Altered ratios of alternatively spliced long and short γ2 subunit mRNAs of the γ-amino butyrate type A receptor in prefrontal cortex of schizophrenics

The relative abundance of alternatively spliced long (γ2L) and short (γ2S) mRNAs of the γ2 subunit of the γ-amino butyrate type A (GABAA) receptor was examined in dorsolateral prefrontal cortex of schizophrenics and matched controls by using in situ hybridization histochemistry and semiquantitative reverse transcription–PCR (RT-PCR) amplification. A cRNA probe identifying both mRNAs showed that the transcripts are normally expressed at moderately high levels in the prefrontal cortex. Consistent with previous studies, overall levels of γ2 transcripts in prefrontal cortex of brains from schizophrenics were reduced by 28.0%, although this reduction did not reach statistical significance. RT-PCR, performed under nonsaturating conditions on total RNA from the same blocks of tissue used for in situ hybridization histochemistry, revealed a marked reduction in the relative proportion of γ2S transcripts in schizophrenic brains compared with controls. In schizophrenics, γ2S transcripts had fallen to 51.7% (±7.9% SE; P < 0.0001) relative to control levels. Levels of γ2L transcripts showed only a small and nonsignificant reduction of 16.9% (±12.0% SE, P > 0.05). These findings indicate differential transcriptional regulation of two functionally distinct isoforms of one of the major GABAA receptor subunits in the prefrontal cortex of schizophrenics. The specific reduction in relative abundance of γ2S mRNAs and the associated relative increase in γ2L mRNAs should result in functionally less active GABAA receptors and have severe consequences for cortical integrative function.

Characterization and tissue-specific expression of the rat basic fibroblast growth factor antisense mRNA and protein

An RNA transcribed from the antisense strand of the FGF-2 gene has been implicated in the regulation of FGF-2 mRNA stability in amphibian oocytes. We have now cloned and characterized a novel 1.1-kb mRNA (fgf-as) from neonatal rat liver. In non-central nervous system (CNS) tissues the fgf-as RNA is abundantly expressed in a developmentally regulated manner. The FGF-AS cDNA contains a consensus polyadenylylation signal and a long open reading frame (ORF) whose deduced amino acid sequence predicts a 35-kDa protein with homology to the MutT family of nucleotide hydrolases. Western blot analysis with antibodies against the deduced peptide sequence demonstrates that the FGF-AS protein is expressed in a broad range of non-CNS tissue in the postnatal period. In the developing brain, the abundance of sense and antisense transcripts are inversely related, suggesting a role for the antisense RNA in posttranscriptional regulation of FGF-2 expression in this tissue.The FGF-AS is complementary to two widely separated regions in the long 3′ untranslated region of the FGF-2 mRNA, in the vicinity of the proximal and distal polyadenylylation sites. These findings demonstrate that the FGF-2 and fgf-as RNAs are coordinately transcribed on a tissue-specific and developmentally regulated basis and suggest that interaction of the sense and antisense RNAs may result in posttranscriptional regulation of FGF-2 in some tissues.

Peptide nucleic acid–DNA duplexes: Long range hole migration from an internally linked anthraquinone

The discovery that peptide nucleic acids (PNA) mimic DNA and RNA by forming complementary duplex structures following Watson–Crick base pairing rules opens fields in biochemistry, diagnostics, and medicine for exploration. Progress requires the development of modified PNA duplexes having unique and well defined properties. We find that anthraquinone groups bound to internal positions of a PNA oligomer intercalate in the PNA–DNA hybrid. Their irradiation with near-UV light leads to electron transfer and oxidative damage at remote GG doublets on the complementary DNA strand. This behavior mimics that observed in related DNA duplexes and provides the first evidence for long range electron (hole) transport in PNA–DNA hybrid. Analysis of the mechanism for electron transport supports hole hopping.

