyses were performed. Three genes in a distal region (zinc finger RNA binding protein [ZFR], natriuretic peptide receptor C, and a disintegrin and metalloproteinase domain with thrombospondin type 1 motif [ADAMTS12]) were associated with BHR, whereas 4 genes in a proximal region (prolactin receptor, IL-7 receptor [IL7R], leukemia inhibitory factor receptor [LIFR], and prostaglandin E4 receptor [PTGER4]) were associated with asthma symptoms in the Hutterites. Furthermore, nearly the entire original linkage signal in the Hutterites was generated by individuals who had the risk-associated alleles in ZFR3, natriuretic peptide receptor C, ADAMTS12, LIFR, and PTGER4. Variation in ADAMTS12, IL7R, and PTGER4 were also associated with asthma in the outbred Germans, and the frequencies of long-range haplotypes composed of SNPs at ZFR, ADAMTS12, IL7R, LIFR, and PTGER4 were significantly different between both the German and Hutterite cases and controls. There is little linkage disequilbrium between alleles in these 2 regions in either population. These results suggest that a broad region on 5p, separated by >9 Mb, harbors at least 2 and possibly 5 asthma or BHR susceptibility loci. These findings are consistent with the hypothesis that regions providing evidence for linkage in multiple populations may, in fact, house more than 1 susceptibility locus, as appears to be the case for the linked region on 5p. Identifying asthma or BHR genes could lead to novel therapeutic approaches.

Antagonistic Functions of Tetradecanoyl Phorbol Acetate-Inducible-Sequence 11b and HuR in the Hormonal Regulation of Vascular Endothelial Growth Factor Messenger Ribonucleic Acid Stability by Adrenocorticotropin

Expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a potent angiogenic factor, is up-regulated by a variety of factors including hypoxia, growth factors, and hormones. In the adrenal cortex, regulation of VEGF expression by the pituitary hormone ACTH ensures the maintenance of the organ vasculature. We have previously shown that ACTH evokes a rapid and transient increase in VEGF mRNA levels in primary adrenocortical cells through transcription-independent mechanisms. We further demonstrated that the zinc finger RNA-binding protein Tis11b (tetradecanoyl phorbol acetate-inducible-sequence 11b) destabilizes VEGF mRNA through its 3'-untranslated region (3'-UTR) and that Tis11b is involved in the decay phase of ACTH-induced VEGF mRNA expression. In the present study, we attempted to determine the mechanisms underlying ACTH-elicited increase in VEGF mRNA levels in adrenocortical cells. We show that ACTH triggers an increase in the levels of the mRNA-stabilizing protein HuR in the cytoplasm and a concomitant decrease in the levels of HuR in the nucleus. This process is accompanied by an increased association of HuR with the nucleocytoplasmic shuttling protein pp32, indicating that ACTH induces HuR translocation from the nuclear to the cytoplasmic compartment. Leptomycin B, a specific inhibitor of CRM1-dependent nuclear export of pp32, significantly reduced ACTH-induced VEGF mRNA levels. Furthermore, RNA interference-mediated depletion of HuR in adrenocortical cells abrogated ACTH-induced VEGF mRNA expression. Finally, we show that Tis11b and HuR exert antagonistic effects on VEGF 3'-UTR in vitro. Although both proteins could bind simultaneously on VEGF 3'-UTR, Tis11b markedly decreases HuR-binding to this RNA sequence. Altogether, these results suggest that the RNA-stabilizing protein HuR is instrumental to ACTH-induced expression of VEGF mRNA and that the nuclear export of HuR is a rate-limiting step in this process. HuR appears to transiently stabilize VEGF transcripts after ACTH stimulation of adrenocortical cells, and Tis11b appears to subsequently trigger their degradation.

A novel mechanism of tumor suppression by destabilizing AU-rich growth factor mRNA.

