Riboregulation in Escherichia coli: DsrA RNA acts by RNA:RNA interactions at multiple loci

DsrA is an 87-nt untranslated RNA that regulates both the global transcriptional silencer and nucleoid protein H-NS and the stationary phase and stress response sigma factor RpoS (σs). We demonstrate that DsrA acts via specific RNA:RNA base pairing interactions at the hns locus to antagonize H-NS translation. We also give evidence that supports a role for RNA:RNA interactions at the rpoS locus to enhance RpoS translation. Negative regulation of hns by DsrA is achieved by the RNA:RNA interaction blocking translation of hns RNA. In contrast, results suggest that positive regulation of rpoS by DsrA occurs by formation of an RNA structure that activates a cis-acting translational operator. Sequences within DsrA complementary to three additional genes, argR, ilvIH, and rbsD, suggest that DsrA is a riboregulator of gene expression that acts coordinately via RNA:RNA interactions at multiple loci.

In vitro activation of the interferon-induced, double-stranded RNA-dependent protein kinase PKR by RNA from the 3' untranslated regions of human alpha-tropomyosin.

The cellular kinase known as PKR (protein kinase RNA-activated) is induced by interferon and activated by RNA. PKR is known to have antiviral properties due to its role in translational control. Active PKR phosphorylates eukaryotic initiation factor 2 alpha and leads to inhibition of translation, including viral translation. PKR is also known to function as a tumor suppressor, presumably by limiting the rate of tumor-cell translation and growth. Recent research has shown that RNA from the 3' untranslated region (3'UTR) of human alpha-tropomyosin has tumor-suppressor properties in vivo [Rastinejad, F., Conboy, M. J., Rando, T. A. & Blau, H. M. (1993) Cell 75, 1107-1117]. Here we report that purified RNA from the 3'UTR of human alpha-tropomyosin can inhibit in vitro translation in a manner consistent with activation of PKR. Inhibition of translation by tropomyosin 3'UTR RNA was observed in a rabbit reticulocyte lysate system, which is known to contain endogenous PKR but was not seen in wheat germ lysate, which is not responsive to a known activator of PKR. A control RNA purified in the same manner as the 3'UTR RNA did not inhibit translation in either system. The inhibition of translation observed in reticulocyte lysates was prevented by the addition of adenovirus virus-associated RNA1 (VA RNAI), an inhibitor of PKR activation. Tropomyosin 3'UTR RNA was bound by immunoprecipitated PKR and activated the enzyme in an in vitro kinase assay. These data suggest that activation of PKR could be the mechanism by which tropomyosin 3'UTR RNA exerts its tumor-suppression activity in vivo.

Single-site cleavage in the 5'-untranslated region of Leishmaniavirus RNA is mediated by the viral capsid protein.

Leishmaniavirus (LRV) is a double-stranded RNA virus that persistently infects the protozoan parasite Leishmania. LRV produces a short RNA transcript, corresponding to the 5' end of positive-sense viral RNA, both in vivo and in in vitro polymerase assays. The short transcript is generated by a single site-specific cleavage event in the 5' untranslated region of the 5.3-kb genome. This cleavage event can be reproduced in vitro with purified viral particles and a substrate RNA transcript possessing the viral cleavage site. A region of nucleotides required for cleavage was identified by analyzing the cleavage sites yielding the short transcripts of various LRV isolates. A 6-nt deletion at this cleavage site completely abolished RNA processing. In an in vitro cleavage assay, baculovirus-expressed capsid protein possessed an endonuclease activity identical to that of native virions, showing that the viral capsid protein is the RNA endonuclease. Identification of the LRV capsid protein as an RNA endonuclease is unprecedented among known viral capsid proteins.