Genomic and cDNA sequence analysis of the cell matrix adhesion regulator gene

The cell matrix adhesion regulator (CMAR) gene has been suggested to be a signal transduction molecule influencing cell adhesion to collagen and, through this, possibly involved in tumor suppression. The originally reported CMAR cDNA was 464 bp long with a tyrosine phosphorylation site at the extreme 3′ end, which mutagenesis studies had shown to be central to the function of this gene. Since the discovery of a 4-bp insertion polymorphism within the originally reported coding region, further sequence information has been obtained. The cDNA has been extended 5′ by ≈2 kb revealing a 559-bp region showing strong homology to the proposed 5′ untranslated sequence of a murine protein kinase receptor family member, variant in kinase (vik). CMAR genomic sequencing has shown the presence of an intron, the intron/exon boundary lying within this region of homology. An RNA transcript for CMAR of ≈2.5 kb has also been identified. The data suggest complex mechanisms for control of expression of two closely associated genes, CMAR and the vik- associated sequence.

Neurotrophin-3 signals redistribute RNA in neurons

The translocation of specific mRNAs to dendrites and their potential for locally regulated translation are likely to serve as an effector in neuronal plasticity. Whether translation in dendrites is regulated by delivery of the RNA to sites of plasticity or a stationary pool of localized RNA undergoes enhanced translational efficiency is not clear. We show that RNA can translocate into dendrites in response to NT-3. RNA granules were visualized in cultured rat cortical neurons using the dye SYTO 14, which labels poly-ribosome complexes. Long before the morphological effects of NT-3 appeared, there was increased distal translocation of labeled complexes. This effect was blocked by K252a, a potent inhibitor of tyrosine kinase receptors. Therefore, neurons can utilize extracellular signals to alter the distribution of protein synthetic machinery via the active transport of RNA granules.

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.

Complex formation between stage-specific oocyte factors and a Xenopus mRNA localization element

It is a long-standing proposal that localization of maternal factors in eggs can provide the basis for pattern formation in the early embryo. The localized information can be stored as RNA, one example being Vg1 RNA, which is localized exclusively to the vegetal hemisphere of Xenopus oocytes and eggs. Localization of Vg1 mRNA is directed by a 340-nt sequence element contained within its 3′ untranslated region. To understand the mechanism of localization, I have tested whether factors from the oocyte interact specifically with the RNA localization sequence. Results presented here show that a set of oocyte proteins form complexes with the localization element both in vitro and in vivo. These proteins are specifically enriched in the stages of oogenesis during which localization occurs and recognize sub-elements of the RNA localization element that are essential for localization in vivo. These data suggest that formation of a localization-specific RNA–protein complex may be the first step in directing Vg1 mRNA to its subcellular destination.

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.

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.

Conserved stem–loop structures in the HIV-1 RNA region containing the A3 3′ splice site and its cis-regulatory element: possible involvement in RNA splicing

The HIV-1 transcript is alternatively spliced to over 30 different mRNAs. Whether RNA secondary structure can influence HIV-1 RNA alternative splicing has not previously been examined. Here we have determined the secondary structure of the HIV-1/BRU RNA segment, containing the alternative A3, A4a, A4b, A4c and A5 3′ splice sites. Site A3, required for tat mRNA production, is contained in the terminal loop of a stem–loop structure (SLS2), which is highly conserved in HIV-1 and related SIVcpz strains. The exon splicing silencer (ESS2) acting on site A3 is located in a long irregular stem–loop structure (SLS3). Two SLS3 domains were protected by nuclear components under splicing condition assays. One contains the A4c branch points and a putative SR protein binding site. The other one is adjacent to ESS2. Unexpectedly, only the 3′ A residue of ESS2 was protected. The suboptimal A3 polypyrimidine tract (PPT) is base paired. Using site-directed mutagenesis and transfection of a mini-HIV-1 cDNA into HeLa cells, we found that, in a wild-type PPT context, a mutation of the A3 downstream sequence that reinforced SLS2 stability decreased site A3 utilization. This was not the case with an optimized PPT. Hence, sequence and secondary structure of the PPT may cooperate in limiting site A3 utilization.

