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At least 667 records · Page 37Linked to original sources
PubMed1997

Changes in plasma HIV RNA levels and CD4+ lymphocyte counts predict both response to antiretroviral therapy and therapeutic failure. VA Cooperative Study Group on AIDS.

BACKGROUND: Markers are needed for assessing response to antiretroviral therapy over time. The CD4+ lymphocyte count is one such surrogate, but it is relatively weak. OBJECTIVE: To assess the association of changes in plasma human immunodeficiency virus (HIV) RNA level and CD4+ lymphocyte count with progression to the acquired immunodeficiency syndrome (AIDS). DESIGN: Analysis of data from a subset of patients in a multicenter, randomized, clinical trial. SETTING: Six Veterans Affairs medical centers and one U.S. Army medical center. PATIENTS: 270 symptomatic HIV-infected patients from the Veterans Affairs Cooperative Study on AIDS. INTERVENTION: Patients were randomly assigned to receive zidovudine or placebo initially; a cross-over protocol was established for patients receiving placebo who had disease progression. MEASUREMENTS: Reverse transcriptase polymerase chain reaction on cryopreserved plasma samples, previously obtained CD4+ lymphocyte counts, and clinical events. RESULTS: For each decrease of 0.5 log10 copies/mL in plasma HIV RNA level, averaged over the 6 months after randomization, the relative risk (RR) for progression to AIDS was 0.67 (P < 0.001). In a subset of 70 treated patients with long-term follow-up, a return to baseline plasma HIV RNA levels within 6 months of randomization was associated with progression to AIDS (RR, 4.28; P = 0.004). Plasma HIV RNA levels or CD4+ lymphocyte counts over time were more strongly associated with progression to AIDS than were baseline levels or counts. CONCLUSIONS: An adequate virologic response after initiation of antiretroviral therapy seems to require a decrease in plasma HIV RNA level of at least 0.5 log10 copies/mL that is sustained for at least 6 months. The independent relation between plasma HIV RNA level and CD4+ lymphocyte count over time and clinical outcome suggests that the measurement of plasma HIV RNA level, in addition to the CD4+ lymphocyte count, has a role in guiding the management of antiretroviral therapy.

Acquired Immunodeficiency Syndrome↗
PubMed2002

The SBP2 and 15.5 kD/Snu13p proteins share the same RNA binding domain: identification of SBP2 amino acids important to SECIS RNA binding.

Selenoprotein synthesis in eukaryotes requires the selenocysteine insertion sequence (SECIS) RNA, a hairpin in the 3' untranslated region of selenoprotein mRNAs. The SECIS RNA is recognized by the SECIS-binding protein 2 (SBP2), which is a key player in this specialized translation machinery. The objective of this work was to obtain structural insight into the SBP2-SECIS RNA complex. Multiple sequence alignment revealed that SBP2 and the U4 snRNA-binding protein 15.5 kD/Snu13p share the same RNA binding domain of the L7A/L30 family, also found in the box H/ACA snoRNP protein Nhp2p and several ribosomal proteins. In corollary, we have detected a similar secondary structure motif in the SECIS and U4 RNAs. Combining the data of the crystal structure of the 15.5 kD-U4 snRNA complex, and the SBP2/15.5 kD sequence similarities, we designed a structure-guided strategy predicting 12 SBP2 amino acids that should be critical for SECIS RNA binding. Alanine substitution of these amino acids followed by gel shift assays of the SBP2 mutant proteins identified four residues whose mutation severely diminished or abolished SECIS RNA binding, the other eight provoking intermediate down effects. In addition to identifying key amino acids for SECIS recognition by SBP2, our findings led to the proposal that some of the recognition principles governing the 15.5 kD-U4 snRNA interaction must be similar in the SBP2-SECIS RNA complex.

Amino Acid Sequence↗
PubMed1996

Isolation of a local tertiary folding transition in the context of a globally folded RNA.

Binding of the Tetrahymena ribozyme's oligonucleotide substrate represents a local folding event in the context of a globally folded RNA. Substrate binding involves P1 duplex formation with the ribozyme's internal guide sequence to give an "open complex', followed by docking of the P1 duplex into tertiary interactions to give a "closed complex'. We have isolated the open complex as a thermodynamically stable species using a site-specific modification and high Na+ concentrations. This has allowed characterization of P1 docking, which represents a folding transition between local secondary and local tertiary structure. P1 docking is entropically driven, possibly accompanied by a release of bound water molecules. Strategies analogous to those described here can be used more generally to study local folding events in large structured RNAs and to explore the structural and energetic landscape for RNA folding.

Animals↗
PubMed2006

Graphical exploratory data analysis of RNA secondary structure dynamics predicted by the massively parallel genetic algorithm.