RNA folding kinetics regulates translation of phage MS2 maturation gene

The gene for the maturation protein of the single-stranded RNA coliphage MS2 is preceded by an untranslated leader of 130 nt, which folds into a cloverleaf, i.e., three stem–loop structures enclosed by a long distance interaction (LDI). This LDI prevents translation because its 3′ moiety contains the Shine–Dalgarno sequence of the maturation gene. Previously, several observations suggested that folding of the cloverleaf is kinetically delayed, providing a time window for ribosomes to access the RNA. Here we present direct evidence for this model. In vitro experiments show that ribosome binding to the maturation gene is faster than refolding of the denatured cloverleaf. This folding delay appears related to special properties of the leader sequence. We have replaced the three stem–loop structures by a single five nt loop. This change does not affect the equilibrium structure of the LDI. Nevertheless, in this construct, the folding delay has virtually disappeared, suggesting that now the RNA folds faster than ribosomes can bind. Perturbation of the cloverleaf by an insertion makes the maturation start permanently accessible. A pseudorevertant that evolved from an infectious clone carrying the insertion had overcome this defect. It showed a wild-type folding delay before closing down the maturation gene. This experiment reveals the biological significance of retarded cloverleaf formation.

Altered phosphorylation and intracellular distribution of a (CUG)n triplet repeat RNA-binding protein in patients with myotonic dystrophy and in myotonin protein kinase knockout mice

Myotonic dystrophy (DM) is associated with expansion of CTG repeats in the 3′-untranslated region of the myotonin protein kinase (DMPK) gene. The molecular mechanism whereby expansion of the (CUG)n repeats in the 3′-untranslated region of DMPK gene induces DM is unknown. We previously isolated a protein with specific binding to CUG repeat sequences (CUG-BP/hNab50) that possibly plays a role in mRNA processing and/or transport. Here we present evidence that the phosphorylation status and intracellular distribution of the RNA CUG-binding protein, identical to hNab50 protein (CUG-BP/hNab50), are altered in homozygous DM patient and that CUG-BP/hNab50 is a substrate for DMPK both in vivo and in vitro. Data from two biological systems with reduced levels of DMPK, homozygous DM patient and DMPK knockout mice, show that DMPK regulates both phosphorylation and intracellular localization of the CUG-BP/hNab50 protein. Decreased levels of DMPK observed in DM patients and DMPK knockout mice led to the elevation of the hypophosphorylated form of CUG-BP/hNab50. Nuclear concentration of the hypophosphorylated CUG-BP/hNab50 isoform is increased in DMPK knockout mice and in homozygous DM patient. DMPK also interacts with and phosphorylates CUG-BP/hNab50 protein in vitro. DMPK-mediated phosphorylation of CUG-BP/hNab50 results in dramatic reduction of the CUG-BP2, hypophosphorylated isoform, accumulation of which was observed in the nuclei of DMPK knockout mice. These data suggest a feedback mechanism whereby decreased levels of DMPK could alter phosphorylation status of CUG-BP/hNab50, thus facilitating nuclear localization of CUG-BP/hNab50. Our results suggest that DM pathophysiology could be, in part, a result of sequestration of CUG-BP/hNab50 and, in part, of lowered DMPK levels, which, in turn, affect processing and transport of specific subclass of mRNAs.

Double-stranded RNA induces mRNA degradation in Trypanosoma brucei

Double-stranded RNA (dsRNA) recently has been shown to give rise to genetic interference in Caenorhabditis elegans and also is likely to be the basis for phenotypic cosuppression in plants in certain instances. While constructing a plasmid vector for transfection of trypanosome cells, we serendipitously discovered that in vivo expression of dsRNA of the α-tubulin mRNA 5′ untranslated region (5′ UTR) led to multinucleated cells with striking morphological alterations and a specific block of cytokinesis. Transfection of synthetic α-tubulin 5′ UTR dsRNA, but not of either strand individually, caused the same phenotype. On dsRNA transfection, tubulin mRNA, but not the corresponding pre-mRNA, was rapidly and specifically degraded, leading to a deficit of α-tubulin synthesis. The transfected cells were no longer capable of carrying out cytokinesis and eventually died. Analysis of cytoskeletal structures from these trypanosomes revealed defects in the microtubules of the flagellar axoneme and of the flagellar attachment zone, a complex cortical structure that we propose is essential for establishing the path of the cleavage furrow at cytokinesis. Last, dsRNA-mediated mRNA degradation is not restricted to α-tubulin mRNA but can be applied to other cellular mRNAs, thus establishing a powerful tool to genetically manipulate these important protozoan parasites.

