Binary vectors for sense and antisense expression of arabidopsis ESTs

Our laboratory is interested in devising methods to identify functions for the vast numbers of arabidopsis genes now available. For this purpose, we have constructed a set of binary vectors that will allow the quick production of transgenic arabidopsis plants containing either sense or antisense copies of EST clones obtained from the PRL2 library. These vectors are based on the pSLJ series containing the bialophos resistance (BAR) gene that confers resistance to the herbicide BASTA. Tn addition, our vectors contain a 35S CaMV promoter-polylinker-nos terminator cassette that allows the direct cloning of arabidopsis ESTs in either antisense (pAOV and pAOV2) or sense (pSOV and pSOV2) orientation. We also describe the construction of two additional vectors conferring BASTA resistance and containing the pBluescript polylinker in both orientations inserted between the 35S CaMV promoter and nos terminator (pKMB and pSMB).

Non-coding transcript in T cells (NTT): Antisense transcript activates PKR and NF-kappa B in human lymphocytes

T cell activation is a complex process involving many steps and the role played by the non-protein-coding RNAs (ncRNAs) in this phenomenon is still unclear. The non-coding T cells transcript (NTT) is differentially expressed during human T cells activation, but its function is unknown. Here, we detected a 426 m NTT transcript by RT-PCR using RNA of human lymphocytes activated with a synthetic peptide of HIV-1. After cloning, the sense and antisense 426 nt NTT transcripts were obtained by in vitro transcription and were sequenced. We found that both transcripts are highly structured and are able to activate PKR. A striking observation was that the antisense 426 nt NTT transcript is significantly more effective in activating PKR than the corresponding sense transcript. The transcription factor NF-kappa B is activated by PKR through phosphorylation and subsequent degradation of its inhibitor I-kappa B beta. We also found that the antisense 426 nt NTT transcript induces more efficiently the degradation Of I-kappa B beta than the sense transcript. Thus, this study suggests that the role played by NTT in the activation of lymphocytes can be mediated by PKR through NF-kappa B activation. However, the physiological significance of the activity of the antisense 426 nt NTT transcript remains unknown. (c) 2007 Elsevier Inc. All rights reserved.

Re-evaluation of the cytokinin receptor role of the Arabidopsis gene GCR1

GCR1 has been tentatively identified in Arabidopsis thaliana as the first plant G-protein coupled receptor (GPCR) (Josefsson and Rask 1997) implicated in the cytokinin sensory pathway (Plakidou-Dymock et al. 1998). A protein fusion of GCR1 and green fluorescent protein has been expressed in Arabidopsis and shown GCR1 to be located on the plasma membrane. Studies of plants with altered GCR1 expression have led us to question GCR1's involvement in cytokinin signaling. Transgenic Arabidopsis plants containing sense and antisense constructs for GCR1 have been produced and over- and under-expression confirmed. The analysis of 12 antisense and 17 sense lines has failed to reveal the previously reported Dainty phenotype or altered cytokinin sensitivity. We have used the Gauntlet approach to test the plants' response to various plant hormones although this has not yet identified a mutant phenotype. The yeast-two hybrid system has been used and so far there is no evidence to suggest GCR1 interacts with heterotrimeric G proteins. Before GCR1 can be identified as genuine G-protein coupled receptor, the identification of a ligand and a proof of association with heterotrimeric G-proteins should be obtained.

A highly conserved c-fms gene intronic element controls macrophage-specific and regulated expression

The c fins gene encodes the receptor for macrophage colony-stimulating factor-1. This gene is expressed selectively in the macrophage cell lineage. Previous studies have implicated sequences in intron 2 that control transcript elongation in tissue-specific and regulated expression of c -fms. Four macrophage-specific deoxyribonuclease I (DNase I)-hypersensitive sites (DHSS) were identified within mouse intron 2. Sequences of these DHSS were found to be highly conserved compared with those in the human gene. A 250-bp region we refer to as the fins intronic regulatory element (FIRE), which is even more highly conserved than the c-fins proximal promoter, contains many consensus binding sites for macrophage-expressed transcription factors including Spl, PU.1, and C/EBP. FIRE was found to act as a macrophage-specific enhancer and as a promoter with an antisense orientation preference in transient transfections. In stable transfections of the macrophage line RAW264, as well as in clones selected for high and low-level c -fms mRNA expression, the presence of intron 2 increased the frequency and level of expression of reporter genes compared with those attained using the promoter alone. Removal of FIRE abolished reporter gene expression, revealing a suppressive activity in the remaining intronic sequences. Hence, FIRE is shown to be a key regulatory element in the fins gene.

