Expression of the human RNA-binding protein HuR in Trypanosoma brucei increases the abundance of mRNAs containing AU-rich regulatory elements

The salivarian trypanosome Trypanosoma brucei infects mammals and is transmitted by tsetse flies. The mammalian ‘bloodstream form’ trypanosome has a variant surface glycoprotein coat and relies on glycolysis while the procyclic form from tsetse flies has EP protein on the surface and has a more developed mitochondrion. We show here that the mRNA for the procyclic-specific cytosolic phosphoglycerate kinase PGKB, like that for EP proteins, contains a regulatory AU-rich element (ARE) that destabilises the mRNA in bloodstream forms. The human HuR protein binds to, and stabilises, mammalian mRNAs containing AREs. Expression of HuR in bloodstream-form trypanosomes resulted in growth arrest and in stabilisation of the EP, PGKB and pyruvate, phosphate dikinase mRNAs, while three bloodstream-specific mRNAs were reduced in abundance. The synthesis and abundance of unregulated mRNAs and proteins were unaffected. Our results suggest that regulation of mRNA stability by AREs arose early in eukaryotic evolution.     

Sex-Specific Effects of Cis-Regulatory Variants in Drosophila melanogaster

Sexual dimorphism at the level of gene expression is common and well documented, but much less is known about how different cis-regulatory alleles interact with the different trans-regulatory environments present in males and females. Here we show that sex-specific effects of cis-regulatory variants are common in Drosophila.     

Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells

The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells.     

Coevolution within and between Regulatory Loci Can Preserve Promoter Function Despite Evolutionary Rate Acceleration

Phenotypes that appear to be conserved could be maintained not only by strong purifying selection on the underlying genetic systems, but also by stabilizing selection acting via compensatory mutations with balanced effects. Such coevolution has been invoked to explain experimental results, but has rarely been the focus of study. Conserved expression driven by the unc-47 promoters of Caenorhabditis elegans and C. briggsae persists despite divergence within a cis-regulatory element and between this element and the trans-regulatory environment. Compensatory changes in cis and trans are revealed when these promoters are used to drive expression in the other species. Functional changes in the C. briggsae promoter, which has experienced accelerated sequence evolution, did not lead to alteration of gene expression in its endogenous environment. Coevolution among promoter elements suggests that complex epistatic interactions within cis-regulatory elements may facilitate their divergence. Our results offer a detailed picture of regulatory evolution in which subtle, lineage-specific, and compensatory modifications of interacting cis and trans regulators together maintain conserved gene expression patterns.     

Independent Pathways Can Transduce the Life-Cycle Differentiation Signal in Trypanosoma brucei

African trypanosomes cause disease in humans and livestock, generating significant health and welfare problems throughout sub-Saharan Africa. When ingested in a tsetse fly bloodmeal, trypanosomes must detect their new environment and initiate the developmental responses that ensure transmission. The best-established environmental signal is citrate/cis aconitate (CCA), this being transmitted through a protein phosphorylation cascade involving two phosphatases: one that inhibits differentiation (TbPTP1) and one that activates differentiation (TbPIP39). Other cues have been also proposed (mild acid, trypsin exposure, glucose depletion) but their physiological relevance and relationship to TbPTP1/TbPIP39 signalling is unknown. Here we demonstrate that mild acid and CCA operate through TbPIP39 phosphorylation, whereas trypsin attack of the parasite surface uses an alternative pathway that is dispensable in tsetse flies. Surprisingly, glucose depletion is not an important signal. Mechanistic analysis through biophysical methods suggests that citrate promotes differentiation by causing TbPTP1 and TbPIP39 to interact.     

POS-1 and GLD-1 repress glp-1 translation through a conserved binding-site cluster

RNA-binding proteins (RBPs) coordinate cell fate specification and differentiation in a variety of systems. RNA regulation is critical during oocyte development and early embryogenesis, in which RBPs control expression from maternal mRNAs encoding key cell fate determinants. The Caenorhabditis elegans Notch homologue glp-1 coordinates germline progenitor cell proliferation and anterior fate specification in embryos. A network of sequence-specific RBPs is required to pattern GLP-1 translation. Here, we map the cis-regulatory elements that guide glp-1 regulation by the CCCH-type tandem zinc finger protein POS-1 and the STAR-domain protein GLD-1. Our results demonstrate that both proteins recognize the glp-1 3′ untranslated region (UTR) through adjacent, overlapping binding sites and that POS-1 binding excludes GLD-1 binding. Both factors are required to repress glp-1 translation in the embryo, suggesting that they function in parallel regulatory pathways. It is intriguing that two equivalent POS-1–binding sites are present in the glp-1 3′ UTR, but only one, which overlaps with a translational derepression element, is functional in vivo. We propose that POS-1 regulates glp-1 mRNA translation by blocking access of other RBPs to a key regulatory sequence.     