Studies indicate that RNA may enter intermediate and multiple conformational states, which may impact gene expression and molecular function. It is known that the biologically functional states of RNA molecules may not correspond to their minimum energy conformations, that kinetic barriers may trap the molecule in a local minimum, that folding often occurs during transcription, and that cases exist in which a molecule will transition between one or more functional conformations. Thus, methods for simulating the folding pathway and dynamic behavior of an RNA molecule are important for the prediction of RNA structure and its associated functions. We have developed several data mining techniques guided by interactive visualization tools associated with our massively parallel genetic algorithm for RNA/DNA secondary structure prediction, MPGAfold, and StructureLab analysis workbench. Most of the methods and tools are also applicable to dynamic programming algorithm (DPA) folding data analysis. When applied to MPGAfold results these methodologies are used to determine the significant intermediate and final structures associated with co-transcriptional and full length RNA folding. Since the genetic algorithm is essentially stochastic, multiple runs are required to develop a consensus understanding of an RNA structure. The interactive visualizations facilitate interpretation of results from sequential or full length individual MPGAfold runs, final results of multiple folding runs, including multiple population sizes, and the results from multiple RNA sequences of one family. This paper describes several of these techniques and shows how they are used to help solve this highly combinatoric problem.

Algorithms↗
PubMed2006

Radiation recall phenomenon secondary to capecitabine: possible role of thymidine phosphorylase.

BACKGROUND: The first reported case of radiation (XRT) recall related to capecitabine described dermatitis in a previously radiated field in a breast cancer patient (Ortman; JCO). We previously reported the first case of recall syndrome manifesting as diffuse gastritis and duodenitis related to capecitabine with prior XRT with 5-FU in a pancreatic cancer patient (Saif; JARCET). We report here another pancreatic cancer patient with a radiation recall receiving capecitabine following capecitabine-XRT. PATIENTS AND METHODS: From April 2004 to June 2005, 20 patients with locally advanced pancreatic adenocarcinoma were treated with capecitabine 1,600 mg/m2 daily with concomitant radiation (5040cGy) Monday-Friday (weekends off) for a total of 6 weeks, followed by capecitabine 2,000 mg/m2 daily for 14 days every 3 weeks. One male patient with tumor in the neck and body of pancreas and not infiltrating the duodenum dropped hemoglobin to 7.3 g/dl at the end of the ninth week, and melena on rectal examination. Specimen of primary pancreatic ductal adenocarcinoma was obtained via EUS-guided biopsy before starting XRT on day 1 and utilized for RNA extraction. TP mRNA level was determined by real-time quantitative PCR (RT-Q-PCR). RESULTS: Upper endoscopy revealed gastritis consistent with radiation toxicity. Colonoscopy was negative. Transfusion of three units of packed red blood cells (PRBCs) was given. The dose of capecitabine was reduced by 25%. His anemia continued to progress, a CT scan revealed 39% decrease in the tumor size (PR). Analysis of tumor specimen prior to the start of capecitabine-XRT showed TP expression of 183.16 (high). In addition to TP, DPD was 7.40, and TNF-alpha 4,114.56. CONCLUSION: We believe this case to be the second case of radiation recall presenting as diffuse gastritis in a patient receiving capecitabine after previous treatment with XRT. Further studies, including the role of TP are warranted into the pathogenesis of this unique phenomenon.

Aged↗
PubMed1999

Guided tours: from precursor snoRNA to functional snoRNP.

Small nucleolar RNAs (snoRNAs) use base pairing to guide modification of conserved nucleotides in functionally important regions of ribosomal RNA. The box C/D snoRNAs direct 2'-O-methylation and the box H/ACA snoRNAs direct pseudouridylation. Each snoRNA interacts with proteins, many of them newly identified. Progress in understanding how snoRNA sequences are stored within genomes, liberated from precursor molecules and targeted to the nucleolus has begun to elucidate each step in the biogenesis of these critical contributors to ribosome formation.

Animals↗
PubMed2005

The complete set of H/ACA snoRNAs that guide rRNA pseudouridylations in Saccharomyces cerevisiae.

Conversion of uridines into pseudouridines (Psis) is the most frequent base modification in ribosomal RNAs (rRNAs). In eukaryotes, the pseudouridylation sites are specified by base-pairing with specific target sequences within H/ACA small nucleolar RNAs (snoRNAs). The yeast rRNAs harbor 44 Psis, but, when this work began, 15 Psis had completely unknown guide snoRNAs. This suggested that many snoRNAs remained to be discovered. To address this problem and further complete the snoRNA assignment to Psi sites, we identified the complete set of RNAs associated with the H/ACA snoRNP specific proteins Gar1p and Nhp2p by coupling TAP-tag purifications with genomic DNA microarrays experiments. Surprisingly, while we identified all the previously known H/ACA snoRNAs, we selected only three new snoRNAs. This suggested that most of the missing Psi guides were present in previously known snoRNAs but had been overlooked. We confirmed this hypothesis by systematically investigating the role of previously known, as well as of the newly identified snoRNAs, in specifying rRNA Psi sites and found all but one missing guide RNAs. During the completion of this work, another study, based on bioinformatic predictions, also reported the identification of most missing guide RNAs. Altogether, all Psi guides are now identified and we can tell that, in budding yeast, the 44 Psis are guided by 28 snoRNAs. Finally, aside from snR30, an atypical small RNA of heterogeneous length and at least one mRNA, all Gar1p and Nhp2p associated RNAs characterized by our work turned out to be snoRNAs involved in rRNA Psi specification.