The expression of tubulin cofactor A (TBCA) is regulated by a noncoding antisense Tbca RNA during testis maturation

Abstract - Recently, long noncoding RNAs have emerged as pivotal molecules for the regulation of coding genes' expression. These molecules might result from antisense transcription of functional genes originating natural antisense transcripts (NATs) or from transcriptional active pseudogenes. TBCA interacts with β-tubulin and is involved in the folding and dimerization of new tubulin heterodimers, the building blocks of microtubules. Methodology/Principal findings: We found that the mouse genome contains two structurally distinct Tbca genes located in chromosomes 13 (Tbca13) and 16 (Tbca16). Interestingly, the two Tbca genes albeit ubiquitously expressed, present differential expression during mouse testis maturation. In fact, as testis maturation progresses Tbca13 mRNA levels increase progressively, while Tbca16 mRNA levels decrease. This suggests a regulatory mechanism between the two genes and prompted us to investigate the presence of the two proteins. However, using tandem mass spectrometry we were unable to identify the TBCA16 protein in testis extracts even in those corresponding to the maturation step with the highest levels of Tbca16 transcripts. These puzzling results led us to re-analyze the expression of Tbca16. We then detected that Tbca16 transcription produces sense and natural antisense transcripts. Strikingly, the specific depletion by RNAi of these transcripts leads to an increase of Tbca13 transcript levels in a mouse spermatocyte cell line. Conclusions/Significance: Our results demonstrate that Tbca13 mRNA levels are post-transcriptionally regulated by the sense and natural antisense Tbca16 mRNA levels. We propose that this regulatory mechanism operates during spermatogenesis, a process that involves microtubule rearrangements, the assembly of specific microtubule structures and requires critical TBCA levels.

Gold nanoparticle-DNA conjugates for oligonucleotide vectorization towards gene silencing

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Interplay of Exoribonucleases, Hfq and Small RNAs Structural Determinants in the Control of Gene Expression

Dissertation presented to obtain the Ph.D degree in Biology

Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.

Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor betaPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.

Disease-causing mutations improving the branch site and polypyrimidine tract: pseudoexon activation of LINE-2 and antisense Alu lacking the poly(T)-tail.

Cryptic exons or pseudoexons are typically activated by point mutations that create GT or AG dinucleotides of new 5' or 3' splice sites in introns, often in repetitive elements. Here we describe two cases of tetrahydrobiopterin deficiency caused by mutations improving the branch point sequence and polypyrimidine tracts of repeat-containing pseudoexons in the PTS gene. In the first case, we demonstrate a novel pathway of antisense Alu exonization, resulting from an intronic deletion that removed the poly(T)-tail of antisense AluSq. The deletion brought a favorable branch point sequence within proximity of the pseudoexon 3' splice site and removed an upstream AG dinucleotide required for the 3' splice site repression on normal alleles. New Alu exons can thus arise in the absence of poly(T)-tails that facilitated inclusion of most transposed elements in mRNAs by serving as polypyrimidine tracts, highlighting extraordinary flexibility of Alu repeats in shaping intron-exon structure. In the other case, a PTS pseudoexon was activated by an A>T substitution 9 nt upstream of its 3' splice site in a LINE-2 sequence, providing the first example of a disease-causing exonization of the most ancient interspersed repeat. These observations expand the spectrum of mutational mechanisms that introduce repetitive sequences in mature transcripts and illustrate the importance of intronic mutations in alternative splicing and phenotypic variability of hereditary disorders.

Down-regulation of NOSII gene expression by iontophoresis of anti-sense oligonucleotide in endotoxin-induced uveitis.

Transcorneoscleral iontophoresis was used to enhance ocular penetration of a 21-bp NH(2) protected anti-NOSII oligonucleotides (ODNs) (fluorescein or infrared-41 labeled) in Lewis rats. Both histochemical localization and acrylamide sequencing gels were used. To evaluate the potential to down-regulate NOSII expression in the rat model of endotoxin-induced uveitis (EIU), anti-sense NOSII ODN, scrambled ODN or saline were iontophorezed into these animals' eyes. Iontophoresis facilitated the penetration of intact ODNs into the intraocular tissues of the rat eye and only the eyes receiving ODNs and electrical current demonstrated intact ODNs within the ocular tissues of both segments of the eye. Iontophoresis of anti-NOSII ODN significantly down-regulated the expression of NOSII expression in iris/ciliary body compared to the saline or scrambled ODN treated eyes. Nitrite production was also significantly reduced in the anti-NOSII applied eyes compared to those treated with saline. Using this system, intraocular delivery of ODNs can be significantly enhanced increasing the potential for successful gene therapy for human eye diseases.

The influence of cationic lipid type on in-vitro release kinetic profiles of antisense oligonucleotide from cationic nanoemulsions.

Novel formulations of cationic nanoemulsions based on three different lipids were developed to strengthen the attraction of the polyanionic oligonucleotide (ODN) macromolecules to the cationic moieties on the oil nanodroplets. These formulations were developed to prolong the release of the ODN from the nanoemulsion under appropriate physiological dilutions as encountered in the eye following topical application. Increasing the concentration of the new cationic lipid exhibiting two cationic amine groups (AOA) in the emulsion from 0.05% to 0.4% did not alter markedly the particle size or zeta potential value of the blank cationic nanoemulsion. The extent of ODN association did not vary significantly when the initial concentration of ODN remained constant at 10 microM irrespective of the cationic lipid nature. However, the zeta potential value dropped consistently with the low concentrations of 0.05% and 0.1% of AOA in the emulsions suggesting that an electrostatic attraction occurred between the cationic lipids and the polyanionic ODN molecules at the o/w interface. Only the nanoemulsion prepared with N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts (DOTAP) remained physically stable over time. DOTAP cationic lipid nanoemulsion was the most efficient formulation capable of retaining the ODN despite the high dilution of 1:100 with simulated tear solution (STS). Less than 10% of the ODN was exchanged in contrast to 40-50% with the other cationic nanoemulsions. The in-vitro release kinetic behavior of ODN exchange with physiological anions present in the STS appears to be complex and difficult to characterize using mathematical fitting model equations. Further pharmacokinetic studies are needed to verify our kinetic assumptions and confirm the in-vitro ODN release profile from DOTAP cationic nanoemulsions.