Hypoxic Regulation of Vascular Endothelial Growth Factor mRNA Stability Requires the Cooperation of Multiple RNA Elements

Vascular endothelial growth factor (VEGF) is a key regulator of developmental, physiological, and tumor angiogenesis. Upregulation of VEGF expression by hypoxia appears to be a critical step in the neovascularization of solid cancers. The VEGF mRNA is intrinsically labile, but in response to hypoxia the mRNA is stabilized. We have systematically analyzed the regions in the VEGF mRNA that are responsible for its lability under normoxic conditions and for stabilization in response to hypoxia. We find that the VEGF mRNA not only contains destabilizing elements in its 3′ untranslated region (3′UTR), but also contains destabilizing elements in the 5′UTR and coding region. Each region can independently promote mRNA degradation, and together they act additively to effect rapid degradation under normoxic conditions. Stabilization of the mRNA in response to hypoxia is completely dependent on the cooperation of elements in each of the 5′UTR, coding region, and 3′UTR. Combinations of any of two of these three regions were completely ineffective in responding to hypoxia, whereas combining all three regions allowed recapitulation of the hypoxic stabilization seen with the endogenous VEGF mRNA. We conclude that multiple regions in the VEGF mRNA cooperate both to ensure the rapid degradation of the mRNA under normoxic conditions and to allow stabilization of the mRNA in response to hypoxia. Our findings highlight the complexity of VEGF gene expression and also reveal a mechanism of gene regulation that could become the target for strategies of therapeutic intervention.

Identification of a Human VPF/VEGF 3′ Untranslated Region Mediating Hypoxia-induced mRNA Stability

Hypoxia is a prominent feature of malignant tumors that are characterized by angiogenesis and vascular hyperpermeability. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) has been shown to be up-regulated in the vicinity of necrotic tumor areas, and hypoxia potently induces VPF/VEGF expression in several tumor cell lines in vitro. Here we report that hypoxia-induced VPF/VEGF expression is mediated by increased transcription and mRNA stability in human M21 melanoma cells. RNA-binding/electrophoretic mobility shift assays identified a single 125-bp AU-rich element in the 3′ untranslated region that formed hypoxia-inducible RNA-protein complexes. Hypoxia-induced expression of chimeric luciferase reporter constructs containing this 125-bp AU-rich hypoxia stability region were significantly higher than constructs containing an adjacent 3′ untranslated region element without RNA-binding activity. Using UV-cross-linking studies, we have identified a series of hypoxia-induced proteins of 90/88 kDa, 72 kDa, 60 kDa, 56 kDa, and 46 kDa that bound to the hypoxia stability region element. The 90/88-kDa and 60-kDa species were specifically competed by excess hypoxia stability region RNA. Thus, increased VPF/VEGF mRNA stability induced by hypoxia is mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3′ untranslated region and distinct mRNA-binding proteins in human tumor cells.

In vivo analysis of the 3′ untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone

Large sections of the 3′ untranslated region (UTR) of hepatitis C virus (HCV) were deleted from an infectious cDNA clone, and the RNA transcripts from seven deletion mutants were tested sequentially for infectivity in a chimpanzee. Mutants lacking all or part of the 3′ terminal conserved region or the poly(U–UC) region were unable to infect the chimpanzee, indicating that both regions are critical for infectivity in vivo. However, the third region, the variable region, was able to tolerate a deletion that destroyed the two putative stem–loop structures within this region. Mutant VR-24 containing a deletion of the proximal 24 nt of the variable region of the 3′ UTR was viable in the chimpanzee and seemed to replicate as well as the undeleted parent virus. The chimpanzee became viremic 1 week after inoculation with mutant VR-24, and the HCV genome titer increased over time during the early acute infection. Therefore, the poly(U–UC) region and the conserved region, but not the variable region, of the 3′ UTR seem to be critical for in vivo infectivity of HCV.

