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At least 757 records · Page 42Linked to original sources
PubMed2004

Evidence for U-tail stabilization of gRNA/mRNA interactions in kinetoplastid RNA editing.

The most dramatic example of RNA editing is found in the mitochondria of trypanosomes. In these organisms, U-insertions/deletions can create mRNAs that are twice as large as the gene that encodes them. Guide RNAs (gRNAs) that are complementary to short stretches of the mature message direct the precise placements of the U residues. The binding of gRNA to mRNA is a fundamental step in RNA editing and understanding the relative importance of the elements that confer affinity and specificity on this interaction is critical to our understanding of the editing process. In this study, we have analyzed the relative binding affinities of two different gRNA/mRNA pairs. The affinity of gA6-14 for its message (ATPase 6) is high, with an apparent K(D) in the 5-10 nM range. In contrast, gCYb-558 has a low affinity for its cognate mRNA. Deletion of the gRNA U-tail caused a significant reduction in the binding affinity for only the gCYb-558 pair, and was observed only under physiological magnesium conditions. These results indicate that the U-tail contribution can differ substantially between the different gRNA/mRNA pairs. In addition, our results suggest that the efficiency of gRNA/mRNA interaction is highly dependent on thermodynamic parameters determined by the local sequences and their adopted structures surrounding the anchor-binding site.

Animals↗
PubMed1998

Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure.

The eukaryotic nucleolus contains a large number of small nucleolar RNAs (snoRNAs) that are involved in preribosomal RNA (pre-rRNA) processing. The H box/ACA-motif (H/ACA) class of snoRNAs has recently been demonstrated to function as guide RNAs targeting specific uridines in the pre-rRNA for pseudouridine (psi) synthesis. To characterize the protein components of this class of snoRNPs, we have purified the snR42 and snR30 snoRNP complexes by anti-m3G-immunoaffinity and Mono-Q chromatography of Saccharomyces cerevisiae extracts. Sequence analysis of the individual polypeptides demonstrated that the three proteins Gar1p, Nhp2p, and Cbf5p are common to both the snR30 and snR42 complexes. Nhp2p is a highly basic protein that belongs to a family of putative RNA-binding proteins. Cbf5p has recently been demonstrated to be involved in ribosome biogenesis and also shows striking homology with known prokaryotic psi synthases. The presence of Cbf5p, a putative psi synthase in each H/ACA snoRNP suggests that this class of RNPs functions as individual modification enzymes. Immunoprecipitation studies using either anti-Cbf5p antibodies or a hemagglutinin-tagged Nhp2p demonstrated that both proteins are associated with all H/ACA-motif snoRNPs. In vivo depletion of Nhp2p results in a reduction in the steady-state levels of all H/ACA snoRNAs. Electron microscopy of purified snR42 and snR30 particles revealed that these two snoRNPs possess a similar bipartite structure that we propose to be a major structural determining principle for all H/ACA snoRNPs.

Amino Acid Sequence↗
PubMed2004

The preferred route for the degradation of silencing target RNAs in transgenic plants depends on pre-established silencing conditions.

RNA silencing can be initiated upon dsRNA accumulation and results in homology-dependent degradation of target RNAs mediated by 21-23 nt small interfering RNAs (siRNAs). These small regulatory RNAs can direct RNA degradation via different routes such as the RdRP/Dicer- and the RNA-induced silencing complex (RISC)-catalysed pathways. The relative contribution of both pathways to degradation of target RNAs is not understood. To gain further insight in the process of target selection and degradation, we analysed production of siRNAs characteristic for Dicer-mediated RNA degradation during silencing of mRNAs and chimeric viral RNAs in protoplasts from plants of a transgenic tobacco silencing model line. We show that small RNA accumulation is limited to silencing target regions during steady-state mRNA silencing. For chimeric viral RNAs, siRNA production appears dependent on pre-established cellular silencing conditions. The observed siRNA accumulation profiles imply that silencing of viral target RNAs in pre-silenced protoplasts occurs mainly via a RISC-mediated pathway, guided by (pre-existing) siRNAs derived from cellular mRNAs. In cells that are not silenced at the time of infection, viral RNA degradation seems to involve Dicer action directly on the viral RNAs. This suggests that the silencing mechanism flexibly deploys different components of the RNA degradation machinery in function of the prevailing silencing status.

