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PubMed2025

Deciphering estrogen receptor alpha-driven transcription in human endometrial stromal cells via transcriptome, cistrome, and integration with chromatin landscape.

OBJECTIVE: To investigate estrogen receptor gene 1 (ESR1) and estrogen-driven transcription in human endometrial stromal cells. DESIGN: RNA sequencing (RNA-seq) and Cleavage Under Targets and Release Using Nuclease (Cut&Run) were performed on telomerase-immortalized human endometrial stromal cells with Clustered Regularly Interspaced Short Palindromic Repeats-mediated ESR1 activation. Hi-C-based chromatin architecture analysis (H3K27ac HiChIP) was conducted in primary endometrial stromal cells. SUBJECTS: Biopsies from two healthy, reproductive-aged volunteers with regular menstrual cycles and no history of gynecological malignancies. EXPOSURE: The ESR1-activated and control endometrial stromal cells were treated with estradiol (E2) or vehicle. Primary endometrial stromal cells were treated with vehicle or a decidualization cocktail. MAIN OUTCOME MEASURES: Differential gene expression analysis (RNA-seq) identified ligand-independent and -dependent ESR1 activity. Cut&Run profiled ESR1 genomic binding in ESR1-activated cells. H3K27ac HiChIP mapped hormone-induced changes in chromatin looping in primary cells. RESULTS: Among seven tested guide RNAs (gRNA), the ESR1-3 gRNA induced robust ESR1 activation and restored E2 responsiveness. Bulk RNA-seq revealed both ligand-dependent and -independent ESR1 transcriptional programs regulating inflammation, proliferation, and cancer-related pathways. Notably, 72% of differentially expressed genes overlapped with genes active in human endometrial tissue during the proliferative estrogen-dominant phase, supporting their physiological relevance. The Cut&Run-seq identified genome-wide ESR1 binding sites, with most binding sites located at distal regulatory elements. Integration of Cut&Run data with H3K27ac HiChIP chromatin loops linked distal ESR1 binding sites to gene promoters, including genes involved in decidualization (e.g., FOXO1) and endometrial cancer (e.g., ERRFI1, NRIP1, and EPAS1). Functional assays showed that ESR1 promotes cell viability and, in the presence of E2, enhances migration. CONCLUSION: The CRISPR-mediated ESR1 activation restores estrogen responsiveness in endometrial stromal cells. Combined transcriptomic, cistromic, and chromatin architecture analyses reveal ESR1's role in regulating decidualization and inflammation-related gene networks, with relevance to endometrial pathologies including endometrial cancer. This model serves as a powerful tool to study estrogen signaling in endometrial stromal cell biology and related pathologies.

Humans↗
PubMed2000

Uridylate addition and RNA ligation contribute to the specificity of kinetoplastid insertion RNA editing.

RNA editing in Trypanosoma brucei inserts and deletes uridylates (U's) in mitochondrial pre-mRNAs under the direction of guide RNAs (gRNAs). We report here the development of a novel in vitro precleaved editing assay and its use to study the gRNA specificity of the U addition and RNA ligation steps in insertion RNA editing. The 5' fragment of substrate RNA accumulated with the number of added U's specified by gRNA, and U addition products with more than the specified number of U's were rare. U addition up to the number specified occurred in the absence of ligation, but accumulation of U addition products was slowed. The 5' fragments with the correct number of added U's were preferentially ligated, apparently by adenylylated RNA ligase since exogenously added ATP was not required and since ligation was eliminated by treatment with pyrophosphate. gRNA-specified U addition was apparent in the absence of ligation when the pre-mRNA immediately upstream of the editing site was single stranded and more so when it was base paired with gRNA. These results suggest that both the U addition and RNA ligation steps contributed to the precision of RNA editing.

Animals↗
PubMed1993

A shortened form of the Tetrahymena thermophila group I intron can catalyze the complete splicing reaction in trans.