Long RNA hairpins that contain inosine are present in Caenorhabditis elegans poly(A)+ RNA

Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that convert adenosine to inosine within double-stranded RNA. In the 12 years since the discovery of ADARs only a few natural substrates have been identified. These substrates were found by chance, when genomically encoded adenosines were identified as guanosines in cDNAs. To advance our understanding of the biological roles of ADARs, we developed a method for systematically identifying ADAR substrates. In our first application of the method, we identified five additional substrates in Caenorhabditis elegans. Four of those substrates are mRNAs edited in untranslated regions, and one is a noncoding RNA edited throughout its length. The edited regions are predicted to form long hairpin structures, and one of the RNAs encodes POP-1, a protein involved in cell fate decisions.

Posttranscriptional regulation of urokinase plasminogen activator receptor messenger RNA levels by leukocyte integrin engagement

As an adhesion receptor, the β2 integrin lymphocyte function-associated antigen-1 (LFA-1) contributes a strong adhesive force to promote T lymphocyte recirculation and interaction with antigen-presenting cells. As a signaling molecule, LFA-1-mediates transmembrane signaling, which leads to the generation of second messengers and costimulation resulting in T cell activation. We recently have demonstrated that, in costimulatory fashion, LFA-1 activation promotes the induction of T cell membrane urokinase plasminogen activator receptor (uPAR) and that this induced uPAR is functional. To investigate the mechanism(s) of this induction, we used the RNA polymerase II inhibitor 5,6-dichloro-1-β-d-ribobenzimidazole and determined that uPAR mRNA degradation is delayed by LFA-1 activation. Cloning of the wild-type, deleted and mutated 3′-untranslated region of the uPAR cDNA into a serum-inducible rabbit β-globin cDNA reporter construct revealed that the AU-rich elements and, in particular the nonameric UUAUUUAUU sequence, are crucial cis-acting elements in uPAR mRNA degradation. Experiments in which Jurkat T cells were transfected with reporter constructs demonstrated that LFA-1 engagement was able to stabilize the unstable reporter mRNA containing the uPAR 3′-untranslated region. Our study reveals a consequence of adhesion receptor-mediated signaling in T cells, which is potentially important in the regulation of T cell activation, including production of cytokines and expression of proto-oncogenes, many of which are controlled through 3′ AU-rich elements.

Polymorphism in the 3′-untranslated region of TNFα mRNA impairs binding of the post-transcriptional regulatory protein HuR to TNFα mRNA

Tumor necrosis factor α (TNFα) acts as a beneficial mediator in the process of host defence. In recent years major interest has focused on the AU-rich elements (AREs) present in the 3′-untranslated region (3′-UTR) of TNFα mRNA as this region plays a pivotal role in post-transcriptional control of TNFα production. Certain stimuli, such as lipopolysaccharides, a component of the Gram-negative bacterial cell wall, have the ability to relinquish the translational suppression of TNFα mRNA imposed by these AREs in macrophages, thereby enabling the efficient production of the TNFα. In this study we show that the polymorphism (GAU trinucleotide insertional mutation) present in the regulatory 3′-UTR of TNFα mRNA of NZW mice results in the hindered binding of RNA-binding proteins, thereby leading to a significantly reduced production of TNFα protein. We also show that the binding of macrophage proteins to the main ARE is also decreased by another trinucleotide (CAU) insertion in the TNFα 3′-UTR. One of the proteins affected by the GAU trinucleotide insertional mutation was identified as HuR, a nucleo-cytoplasmic shuttling protein previously shown to play a prominent role in the stability and translatability of mRNA containing AREs. Since binding of this protein most likely modulates the stability, translational efficiency and transport of TNFα mRNA, these results suggest that mutations in the ARE of TNFα mRNA decrease the production of TNFα protein in macrophages by hindering the binding of HuR to the ARE.