Cell Line↗
PubMed1996

Requirements for cleavage by a modified RNase P of a small model substrate.

M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, has been covalently linked at its 3' terminus to oligonucleotides (guide sequences) that guide the enzyme to target RNAs through hybridization with the target sequences. These constructs (M1GS RNAs) have been used to determine some minimal features of model substrates. As few as 3 bp on the 3' side of the site of cleavage in a substrate complex and 1 nt on the 5' side are required for cleavage to occur. The cytosines in the 3' terminal CCA sequence of the model substrates are important for cleavage efficiency but not cleavage site selection. A purine (base-paired or not) at the 3' side of the cleavage site is important both for cleavage site selection and efficiency. M1GS RNAs provide both a simple system for characterization of the reaction governed by M1 RNA and a tool for gene therapy.

Base Sequence↗
PubMed2002

Identification and characterization of a novel U14 small nucleolar RNA gene cluster in Oryza sativa.

Small nucleolar RNAs (snoRNAs) are required for ribose 2'-O-methylation of eukaryotic ribosomal RNA. Through computer search in international rice genome database, a novel U14 snoRNA gene cluster, consisting of two U14 snoRNA gene candidates, was found on rice chromosome II. They both have box C/D sequences and a 14 nucleotides (nt)-long complementarity to rice 18S ribosomal RNA (rRNA). Functional analysis of this gene cluster indicated that both were transcribed in vivo and might guide the methylations of C418 in rice 18S rRNA. By using primer extension, 5' and 3' rapid amplification of cDNA ends, the 5' and 3' ends of two snoRNAs were determined. The 52 nt long intergenic spacer of the gene cluster is rich in uridine. The absence of a conserved promoter element in this spacer, the proximity of the genes and the detection of transcripts containing linked U14 snoRNAs by reverse transcript polymerase chain reaction suggest that the rice U14 snoRNAs encoded in the cluster are transcribed as a polycistron under an upstream promoter, and individual U14 snoRNAs are released after processing of the precursor RNAs.

Base Sequence↗
PubMed2002

RNA editing: complexity and complications.

RNA editing in Trypanosomatids creates functional mitochondrial mRNAs by extensive uridylate (U) insertion and deletion as specified by small guide RNAs (gRNAs). Editing is catalysed by the multiprotein editosome. Over 20 of its protein components have been identified and additional proteins are likely to function in editing and its regulation. The functions of only a few editosome proteins have been determined. Surprisingly, there are related pairs or sets of editosome proteins, and insertion and deletion editing appear to be functionally and perhaps spatially separate. A model for the editosome is proposed, which has a catalysis domain with separate sectors for insertion and deletion editing. It also contains domains for anchor duplex and upstream RNA binding, which position the sequence to be edited in the catalysis domain.

Animals↗
PubMed2003

Generalized RNA-directed recombination of RNA.

RNA strand exchange through phosphor-nucleotidyl transfer reactions is an intrinsic chemistry promoted by group I intron ribozymes. We show here that Tetrahymena and Azoarcus ribozymes can promote RNA oligonucleotide recombination in either two-pot or one-pot schemes. These ribozymes bind one oligonucleotide, cleave following a guide sequence, transfer the 3' portion of the oligo to their own 3' end, bind a second oligo, and catalyze another transfer reaction to generate recombinant oligos. Recombination is most effective with the Azoarcus ribozyme in a single reaction vessel in which over 75% of the second oligo can be rapidly converted to recombinant product. The Azoarcus ribozyme can also create a new functional RNA, a hammerhead ribozyme, which can be constructed via recombination and then immediately promote its own catalysis in a homogeneous milieu, mimicking events in a prebiotic soup.