The group I intron from Tetrahymena thermophila is able to catalyze its own excision from a precursor RNA. The intron recognizes the splice sites through an intron-encoded sequence called the internal guide sequence, or IGS. The 5' and 3' exons are thought to align on the IGS and form a pseudoknot structure consisting of two stems (P1 and P10). We created a shortened form of the intron that lacks the exon sequences and the entire IGS. This RNA is unable to react upon itself. It can catalyze a sequential two-step transesterification reaction on a P1P10 substrate added in trans that completely mimics splicing. The reaction works for different substrates that contain a U.G base-pair preceding the 5' cleavage site and a guanosine base preceding the 3' cleavage site, but that are otherwise unrelated in sequence. The ribozyme uses primarily the correct 5' and 3' splice sites even in the presence of potential cryptic splice sites, and therefore it must rely on the structure of the substrate (formation of the P1 and P10 helices) for correct splice site recognition. A C-G base-pair after the 5' splice site in P1 decreases activity while a U.G or G.U base-pair enhances activity. The relative position in P1 of the U.G base-pair preceding the 5' splice site is an important determinant. The ability of the intron to recognize primarily a specific structure, rather than a sequence, has ramifications for splice-site selection, for molecular modeling of the group I intron, and for ribozyme-based gene targeting.

Animals↗
PubMed2000

Persisting long-term benefit of genotype-guided treatment for HIV-infected patients failing HAART. The Viradapt Study: week 48 follow-up.

OBJECTIVE: We report the 12 months follow-up of the patients who participated in the Viradapt study. METHODS: A total of 108 HIV-infected patients failing antiretroviral (ARV) therapy (HIV RNA > 10,000 copies/ml, therapy > 6 months with nucleoside reverse transcriptase inhibitors, > 3 months with protease inhibitors (PIs) were randomized into two arms: standard of care in the control arm, and treatment according to the resistance mutations in the protease and reverse transcriptase genes in the study arm. After the first 6 months of the randomized study, open-label, genotype-guided treatment was offered in both arms. A multivariate analysis was performed to assess the predictive factors of treatment success (HIV RNA < 200 copies/ml). RESULTS: The two arms were comparable in terms of risk factors, age, sex, previous treatments, CD4 cell count and log10 HIV-1 RNA at baseline. At week 24, an interim combined analysis showed a statistically significant difference in the drop in viral load at months 3 and 6 (P = 0.015, repeated measures analysis of variance) in favour of the genotype group. Patients in both arms were then offered open-label genotyping. Genotype analysis was performed every 3 months, and treatment changes could accordingly be made. As some of the patients in the control arm had already progressed to months 9 or 12, only 69% (30/43) of these patients received genotype-guided treatment changes. In the genotype arm, the mean drop in HIV RNA of 1.15 log10 copies/ml, obtained at month 6, persisted at months 9 and 12 (1.15 log10 copies/ml +/- 0.17). In the control arm, an additional drop in HIV RNA to 0.98 log10 +/- 0.22 copies/ml was observed by month 12. In control patients receiving open-label genotype, the percentage of patients with HIV-1 RNA levels below detection limit (200 copies/ml) rose from 14% at month 6 to 30.5% at month 12. This percentage in the study arm remained stable at 31.3% and 30% at months 9 and 12, respectively. Genotype-guided therapy, primary protease mutations and PI plasma concentrations were significantly correlated with virological success. CONCLUSION: In this heavily pretreated patient population, genotype-guided therapy resulted in a sustained reduction in HIV RNA of greater than one log10 throughout a 1 year follow-up period. Performance of genotype-guided therapy may have contributed to the additional viral load reduction seen in patients in the control group who received open-label genotyping after the 6 months point. Multivariate analysis showed that the presence of primary protease mutations, performance of genotype-guided treatment changes and PI plasma concentrations independently affected virological response.

Anti-HIV Agents↗
PubMed2006

Toward building a database of bifunctional probes for the MS3D investigation of nucleic acids structures.