A rice cis-natural antisense RNA acts as a translational enhancer for its cognate mRNA and contributes to phosphate homeostasis and plant fitness.

cis-natural antisense transcripts (cis-NATs) are widespread in plants and are often associated with downregulation of their associated sense genes. We found that a cis-NAT positively regulates the level of a protein critical for phosphate homeostasis in rice (Oryza sativa). PHOSPHATE1;2 (PHO1;2), a gene involved in phosphate loading into the xylem in rice, and its associated cis-NATPHO1;2 are both controlled by promoters active in the vascular cylinder of roots and leaves. While the PHO1;2 promoter is unresponsive to the plant phosphate status, the cis-NATPHO1;2 promoter is strongly upregulated under phosphate deficiency. Expression of both cis-NATPHO1;2 and the PHO1;2 protein increased in phosphate-deficient plants, while the PHO1;2 mRNA level remained stable. Downregulation of cis-NATPHO1;2 expression by RNA interference resulted in a decrease in PHO1;2 protein, impaired the transfer of phosphate from root to shoot, and decreased seed yield. Constitutive overexpression of NATPHO1;2 in trans led to a strong increase of PHO1;2, even under phosphate-sufficient conditions. Under all conditions, no changes occurred in the level of expression, sequence, or nuclear export of PHO1;2 mRNA. However, expression of cis-NATPHO1;2 was associated with a shift of both PHO1;2 and cis-NATPHO1;2 toward the polysomes. These findings reveal an unexpected role for cis-NATPHO1;2 in promoting PHO1;2 translation and affecting phosphate homeostasis and plant fitness.

Characterization of the rice PHO1 gene family reveals a key role for OsPHO1;2 in phosphate homeostasis and the evolution of a distinct clade in dicotyledons.

Phosphate homeostasis was studied in a monocotyledonous model plant through the characterization of the PHO1 gene family in rice (Oryza sativa). Bioinformatics and phylogenetic analysis showed that the rice genome has three PHO1 homologs, which cluster with the Arabidopsis (Arabidopsis thaliana) AtPHO1 and AtPHO1;H1, the only two genes known to be involved in root-to-shoot transfer of phosphate. In contrast to the Arabidopsis PHO1 gene family, all three rice PHO1 genes have a cis-natural antisense transcript located at the 5 ' end of the genes. Strand-specific quantitative reverse transcription-PCR analyses revealed distinct patterns of expression for sense and antisense transcripts for all three genes, both at the level of tissue expression and in response to nutrient stress. The most abundantly expressed gene was OsPHO1;2 in the roots, for both sense and antisense transcripts. However, while the OsPHO1;2 sense transcript was relatively stable under various nutrient deficiencies, the antisense transcript was highly induced by inorganic phosphate (Pi) deficiency. Characterization of Ospho1;1 and Ospho1;2 insertion mutants revealed that only Ospho1;2 mutants had defects in Pi homeostasis, namely strong reduction in Pi transfer from root to shoot, which was accompanied by low-shoot and high-root Pi. Our data identify OsPHO1;2 as playing a key role in the transfer of Pi from roots to shoots in rice, and indicate that this gene could be regulated by its cis-natural antisense transcripts. Furthermore, phylogenetic analysis of PHO1 homologs in monocotyledons and dicotyledons revealed the emergence of a distinct clade of PHO1 genes in dicotyledons, which include members having roles other than long-distance Pi transport.

Role of Sam68 in post-transcriptional gene regulation.

The STAR family of proteins links signaling pathways to various aspects of post-transcriptional regulation and processing of RNAs. Sam68 belongs to this class of heteronuclear ribonucleoprotein particle K (hnRNP K) homology (KH) single domain-containing family of RNA-binding proteins that also contains some domains predicted to bind critical components in signal transduction pathways. In response to phosphorylation and other post-transcriptional modifications, Sam68 has been shown to have the ability to link signal transduction pathways to downstream effects regulating RNA metabolism, including transcription, alternative splicing or RNA transport. In addition to its function as a docking protein in some signaling pathways, this prototypic STAR protein has been identified to have a nuclear localization and to take part in the formation of both nuclear and cytosolic multi-molecular complexes such as Sam68 nuclear bodies and stress granules. Coupling with other proteins and RNA targets, Sam68 may play a role in the regulation of differential expression and mRNA processing and translation according to internal and external signals, thus mediating important physiological functions, such as cell death, proliferation or cell differentiation.