Animals↗
PubMed2002

Bi-functional, substrate mimicking RNA inhibits MSK1-mediated cAMP-response element-binding protein phosphorylation and reveals magnesium ion-dependent conformational changes of the kinase.

The design of specific inhibitors for protein kinases is an important step toward elucidation of intracellular signal transduction pathways and to guide drug discovery programs. We devised a model approach to generate specific, competitive kinase inhibitors by isolating substrate mimics containing two independent binding sites with an anti-idiotype strategy from combinatorial RNA libraries. As a general test for the ability to generate highly specific kinase inhibitors, we selected the transcription factor cAMP-response element-binding protein (CREB) that is phosphorylated on the same serine residue by the protein kinase MSK1 as well as by RSK1. The sequences and structures of these kinases are very similar, about 60% of their amino acids are identical. Nevertheless, we can demonstrate that the selected RNA inhibitors inhibit specifically CREB phosphorylation by MSK1 but do not affect CREB phosphorylation by RSK1. The inhibitors interact preferentially with the inactive form of MSK1. Furthermore, we demonstrate that RNA ligands can be conformation-specific probes, and this feature allowed us to describe magnesium ion-dependent conformational changes of MSK1 upon activation.

Base Sequence↗
PubMed1991

Evidence for a substrate-binding subsite in ribonuclease T1. Crystal structure of the complex with two guanosines, and model building of the complex with the substrate guanylyl-3',5'-guanosine.

The enzyme ribonuclease T1 cleaves single-stranded RNA at the 3'-side of guanosine. The structure of the complex with two guanosines has been analyzed at 1.8-A resolution and refined to a crystallographic R value of 14.0%. One guanosine occupies the guanosine recognition site as observed in previously analyzed complexes of ribonuclease T1 with guanosine phosphates. The other is bound to a base-unspecific subsite marking the binding locus of the nucleoside 3'-proximal to guanosine in a cleavable RNA chain. The positions of the guanosine bound to the recognition site and of the guanine base at the subsite were used to guide model building of the substrate guanylyl-3',5'-guanosine bound to the active site of ribonuclease T1. After energy minimization and a 7-ps stochastic dynamics simulation, a plausible model of the enzyme-substrate complex was obtained which may serve as a reference point in consideration of the mechanisms of RNA hydrolysis by ribonuclease T1.

Crystallization↗
PubMed2006

[Trans-acting short interfering RNAs].

RNA is now considered a key factor in the regulation of gene expression. There are several classes of small regulatory RNAs in plants, functioning in posttranscriptional gene silencing (PTGS) or epigenetic DNA modification. Trans-acting short interfering RNAs (tasiRNAs) form a class of small regulatory RNAs which has been distinguished only recently. To date, five genes encoding tasiRNAs have been identified in Arabidopsis thaliana. TasiRNAs derive from non-coding RNA precursors which are initially targeted for cleavage by a miRNA. Cleavage products are then converted into dsRNAs by a RNA dependent RNA polymerase and sequentially cleaved into 21-nt tasiRNAs. Like the majority of plant miRNAs, tasiRNAs regulate gene expression at the posttranscriptional level, guiding cleavage of ARF and PPR transcripts. Here, we briefly present tasiRNAs and speculate whether they form a homogeneous class of siRNAs.

Gene Expression Profiling↗
PubMed1999

Disappearance of serum HCV-RNA after short-term prednisolone therapy in a patient with chronic hepatitis C associated with autoimmune hepatitis-like serological manifestations.