Functional Analysis of the Human Immunodeficiency Virus Type 1 Rev Protein Oligomerization Interface

The expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the action of the viral trans-regulatory protein Rev. Rev is a nuclear shuttle protein that directly binds to its cis-acting Rev response element (RRE) RNA target sequence. Subsequent oligomerization of Rev monomers on the RRE and interaction of Rev with a cellular cofactor(s) result in the cytoplasmic accumulation of RRE-containing viral mRNAs. Moreover, Rev by itself is exported from the nucleus to the cytoplasm. Although it has been demonstrated that Rev multimerization is critically required for Rev activity and hence for HIV-1 replication, the number of Rev monomers required to form a trans-activation-competent complex on the RRE is unknown. Here we report a systematic analysis of the putative multimerization domains within the Rev trans-activator protein. We identify the amino acid residues which are part of the proposed single hydrophobic surface patch in the Rev amino terminus that mediates intermolecular interactions. Furthermore, we show that the expression of a multimerization-deficient Rev mutant blocks HIV-1 replication in a trans-dominant (dominant-negative) fashion.     

The interaction of platinum complexes with nucleosomes investigated with fluorescent probes

The fluorescent probes, N-(3-pyrene)maleimide, which is specific for histone H3, and terbium (Tb³⁺), which is specific for guanine single-stranded residues in DNA, are used to investigate the interaction of platinum complexes (cis- and trans-dichlorodiammineplatinum(II)) with rat liver and calf thymus nucleosomes. At low concentrations of the drug, lower than most of those reported previously in studies investigating the interaction of the drugs with isolated DNA, N-(3-pyrene)maleimide studies show that profound modifications occur near or in the cysteinyl binding site of histone H3. H3 dimer formation appears to be the cause of the change induced by trans-DDP; however, the effects observed with the cis-isomer do not seem to be correlated with dimer formation. At short incubation times, Tb³⁺ fluorescence shows small changes in DNA conformation, but they are slight when compared to the effect observed with proteins at the same length of incubation. SDS-polyacrylamide gels indicate some changes in protein composition, and agarose gels display a decrease in ethidium bromide staining of the cis-treated DNA. The results suggest that the protein portion, predominantly histone H3, as well as DNA are targets for the platinum derivatives in the nucleosome.     

Insulin increases tristetraprolin and decreases VEGF gene expression in mouse 3T3-L1 adipocytes

Objectives: Tristetraprolin (TTP) family proteins (TTP/ZFP36; ZFP36L1, ZFP36L2, ZFP36L3) destabilize adenylate uridylate‐rich element‐containing mRNAs encoding cytokines, such as tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF). Little is known about the expression and insulin regulation of TTP and related genes in adipocytes. We analyzed the relative abundance of TTP family mRNAs in 3T3‐L1 adipocytes compared to RAW264.7 macrophages and investigated insulin effects on the expression of 43 genes in 3T3‐L1 adipocytes. Methods and Procedures: Insulin was added to mouse 3T3‐L1 adipocytes. Relative abundance of mRNA levels was determined by quantitative real‐time PCR. TTP and ZFP36L1 proteins were detected by immunoblotting. Results: Zfp36l1 and Zfp36l2 genes were expressed at eight‐ to tenfold higher than Ttp in adipocytes. Zfp36l3 mRNA was detected at ～1% of Ttp mRNA levels in adipocytes and its low level expression was confirmed in RAW cells. Insulin at 10 and 100 nmol/l increased Ttp mRNA levels by five‐ to sevenfold, but decreased those of Zfp36l3 by 40% in adipocytes after a 30‐min treatment. Immunoblotting showed that insulin induced TTP but did not affect ZFP36L1 protein levels in adipocytes. Insulin decreased mRNA levels of Vegf and a number of other genes in adipocytes. Discussion: Insulin induced Ttp mRNA and protein expression and decreased Vegf mRNA levels in adipocytes. Zfp36l3 mRNA was detected, for the first time, in cells other than mouse placenta and extraembryonic tissues. This study established a basis for the investigation of TTP and VEGF genes in the regulation of obesity and suggested that Vegf mRNA may be a target of TTP in fat cells.