This report illustrates the approaches employed to investigate critical aspects of the activity of crosslinking reagents toward nucleic acid substrates, which should be evaluated to identify candidate probes for mass spectrometric 3D (MS3D) investigations of biomolecules and macromolecular complexes. Representative members of different classes of bifunctional reagents were taken into consideration, including bikethoxal and phenyl-diglyoxal [bis-(1,2-dicarbonyls)], cisplatin (coordinative binding agents), chlorambucil and nitrogen mustard [bis-(2-chloroethyl)amines], and sym-triazine trichloride (triazines). Nanospray-Fourier transform mass spectrometry (FTMS) was applied without desalting or separation procedures to characterize the covalent products obtained by probing dinucleotide and trinucleotide substrates under a variety of experimental conditions in vitro. The carefully controlled composition of these substrates enabled us to obtain valid comparisons of probe activity toward individual nucleotides and evaluate possible base-specific effects, including the stability of the different adducts in solution under the selected reaction conditions. The gas-phase behavior of the observed products was investigated using sustained off-resonance irradiation collision-induced dissociation (SORI-CID) to obtain valuable information for guiding the design of sequencing experiments and helping the data interpretation. Structured RNA substrates, such as HIV-1 stemloop 1, were finally employed to investigate the structural determinant of adduct formation and highlight the different nature of the spatial information provided by the various candidate probes.

Cross-Linking Reagents↗
PubMed2006

Two structurally atypical HEAT domains in the C-terminal portion of human eIF4G support binding to eIF4A and Mnk1.

The X-ray structure of the C-terminal region of human eukaryotic translation initiation factor 4G (eIF4G) has been determined at 2.2 A resolution, revealing two atypical HEAT-repeat domains. eIF4G recruits various translation factors and the 40S ribosomal subunit to the mRNA 5' end. In higher eukaryotes, the C terminus of eIF4G (4G/C) supports translational regulation by recruiting eIF4A, an RNA helicase, and Mnk1, the kinase responsible for phosphorylating eIF4E. Structure-guided surface mutagenesis and protein-protein interaction assays were used to identify binding sites for eIF4A and Mnk1 within the HEAT-repeats of 4G/C. p97/DAP5, a translational modulator homologous to eIF4G, lacks an eIF4A binding site in the corresponding region. The second atypical HEAT domain of the 4G/C binds Mnk1 using two conserved aromatic/acidic-box (AA-box) motifs. Within the first AA-box, the aromatic residues contribute to the hydrophobic core of the domain, while the acidic residues form a negatively charged surface feature suitable for electrostatic interactions with basic residues in Mnk1.

Amino Acid Sequence↗
PubMed2006

Cyclooxygenase-2 inhibition selectively attenuates bone morphogenetic protein-6 synthesis and bone formation during guided tissue regeneration in a rat model.

OBJECTIVES: Bone formation during guided tissue regeneration is a tightly regulated process involving cells, extracellular matrix and growth factors. The aims of this study were (i) to examine the expression of cyclooxygenase-2 (COX-2) during bone regeneration and (ii) the effects of selective COX-2 inhibition on osseous regeneration and growth factor expression in the rodent femur model. MATERIAL AND METHODS: A standardized transcortical defect of 5 x 1.5 mm was prepared in the femur of 12 male rats and a closed half-cylindrical titanium chamber was placed over the defect. The expression of COX-2 and of platelet-derived growth factor-B (PDGF-B), bone morphogenetic protein-6 (BMP-6) and insulin-like growth factor-I/II (IGF-I/II) was analyzed at Days 3, 7, 21 and 28 semiquantitatively by reverse transcriptase-polymerase chain reaction and immunohistochemistry. The effects of COX-2 inhibition by intraperitoneal injection of NS-398 (3 mg/kg/day) were analyzed in five additional animals sacrificed at Day 14. RESULTS: Histomorphometry revealed that new bone formation occurred in the cortical defect area as well as in the supracortical region, i.e. region within the chamber by Day 7 and increased through Day 28. Immunohistochemical evidence of COX-2 and PDGF-B levels were observed early (i.e. Day 3) and decreased rapidly by Day 7. BMP-6 expression was maximal at Day 3 and slowly declined by Day 28. In contrast, IGF-I/II expression gradually increased during the 28-day period. Systemic administration NS-398 caused a statistically significant reduction (P<0.05) in new bone formation (25-30%) and was associated with a statistically significant reduction in BMP-6 protein and mRNA expression (50% and 65% at P<0.05 and P<0.01, respectively). PDGF-B mRNA or protein expression was not affected by NS-398 treatment. CONCLUSION: COX-2 inhibition resulted in reduced BMP-6 expression and impaired osseous regeneration suggesting an important role for COX-2-induced signaling in BMP synthesis and new bone formation.