We report a 70-year-old woman with chronic hepatitis C associated with autoimmune hepatitis (AIH)-like serological manifestations, in whom elimination of hepatitis C virus (HCV) was observed after corticosteroid treatment. The patient was infected with HCV, genotype Ib, but had several laboratory findings, such as markedly elevated serum gamma-globulin and IgG, characteristic of AIH, as well as a high titer of an anti-nuclear antibody. An ultrasound (US)-guided liver biopsy disclosed chronic active hepatitis F3. Corticosteroid worsened her liver function test results and raised amounts of HCV-RNA in the serum. Withdrawal of the corticosteroid led to prompt normalization of transaminase levels and the disappearance of serum HCV-RNA, determined by reverse transcription-polymerase chain reaction (RT-PCR). For 4 years, up to the time of this study, her transaminase values have been normal and HCV viremia was not detected by repeated RT-PCR. We believe this to be the first reported case in which eradication of HCV was achieved by corticosteroid therapy alone, without the introduction of interferon.

Aged↗
PubMed2006

Application of oligonucleotide microarrays to assess the biological effects of neoadjuvant imatinib mesylate treatment for localized prostate cancer.

PURPOSE: Neoadjuvant administration of antineoplastic therapies is used to rapidly assess the clinical and biological activity of novel systemic treatments. To assess the feasibility of using microarrays to assess molecular end points following targeted treatment in a heterogeneous tumor, we measured global gene expression in localized prostate cancer before and following neoadjuvant treatment with imatinib mesylate. PATIENTS AND METHODS: Patients with intermediate-risk to high-risk prostate cancer were treated for 6 weeks with 200 to 300 mg of oral imatinib mesylate. Frozen tissue was obtained from pretreatment ultrasound-guided biopsies and posttreatment radical prostatectomy specimens. Oligonucleotide microarray analysis following laser capture microdissection (LCM) and RNA amplification was used to assess gene expression changes associated with imatinib mesylate therapy. Immunohistochemistry was used to measure protein expression of MKP1 and CD31 and to assess cellular apoptosis. RESULTS: Of the 11 patients enrolled, high-quality microarray data was obtained from both biopsies (n = 7) and radical prostatectomy specimens (n = 9). Technically introduced intrasample gene expression variability was found to be significantly less than intertumor biological variability. Large gene expression differences were observed, and the gene with the most consistent differential expression (MKP1) was validated by immunohistochemistry. Gene set enrichment analysis suggests that imatinib mesylate therapy results in apoptosis of microvascular endothelial cells, an observation anecdotally supported by immunohistochemistry. CONCLUSIONS: This study shows that high-quality microarray data can be generated using LCM and RNA amplification to discover potential mechanisms of targeted therapy in cancer.

Adenocarcinoma↗
PubMed2026

Comprehensive Multiplatform Tyrosine Kinase Profiling Reveals Novel Actionable FGFR Aberrations across Sarcomas Affecting the Young.

Limited targeted agents are approved for pediatric sarcomas. Tyrosine kinase (TK) inhibitors (TKi) have shown clinical efficacy in some, but not all, young patients with sarcoma. A major obstacle preventing further advances and clinical implementation is the lack of predictive response biomarkers to guide TK-targeted treatments. TK-activating fusions or mutations are rare in these patients. RNA overexpression of TKs is a frequent feature. The unresolved question is when upregulated TK expression is associated with kinase activation and signaling dependence. We explored the TK molecular landscape of 107 patients with sarcoma from the ZERO Childhood Cancer Precision Medicine Program (ZERO) using whole-genome and -transcriptome sequencing. Phosphoproteomic analyses of tyrosine phosphorylation (pY) and functional in vitro and in vivo assays were performed in cell lines and patient-derived xenografts (PDX). Our analysis shows that although novel genomic driver lesions are rare, when present they are therapeutically actionable as exemplified by a novel LSM1-FGFR1 fusion identified in a patient with osteosarcoma. We further show that in certain contexts, TK RNA expression can indicate TK pathway activity and predict TKi sensitivity. We highlight the utility of FGFR inhibitors in PAX3-FOXO1 fusion-positive rhabdomyosarcomas (FP-RMS) characterized by high FGFR4 and FGF8 RNA expression levels and FGFR4 activation (FGFR4_pY). We demonstrate marked tumor growth inhibition in all FP-RMS PDXs treated with single-agent FGF401 (FGFR4-specific inhibitor) and single-agent lenvatinib (multikinase FGFR inhibitor) and report a clinical response to lenvatinib in a patient with relapsed metastatic FP-RMS. Altogether, we identified new patients with sarcoma who may benefit from FGFR inhibitors, most notably FP-RMS via FGFR4/FGF8 coexpression.