The occurrence of pathologically stable mRNAs of proto-oncogenes, growth factors and cyclins has been proposed to contribute to experimental and human oncogenesis. In normal resting cells, mRNAs containing an AU-rich element (ARE) in their 3' untranslated region are subjected to rapid degradation. Tristetraprolin (TTP) is an RNA-binding zinc-finger protein that promotes decay of ARE-containing mRNAs. Here we report that TTP acts as a potent tumor suppressor in a v-H-ras-dependent mast cell tumor model, where tumors express abnormally stable interleukin-3 (IL-3) mRNA as part of an oncogenic autocrine loop. Premalignant v-H-ras cells were transfected with TTP and injected into syngeneic mice. TTP expression delayed tumor progression by 4 weeks, and late appearing tumors escaped suppression by loss of TTP. When transfected into a fully established tumor line, TTP reduced cloning efficiency in vitro and growth of the inoculated cells in vivo. Transgenic TTP interfered with the autocrine loop by enhancing the degradation of IL-3 mRNA with concomitant reduction of IL-3 secretion. Our data establish the ARE as an antioncogenic target in a model situation, underline the importance of mRNA stabilization in oncogenesis and show for the first time that tumor suppression can be achieved by interfering with mRNA turnover.

Two RNA Binding Proteins, HEN4 and HUA1, Act in the Processing of AGAMOUS Pre-mRNA in Arabidopsis thaliana

AGAMOUS, a key player in floral morphogenesis, specifies reproductive organ identities and regulates the timely termination of stem cell fates in the floral meristem. Here, we report that strains carrying mutations in three genes, HUA1, HUA2, and HUA ENHANCER4 ( HEN4), exhibit floral defects similar to those in agamous mutants: reproductive-to-perianth organ transformation and loss of floral determinacy. HEN4 codes for a K homology (KH) domain-containing, putative RNA binding protein that interacts with HUA1, a CCCH zinc finger RNA binding protein in the nucleus. We show that HUA1 binds AGAMOUS pre-mRNA in vitro and that HEN4, HUA1, and HUA2 act in floral morphogenesis by specifically promoting the processing of AGAMOUS pre-mRNA. Our studies underscore the importance of RNA processing in modulating plant development.

The RRM Protein NonA from Drosophila Forms a Complex with the RRM Proteins Hrb87F and S5 and the Zn Finger Protein PEP on hnRNA

The RRM protein NonA, an ubiquitous nuclear protein present in puffs on polytene chromosomes, has been immunopurified as a RNA–protein complex from Drosophila Kc cells. Three other proteins present in the complex have been identified: X4/PEP (protein on ecdysone puffs), a 100-kDa zinc finger RNA-binding protein; the 70-kDa S5 protein, an as yet uncharacterized RNA-binding protein; and P11/Hrb87F, a 38-kDa RRM protein homologous to hnRNP protein A1 from mammals. Monoclonal antibodies against any of the protein components coprecipitate all four proteins although at different ratios. NonA does not coprecipitate with the hrp40 hnRNP proteins and immunolocalizes in a pattern distinct of major hnRNP proteins. Like NonA, X4/PEP, S5, and P11/Hrb87F are present on active sites on polytene chromosomes. The precipitated NonA complex is enriched for certain protein encoding RNAs, notably, histone H3 and H4 RNA.

Structure, expression and in vitro functional characterization of a novel RNA binding zinc finger protein from Xenopus.

Large multigene families of zinc finger proteins are expressed in vertebrates. One way of approaching their function is to characterize their structure, expression and biochemical properties. XFG 5-1 is a Xenopus zinc finger protein which is widely transcribed in oocytes, embryos and adult tissues. It carries a novel, non-finger repeat structure, which is common to a subfamily of Xenopus zinc finger proteins. The bacterially expressed protein exhibits specific RNA homopolymer binding activities with the zinc finger domain being sufficient for this ability. These findings suggest that XFG 5-1 serves a general biological function involving its RNA binding capacity.