Animals↗
PubMedDate not supplied

The MXI1 tumor suppressor gene is not mutated in primary prostate cancer.

For prostate cancer, allelic deletions from the long arm of chromosome 10 (#10q23-25), the locus of the putative tumor suppressor gene MXI1 (#10q24-25), have been identified as a frequently occurring genetic event. During the development of several human malignancies, the c-myc proto-oncogene has been identified to enhance cellular transformation, mitogenesis and cell proliferation. The MXI1 gene, belonging to the helix-loop-helix (bHLH) gene family, was demonstrated to display tumor suppressor function by antagonizing c-myc induced transcriptional activities. Due to the detection of point mutations in the retained alleles of four primary adenocarcinomas of the prostate, MXI1 gene alterations have been suggested to be involved in the development and/or the progression of prostate cancer. To evaluate the role of MXI1 gene alterations for the development of adenocarcinoma of the prostate, 42 primary prostate cancers of different stage (T1-4) and histological grade (G1-3) were investigated for alterations within exons 4 and 5 of the MXI1 gene (spanning 6 exons in total), encoding for the functional HLH-Zip domain, by RNA-SSCP analysis and direct PCR-DNA-sequencing following the microscopically guided tumor cell dissection from 5 microm fresh-frozen buffer-soaked tissue sections. Even by application of this highly elaborated technical approach, MXI1 gene alterations could not be deleted in any of the tumor specimens investigated. Therefore, a substantial involvement of MXI1 gene alterations in the development of prostate cancer appears unlikely. The newly identified putative tumor suppressor gene PTEN, located at #10q23, might be responsible for the frequently observed allelic deletions from #10q23-25 in prostate cancer.

Adenocarcinoma↗
PubMed2004

RNAi expression mediated inhibition of HCV replication.

The RNA interference (RNAi) mechanism is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of an mRNA containing the same sequence. The 21-23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that vector derived dsRNA specifically inhibit the replication of HCV RNA in HCV replicon cells. We designed a long dsRNA targeted to the full length HCV IRES region (1-377 nt). Real Time RT-PCR was performed with a TaqMan RT-PCR, to solely amplify and enable quantification of HCV RNA. Our results indicated HCV replication reduction to near background levels in a sequence-specific manner by the long-dsRNAs in the HCV replicon cells. Our results support the potential of using siRNA gene therapy to inhibit HCV replication, which may prove to be valuable in the treatment of hepatitis C.

Cell Line, Tumor↗
PubMed2005

Combining RNA interference and kinase inhibitors against cell signalling components involved in cancer.