Journal Article↗
PubMed2003

[Novel approaches; improved diagnostics and therapeutics with DNA microarrays. II. Applications].

The most important (future) applications of DNA microarrays in medicine are based on three different molecular analyses: gene expression analysis, genotyping with a specific type of microarray (single nucleotide polymorphism chip (SNP chip)), and DNA sequencing to search for a well-known mutation in a certain gene. With gene expression analysis one can obtain a 'molecular signature' of a tissue by allowing tissue RNA to react with a DNA microarray. This information may help to refine disease classifications, to guide the choice of therapy and to find new therapeutic targets. Genotyping utilizes genomic DNA that, after digestion, reacts with a SNP chip to obtain an individual SNP pattern. An SNP is a change of a single base pair within a gene sequence that can sometimes influence the function of the gene product. SNPs are considered to be variations in the genome and should be distinguished from mutations. These variations can for instance provide information about the probability of a certain disease, or the effectiveness or side effect of a certain drug. Sequencing well characterized genes to search for mutations is used to screen for genetic abnormalities. This process can be accelerated with the aid of oligonucleotide arrays. With this tool an individual's risk of developing certain forms of cancer or monogenetic diseases can be estimated.

Diagnostic Techniques and Procedures↗
PubMed2004

Complete cycles of bloodstream trypanosome RNA editing in vitro.

RNA editing in kinetoplastid protists is required for the maturation of mitochondrial pre-mRNAs and occurs by protein-catalyzed cycles of uridylate insertion and deletion. During the complex life cycle of Trypanosoma brucei this process is differentially regulated in the mammalian bloodstream and insect procyclic stages. Complementary guide RNAs (gRNAs) direct editing, but the abundance of these transcripts is not developmentally controlled. The establishment of in vitro systems that recreate efficient RNA editing in bloodstream T. brucei would be valuable for mechanistic studies of regulation. Here we describe a robust in vitro system that reconstitutes full cycles of both U insertion and U deletion in bloodstream trypanosomes, and the first direct comparisons of the in vitro systems for strains of mammalian and insect stages.

Animals↗
PubMed2004

Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step.

RNA silencing suppressors from different plant viruses are structurally diverse. In addition to inhibiting the antiviral silencing response to condition susceptibility, many suppressors are pathogenicity factors that cause disease or developmental abnormalities. Here, unrelated suppressors from multiple viruses were shown to inhibit microRNA (miRNA) activities and trigger an overlapping series of severe developmental defects in transgenic Arabidopsis thaliana. This suggests that interference with miRNA-directed processes may be a general feature contributing to pathogenicity of many viruses. A normally labile intermediate in the miRNA biogenesis/RNA-induced silencing complex (RISC) assembly pathway, miRNA*, accumulated specifically in the presence of suppressors (P1/HC-Pro, p21, or p19) that inhibited miRNA-guided cleavage of target mRNAs. Both p21 and p19, but not P1/HC-Pro, interacted with miRNA/miRNA* complexes and hairpin RNA-derived short interfering RNAs (siRNAs) in vivo. In addition, p21 bound to synthetic miRNA/miRNA* and siRNA duplexes in vitro. We propose that several different suppressors act by distinct mechanisms to inhibit the incorporation of small RNAs into active RISCs.

Arabidopsis↗
PubMed2006

Comparison of a rule-based algorithm with a phenotype-based algorithm for the interpretation of HIV genotypes in guiding salvage regimens in HIV-infected patients by a randomized clinical trial: the mutations and salvage study.