RNA: a method to specifically inhibit PCR amplification of known members of a multigene family by degenerate primers

The polymerase chain reaction (PCR) is a versatile method to amplify specific DNA with oligonucleotide primers. By designing degenerate PCR primers based on amino acid sequences that are highly conserved among all known gene family members, new members of a multigene family can be identified. The inherent weakness of this approach is that the degenerate primers will amplify previously identified, in addition to new, family members. To specifically address this problem, we synthesized a specific RNA for each known family member so that it hybridized to one strand of the template, adjacent to the 3′-end of the primer, allowing the degenerate primer to bind yet preventing extension by DNA polymerase. To test our strategy, we used known members of the soluble, nitric oxide-sensitive guanylyl cyclase family as our templates and degenerate primers that discriminate this family from other guanylyl cyclases. We demonstrate that amplification of known members of this family is effectively and specifically inhibited by the corresponding RNAs, alone or in combination. This robust method can be adapted to any application where multiple PCR products are amplified, as long as the sequence of the desired and the undesired PCR product(s) is sufficiently distinct between the primers.

Trypanosome spliced leader RNA genes contain the first identified RNA polymerase II gene promoter in these organisms

Typical general transcription factors, such as TATA binding protein and TFII B, have not yet been identified in any member of the Trypanosomatidae family of parasitic protozoa. Interestingly, mRNA coding genes do not appear to have discrete transcriptional start sites, although in most cases they require an RNA polymerase that has the biochemical properties of eukaryotic RNA polymerase II. A discrete transcription initiation site may not be necessary for mRNA synthesis since the sequences upstream of each transcribed coding region are trimmed from the nascent transcript when a short m7G-capped RNA is added during mRNA maturation. This short 39 nt m7G-capped RNA, the spliced leader (SL) sequence, is expressed as an ∼100 nt long RNA from a set of reiterated, though independently transcribed, genes in the trypanosome genome. Punctuation of the 5′ end of mRNAs by a m7G cap-containing spliced leader is a developing theme in the lower eukaryotic world; organisms as diverse as Euglena and nematode worms, including Caenorhabditis elegans, utilize SL RNA in their mRNA maturation programs. Towards understanding the coordination of SL RNA and mRNA expression in trypanosomes, we have begun by characterizing SL RNA gene expression in the model trypanosome Leptomonas seymouri. Using a homologous in vitro transcription system, we demonstrate in this study that the SL RNA is transcribed by RNA polymerase II. During SL RNA transcription, accurate initiation is determined by an initiator element with a loose consensus of CYAC/AYR(+1). This element, as well as two additional basal promoter elements, is divergent in sequence from the basal transcription elements seen in other eukaryotic gene promoters. We show here that the in vitro transcription extract contains a binding activity that is specific for the initiator element and thus may participate in recruiting RNA polymerase II to the SL RNA gene promoter.

An Allele of the Ripening-Specific 1-Aminocyclopropane-1-Carboxylic Acid Synthase Gene (ACS1) in Apple Fruit with a Long Storage Life1

An allele of the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene (Md-ACS1), the transcript and translated product of which have been identified in ripening apples (Malus domestica), was isolated from a genomic library of the apple cultivar, Golden Delicious. The predicted coding region of this allele (ACS1-2) showed that seven nucleotide substitutions in the corresponding region of ACS1-1 resulted in just one amino acid transition. A 162-bp sequence characterized as a short interspersed repetitive element retrotransposon was inserted in the 5′-flanking region of ACS1-2 corresponding to position −781 in ACS1-1. The XhoI site located near the 3′ end of the predicted coding region of ACS1-2 was absent from the reverse transcriptase-polymerase chain reaction product, revealing that exclusive transcription from ACS1-1 occurs during ripening of cv Golden Delicious fruit. DNA gel-blot and polymerase chain reaction analyses of genomic DNAs showed clearly that apple cultivars were either heterozygous for ACS1-1 and ACS1-2 or homozygous for each type. RNA gel-blot analysis of the ACS1-2 homozygous Fuji apple, which produces little ethylene and has a long storage life, demonstrated that the level of transcription from ACS1-2 during the ripening stage was very low.