BACKGROUND: The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including oncogenic transformation. The tyrosine kinases of the epidermal growth factor receptor (EGFR) constitute the beginning of one signal transduction cascade leading to AP-1 activation and are known to control cell proliferation and differentiation. Drug discovery efforts targeting this receptor and other pathway components have centred on monoclonal antibodies and small molecule inhibitors. Resistance to such inhibitors has already been observed, guiding the prediction of their use in combination therapies with other targeted agents such as RNA interference (RNAi). This study examines the use of RNAi and kinase inhibitors for qualification of components involved in the EGFR/AP-1 pathway of ME180 cells, and their inhibitory effects when evaluated individually or in tandem against multiple components of this important disease-related pathway. METHODS: AP-1 activation was assessed using an ME180 cell line stably transfected with a beta-lactamase reporter gene under the control of AP-1 response element following epidermal growth factor (EGF) stimulation. Immunocytochemistry allowed for further quantification of small molecule inhibition on a cellular protein level. RNAi and RT-qPCR experiments were performed to assess the amount of knockdown on an mRNA level, and immunocytochemistry was used to reveal cellular protein levels for the targeted pathway components. RESULTS: Increased potency of kinase inhibitors was shown by combining RNAi directed towards EGFR and small molecule inhibitors acting at proximal or distal points in the pathway. After cellular stimulation with EGF and analysis at the level of AP-1 activation using a beta-lactamase reporter gene, a 10-12 fold shift or 2.5-3 fold shift toward greater potency in the IC50 was observed for EGFR and MEK-1 inhibitors, respectively, in the presence of RNAi targeting EGFR. CONCLUSION: EGFR pathway components were qualified as targets for inhibition of AP-1 activation using RNAi and small molecule inhibitors. The combination of these two targeted agents was shown to increase the efficacy of EGFR and MEK-1 kinase inhibitors, leading to possible implications for overcoming or preventing drug resistance, lowering effective drug doses, and providing new strategies for interrogating cellular signalling pathways.

Antibodies, Monoclonal↗
PubMed1992

Important 2'-hydroxyl groups in model substrates for M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli.

The role of 2'-hydroxyl groups in a model substrate for RNase P from Escherichia coli was studied using mixed DNA/RNA derivatives of such a substrate. The presence of the 2'-hydroxyl groups of nucleotides at positions -1 and -2 in the leader sequence and at position 1, as well as at the first C in the 3'-terminal CCA sequence, are important but not absolutely essential for efficient cleavage of the substrate by RNase P or its catalytic RNA subunit, M1 RNA. The 2'-hydroxyl groups in the substrate that are important for efficient cleavage also participate in the binding of Mg2+. An all-DNA external guide sequence (EGS) can efficiently render a potential substrate, derived from the model substrate, susceptible to cleavage by the enzyme or its catalytic RNA subunit. Furthermore, both DNA and RNA EGSs turn over during the reaction with RNase P in vitro. The identity of the nucleotide at position 1 in the substrate, the adjacent Mg(2+)-binding site in the leader sequence, and the junction of the single and double-stranded regions are the important elements in the recognition of model substrates, as well as in the identification of the sites of cleavage in those model substrates.

Base Sequence↗
PubMed2005

Human RISC couples microRNA biogenesis and posttranscriptional gene silencing.

RNA interference is implemented through the action of the RNA-induced silencing complex (RISC). Although Argonaute2 has been identified as the catalytic center of RISC, the RISC polypeptide composition and assembly using short interfering RNA (siRNA) duplexes has remained elusive. Here we show that RISC is composed of Dicer, the double-stranded RNA binding protein TRBP, and Argonaute2. We demonstrate that this complex can cleave target RNA using precursor microRNA (pre-miRNA) hairpin as the source of siRNA. Although RISC can also utilize duplex siRNA, it displays a nearly 10-fold greater activity using the pre-miRNA Dicer substrate. RISC distinguishes the guide strand of the siRNA from the passenger strand and specifically incorporates the guide strand. Importantly, ATP is not required for miRNA processing, RISC assembly, or multiple rounds of target-RNA cleavage. These results define the composition of RISC and demonstrate that miRNA processing and target-RNA cleavage are coupled.

Adenosine Triphosphate↗
PubMed2005

Small interfering RNAs that trigger posttranscriptional gene silencing are not required for the histone H3 Lys9 methylation necessary for transgenic tandem repeat stabilization in Neurospora crassa.