RNA binding by the Wilms tumor suppressor zinc finger proteins.

The Wilms tumor suppressor gene WT1 is implicated in the ontogeny of genito-urinary abnormalities, including Denys-Drash syndrome and Wilms tumor of the kidney. WT1 encodes Kruppel-type zinc finger proteins that can regulate the expression of several growth-related genes, apparently by binding to specific DNA sites located within 5' untranslated leader regions as well as 5' promoter sequences. Both WT1 and a closely related early growth response factor, EGR1, can bind the same DNA sequences from the mouse gene encoding insulin-like growth factor 2 (Igf-2). We report that WT1, but not EGR1, can bind specific Igf-2 exonic RNA sequences, and that the zinc fingers are required for this interaction. WT1 zinc finger 1, which is not represented in EGR1, plays a more significant role in RNA binding than zinc finger 4, which does have a counterpart in EGR1. Furthermore, the normal subnuclear localization of WT1 proteins is shown to be RNase, but not DNase, sensitive. Therefore, WT1 might, like the Kruppel-type zinc finger protein TFIIIA, regulate gene expression by both transcriptional and posttranscriptional mechanisms.

Antisense globin RNA in mouse erythroid tissues: structure, origin, and possible function.

The aim of the experiments described in this paper was to test for the presence of antisense globin RNA in mouse erythroid tissues and, if found, to characterize these molecules. The present study made use of a multistep procedure in which a molecular tag is attached to cellular RNA by ligation with a defined ribooligonucleotide. The act of ligation preserves the termini of RNA molecules, which become the junctions between cellular RNAs and the ligated ribooligonucleotide. It also unambiguously preserves the identity of cellular RNA as a sense or antisense molecule through all subsequent manipulations. Using this approach, we identified and characterized antisense beta-globin RNA in erythroid spleen cells and reticulocytes from anemic mice. We show in this paper that the antisense globin RNA is fully complementary to spliced globin mRNA, indicative of the template/transcript relationship. It terminates at the 5' end with a uridylate stretch, reflecting the presence of poly(A) at the 3' end of the sense globin mRNA. With respect to the structure of their 3' termini, antisense globin RNA can be divided into three categories: full-size molecules corresponding precisely to globin mRNA, truncated molecules lacking predominantly 14 3'-terminal nucleotides, and extended antisense RNA containing 17 additional 3'-terminal nucleotides. The full-size antisense globin RNA contains two 14-nt-long complementary sequences within its 3'-terminal segment corresponding to the 5'-untranslated region of globin mRNA. This, together with the nature of the predominant truncation, suggests a mechanism by which antisense RNA might give rise to new sense-strand globin mRNA.

Testis/brain RNA-binding protein attaches translationally repressed and transported mRNAs to microtubules.

We have previously identified a testicular phosphoprotein that binds to highly conserved sequences (Y and H elements) in the 3' untranslated regions (UTRs) of testicular mRNAs and suppresses in vitro translation of mRNA constructs that contain these sequences. This protein, testis/brain RNA-binding protein (TB-RBP) also is abundant in brain and binds to brain mRNAs whose 3' UTRs contain similar sequences. Here we show that TB-RBP binds specific mRNAs to microtubules (MTs) in vitro. When TB-RBP is added to MTs reassembled from either crude brain extracts or from purified tubulin, most of the TB-RBP binds to MTs. The association of TB-RBP with MTs requires the assembly of MTs and is diminished by colcemid, cytochalasin D, and high levels of salt. Transcripts from the 3' UTRs of three mRNAs that contain the conserved sequence elements (transcripts for protamine 2, tau protein, and myelin basic protein) are linked by TB-RBP to MTs, whereas transcripts that lack the conserved sequences do not bind TB-RBP. We conclude that TB-RBP serves as an attachment protein for the MT association of specific mRNAs. Considering its ability to arrest translation in vitro, we propose that TB-RBP functions in the storage and transportation of mRNAs to specific intracellular sites where they are translated.