BACKGROUND: There is still considerable uncertainty as to the best algorithm for interpreting human immunodeficiency virus (HIV) genotyping results. METHODS: A total of 318 subjects with HIV RNA levels of >1000 copies/mL were enrolled in 41 centers throughout Italy from 2001 through 2003, stratified on the basis of their drug history, randomized (1:1) to 2 arms to have their treatments modified on the basis of the results of HIV genotyping (as interpreted by virtual phenotype analysis or with use of a rule-based interpretation system), and followed up for 48 weeks. At least 1 nucleoside reverse-transcriptase inhibitor and 1 protease inhibitor had to be included in any new regimen; nonnucleoside reverse-transcriptase inhibitor-naive patients were also prescribed a nonnucleoside reverse-transcriptase inhibitor. Only drugs licensed in Italy were allowed. The primary end point was a decrease in HIV RNA level to <400 copies/mL by week 12 according to on-treatment analysis. RESULTS: The mean (+/- standard deviation) values at baseline were as follows: HIV RNA level, 4.1+/-0.74 log(10) copies/mL; CD4(+) T lymphocyte count, 410+/-262 cells/microL; reverse-transcriptase mutations, 4.8+/-2.9; and protease mutations, 2.8+/-2.5. There were 133 patients (41.8%) who were nonnucleoside reverse-transcriptase inhibitor naive and protease inhibitor experienced, 63 patients (19.8%) who were nonnucleoside reverse-transcriptase inhibitor experienced and protease inhibitor naive, and 122 patients (38.4%) who were 3-class experienced. A total of 192 patients completed 12 weeks of the treatment regimen assigned at baseline; at 12 weeks, 66.3% of patients in the virtual phenotype arm and 71.3% of patients in the rule-based interpretation arm had HIV RNA levels of <400 copies/mL (P = .46). No statistically significant difference between arms was observed by intention-to-treat analysis. CONCLUSION: Both the virtual phenotype and rule-based interpretation methods of HIV genotyping can guide the selection of effective antiretroviral drugs for a salvage regimen.

Adult↗
PubMed1999

RNA tertiary folding monitored by fluorescence of covalently attached pyrene.

The pathways by which large RNAs adopt tertiary structure are just beginning to be explored, and new methods that reveal RNA folding are highly desirable. Here we report an assay for RNA tertiary folding in which the fluorescence of a covalently incorporated chromophore is monitored. Folding of the 160-nucleotide Tetrahymena group I intron P4-P6 domain was used as a test system. Guided by the P4-P6 X-ray crystal structure, we chose a nucleotide (U107) for which derivatization at the 2'-position should not perturb the folded conformation. A 15-mer RNA oligonucleotide with a 2'-amino substitution at U107 was derivatized with a pyrene chromophore on a variable-length tether, and then ligated to the remainder of P4-P6, providing a site-specifically pyrene-labeled P4-P6 derivative. Upon titration of the pyrene-derivatized P4-P6 with Mg(2+), the equilibrium fluorescence intensity reversibly increased several-fold, as expected if the probe's chemical microenvironment changes as the RNA to which it is attached folds. The concentration and specificity of divalent ions required to induce the fluorescence change (Mg(2+) approximately Ca(2+) > Sr(2+)) correlated well with biochemical folding assays that involve nondenaturing gel electrophoresis. Furthermore, mutations in P4-P6 remote from the chromophore that shifted the Mg(2+) folding requirement on nondenaturing gels also affected in a predictable way the Mg(2+) requirement for the fluorescence increase. Initial stopped-flow studies with millisecond time resolution suggest that this fluorescence method will be useful for following the kinetics of P4-P6 tertiary folding. We conclude that a single site-specifically tethered chromophore can report the formation of global structure of a large RNA molecule, allowing one to monitor both the equilibrium progress and the real-time kinetics of RNA tertiary folding.

Animals↗