In Neurospora crassa, the introduction of a transgene can lead to small interfering RNA (siRNA)-mediated posttranscriptional gene silencing (PTGS) of homologous genes. siRNAs can also guide locus-specific methylation of Lys9 of histone H3 (Lys9H3) in Schizosaccharomyces pombe. Here we tested the hypothesis that transgenically derived siRNAs may contemporaneously both activate the PTGS mechanism and induce chromatin modifications at the transgene and the homologous endogenous gene. We carried out chromatin immunoprecipitation using a previously characterized albino-1 (al-1) silenced strain but detected no alterations in the pattern of histone modifications at the endogenous al-1 locus, suggesting that siRNAs produced from the transgenic locus do not trigger modifications in trans of those histones tested. Instead, we found that the transgenic locus was hypermethylated at Lys9H3 in our silenced strain and remained hypermethylated in the quelling defective mutants (qde), further demonstrating that the PTGS machinery is dispensable for Lys9H3 methylation. However, we found that a mutant in the histone Lys9H3 methyltransferase dim-5 was unable to maintain PTGS, with transgenic copies being rapidly lost, resulting in reversion of the silenced phenotype. These results indicate that the defect in PTGS of the Deltadim-5 strain is due to the inability to maintain the transgene in tandem, suggesting a role for DIM-5 in stabilizing such repeated sequences. We conclude that in Neurospora, siRNAs produced from the transgenic locus are used in the RNA-induced silencing complex-mediated PTGS pathway and do not communicate with an RNAi-induced initiation of transcriptional gene silencing complex to effect chromatin-based silencing.

Chromatin Immunoprecipitation↗
PubMed1994

Catalysis of RNA cleavage by a ribozyme derived from the group I intron of Anabaena pre-tRNA(Leu).

In the cyanobacterium Anabaena PCC7120, the precursor to tRNA(Leu) contains a 249-nucleotide group I intron that undergoes efficient self-splicing in vitro. By deleting the 5' and 3' splice sites, this intron has now been converted to an RNA enzyme that uses a guanosine nucleophile to cleave substrate RNAs (S) with multiple turnover. This Anabaena ribozyme has a second-order rate constant for RNA cleavage (kcat/Km)S that is 250-500-fold smaller than that of the Tetrahymena ribozyme, and a multiple-turnover rate constant at saturating S [kcat(mt)] that is approximately 400-fold larger. Several lines of evidence, including kinetic analysis of cleavage of phosphorothioate- and deoxynucleotide-substituted substrates and pH dependence, support the conclusion that both (kcat/Km)S and kcat(mt) are limited by the actual chemical cleavage step. In contrast, for the Tetrahymena ribozyme, it has been shown that neither of these rate constants reflects the chemical step. These kinetic differences are expected from the shorter guide sequence-substrate pairing of the Anabaena ribozyme; for example, weaker binding of RNA speeds product release during multiple turnover and thereby overcomes the rate-limiting product release observed for the Tetrahymena ribozyme. Thus, the large kinetic differences represent superficial rather than fundamental differences between these ribozymes. Furthermore, the strength of the guanosine-binding interaction, the stereospecificity for Rp-phosphorothioate at the cleavage site, and the 10(3)-fold slower cleavage with a deoxyribonucleoside leaving group are properties conserved between the Anabaena and Tetrahymena ribozymes. Finally, log(kcat/Km)S increases linearly with pH in the acid range where chemistry is rate-limiting and becomes pH-independent above pH 7, perhaps because a conformational step becomes rate-limiting; again, these are characteristics shared with the Tetrahymena ribozyme. We conclude that two group I ribozymes, although differing in the identity of many of their active site nucleotides, nevertheless provide functionally similar active sites for sequence-specific RNA cleavage.

Anabaena↗
PubMed1980

Proteolytic removal of the COOH terminus of the T4 gene 32 helix-destabilizing protein alters the T4 in vitro replication complex.

The proteolytic removal of about 60 amino acids from the COOH terminus of the bacteriophage T4 helix-destabilizing protein (gene 32 protein) produces 32*I, a 27,000-dalton fragment which still binds tightly and cooperatively to single-stranded DNA. The substitution of 32*I protein for intact 32 protein in the seven-protein T4 replication complex results in dramatic changes in some of the reactions catalyzed by this in vitro DNA replication system, while leaving others largely unperturbed. 1. Like intact 32 protein, the 32*I protein promotes DNA synthesis by the DNA polymerase when the T4 polymerase accessory proteins (gene 44/62 and 45 proteins) are also present. The host helix-destabilizing protein (Escherichia coli ssb protein) cannot replace the 32I protein for this synthesis. 2. Unlike intact 32 protein, 32*I protein strongly inhibits DNA synthesis catalyzed by the T4 DNA polymerase alone on a primed single-stranded DNA template. 3. Unlike intact 32 protein, the 32*I protein strongly inhibits RNA primer synthesis catalyzed by the T4 gene 41 and 61 proteins and also reduces the efficiency of RNA primer utilization. As a result, de novo DNA chain starts are blocked completely in the complete T4 replication system, and no lagging strand DNA synthesis occurs. 4. The 32*I protein does not bind to either the T4 DNA polymerase or to the T4 gene 61 protein in the absence of DNA; these associations (detected with intact 32 protein) would therefore appear to be essential for the normal control of 32 protein activity, and to account at least in part for observations 2 and 3, above. We propose that the COOH-terminal domain of intact 32 protein functions to guide its interactions with the T4 DNA polymerase and the T4 gene 61 RNA-priming protein. When this domain is removed, as in 32*I protein, the helix destabilization induced by the protein is controlled inadequately, so that polymerizing enzymes tend to be displaced from the growing 3'-OH end of a polynucleotide chain and are thereby inhibited. Eukaryotic helix-destabilizing proteins may also have similar functional domains essential for the control of their activities.

Coliphages↗
PubMed1998

RNA editing: trypanosomes rewrite the genetic code.

The understanding of how genetic information is stored and expressed has advanced considerably since the "central dogma" asserted that genetic information flows from the nucleotide sequence of DNA to that of messenger RNA (mRNA) which in turn specifies the amino acid sequence of a protein. It was found that genetic information can be stored as RNA (e.g. in RNA viruses) and can flow from RNA to DNA by reverse transcriptase enzyme activity. In addition, some genes contain introns, nucleotide sequences that are removed from their RNA (by RNA splicing) and thus are not represented in the resultant protein. Furthermore, alternative splicing was found to produce variant proteins from a single gene. More recently, the study of trypanosome parasites revealed an unexpected and indeed counter-intuitive genetic complexity. Genetic information for a single protein can be dispersed among several (DNA) genes in these organisms. One of these genes specifies an encrypted precursor mRNA that is converted to a functional mRNA by a process called RNA editing that inserts and deletes uridylate nucleotides. The sequence of the edited mRNA is specified by multiple small RNAs, named guide RNAs, (gRNAs) each of which is encoded in a separate gene. Thus, edited mRNA sequences are assembled from multiple genes by the transfer of information from one type of RNA to another. The existence of editing was surprising but has stimulated the discovery of other types of RNA editing. The Stuart laboratory has been exploring RNA editing in trypanosomes from the time of its discovery. They found dramatic differences between the mitochondrial gene sequences and those of the corresponding mRNAs, which indicated editing by the insertion and deletion of uridylates. Some editing was modest; simply eliminating shifts in sequence register of minimally extending the protein coding sequence. However, editing of many mRNAs was startingly extensive. The RNA sequence was essentially entirely remodeled with its sequence more the result of editing than the gene sequence. The identities of genes for such extensively edited RNA were not recognizable from the DNA sequence but they were readily identifiable from the edited mRNA sequence. Thus, despite the complex and extensive editing the resultant mRNA sequence is precise. Characterization of partially edited RNAs indicated that editing proceeds in the direction opposite to that used to specify the protein which reflects the use of the gRNAs. The numerous gRNAs that are used for editing are encoded in the DNA molecules whose role was previously a mystery. Using information gained in our earlier studies, the Stuart group developed an in vitro system that reproduces the fundamental process of editing in order to resolve the mechanism by which it occurs. They determined that editing entails a series of enzymatic steps rather than the mechanism used in RNA splicing. They also showed that chimeric gRNA-mRNA molecules are aberrant by-products of editing rather than intermediates in the process as had been proposed. Additional studies are exploring precisely how the number of added and deleted uridylates is specified by the gRNA. The Stuart laboratory showed that editing is performed by an aggregation of enzymes that catalyze the separate steps of editing. It also developed a method to purify this multimolecule complex that contains several, perhaps tens of, proteins. This will allow the study of its composition and the functions of its component parts. Indeed, the gene for one component has been identified and its detailed characterization begun. These studies are developing tools to explore related processes. An early finding in the lab was that the various mRNAs are differentially edited during the life cycle of the parasite. The pattern of this editing indicates that editing serves to regulate the alternation between two modes of energy generation. This regulation is coordinated with other events that are occurring during the life c

Animals↗
PubMed2002

A randomized trial assessing the impact of phenotypic resistance testing on antiretroviral therapy.

OBJECTIVE: To compare the effect of treatment decisions guided by phenotypic resistance testing (PRT) or standard of care (SOC) on short-term virological response. DESIGN: A prospective, randomized, controlled clinical trial conducted in 25 university and private practice centers in the United States. PARTICIPANTS: A total of 272 subjects who failed to achieve or maintain virological suppression (HIV-1-RNA plasma level > 2000 copies/ml) with previous exposure to two or more nucleoside reverse transcriptase inhibitors and one protease inhibitor. INTERVENTIONS: Randomization was to antiretroviral therapy guided by PRT or SOC. MAIN OUTCOME MEASURES: The percentage of subjects with HIV-1-RNA plasma levels less than 400 copies/ml at week 16 (primary); change from baseline in HIV-1-RNA plasma levels and number of "active" (less than fourfold resistance) antiretroviral agents used (secondary). RESULTS: At week 16, using intent-to-treat (ITT) analysis, a greater proportion of subjects had HIV-1-RNA levels less than 400 copies/ml in the PRT than in the SOC arm (P = 0.036, ITT observed; P = 0.079, ITT missing equals failure). An ITT observed analysis showed that subjects in the PRT arm had a significantly greater median reduction in HIV-1-RNA levels from baseline than the SOC arm (P = 0.005 for 400 copies/ml; P = 0.049 for 50 copies/ml assay detection limit). Significantly more subjects in the PRT arm were treated with two or more "active" antiretroviral agents than in the SOC arm (P = 0.003). CONCLUSION: Antiretroviral treatment guided prospectively by PRT led to the increased use of "active" antiretroviral agents and was associated with a significantly better virological response.

Adult↗
PubMed2002

Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus.

In a specialized cDNA library from the archaeon Archaeoglobus fulgidus we have identified a total of 86 different expressed RNA sequences potentially encoding previously uncharacterized small non-messenger RNA (snmRNA) species. Ten of these RNAs resemble eukaryotic small nucleolar RNAs, which guide rRNA 2'-O-methylations (C/D-box type) and pseudouridylations (H/ACA-box type). Thereby, we identified four candidates for H/ACA small RNAs in an archaeal species that are predicted to guide a total of six rRNA pseudouridylations. Furthermore, we have verified the presence of the six predicted pseudouridines experimentally. We demonstrate that 22 snmRNAs are transcribed from a family of short tandem repeats conserved in most archaeal genomes and shown previously to be potentially involved in replicon partitioning. In addition, four snmRNAs derived from the rRNA operon of A. fulgidus were identified and shown to be generated by a splicing/processing pathway of pre-rRNAs. The remaining 50 RNAs could not be assigned to a known class of snmRNAs because of the lack of known structure and/or sequence motifs. Regarding their location on the genome, only nine were located in intergenic regions, whereas 33 were complementary to an ORF, five were overlapping an ORF, and three were derived from the sense orientation within an ORF. Our study further supports the importance of snmRNAs in all three domains of life.

Archaeoglobus fulgidus↗