Base Sequence↗
PubMed2005

A nomenclature for all signal recognition particle RNAs.

The signal recognition particle (SRP) is a cytosolic ribonucleoprotein complex that guides secretory proteins to biological membranes in all organisms. The SRP RNA is at the center of the structure and function of the SRP. The comparison of the growing number of SRP RNA sequences provides a rich source for gaining valuable insight into the composition, assembly, and phylogeny of the SRP. In order to assist in the continuation of these studies, we propose an SRP RNA nomenclature applicable to the three divisions of life.

Animals↗
PubMed2004

Microbead-based affinity chromatography chip using RNA aptamer modified with photocleavable linker.

A microbead-based affinity chromatography chip (micro-BACC) controlling hundreds of nanoliters of reaction volume was developed to separate and analyze hepatitis C virus (HCV) RNA polymerase protein by immobilization of an RNA aptamer on beads. A photocleavable linker was conjugated in between the beads and the aptamer to elute the bound RNA polymerase from the RNA aptamer in one step by UV irradiation, resulting in an efficient method to elute and identify the target molecule bound on RNA using a mass spectrometer. This linker showed a cleavage activity over 70% upon UV irradiation at 1050 mW/cm2 for more than 5 min. The photoelution method could prevent the target molecule from contaminations in affinity chromatography caused by elution solutions of high salt concentration, extreme pH and detergent, respectively. In this chip, sample reagents up to 800 nL could be metered quantitatively into the bead chamber using a nanoliter dispenser working, based on surface-guided flow control and pneumatic control by external air pressure on the chip. RNA polymerase eluted after UV irradiation was successfully analyzed by trypsin treatment without additional purification. As a result, using the aptamer, we could detect RNA polymerase from 800 nL hepatitis C patient serum containing 96 fmol HCV RNA polymerase. The detection limit of this system was estimated to be 9.6 fmol HCV RNA polymerase.

Chromatography, Affinity↗
PubMed1984

Assessment of a model for intron RNA secondary structure relevant to RNA self-splicing--a review.

A widespread class of introns is characterized by a particular RNA secondary structure, based upon four conserved nucleotide sequences. Among such "class I" introns are found the majority of introns in fungal mitochondrial genes and the self-splicing intron of the large ribosomal RNA of several species of Tetrahymena. A model of the RNA secondary structure, which must underlie the self-splicing activity, is here evaluated in the light of data on 16 further introns. The main body or "core structure" of the intron always consists of the base-paired regions P3 to P9 with the associated single-stranded loops, with P2 present also in most cases. Two minority sub-classes of core structure occur, one of which is typical of introns in fungal ribosomal RNA. Introns in which the core structure is close to the 5' splice site all have an internal guide sequence (IGS) which can pair with exon sequences adjacent to the 5' and 3' splice sites to align them precisely, as proposed by Davies et al. [Nature 300 (1982) 719-724]. In these cases, the internal guide model allows us to predict correctly the exact location of splice sites. All other introns probably use other mechanisms of alignment. This analysis provides strong support for the RNA splicing model which we have developed.

Base Sequence↗
PubMed1997

Early detection of perinatal human immunodeficiency virus (HIV) type 1 infection using HIV RNA amplification and detection. New York City Perinatal HIV Transmission Collaborative Study.

Early diagnosis of perinatally transmitted human immunodeficiency virus type 1 (HIV) infection can guide early interventions. HIV coculture and DNA polymerase chain reaction (DNA-PCR) detect few HIV-infected infants at birth and 90%-100% by age 3 months. Because extracellular HIV RNA may appear soon after infection, a plasma HIV RNA assay was compared with DNA-PCR for early detection of perinatally infected infants. Blood-draw specimens (108) obtained at the same time from 49 HIV-infected infants and 10 specimens from 8 uninfected infants were tested. HIV RNA and DNA-PCR positivity rates were 56% and 33%, respectively, in 36 specimens from 36 infants <28 days of age (binomial test, P = .001). Among 81 specimens obtained after age 14 days, 79 (98%) were positive by HIV RNA testing. No HIV-infected infant specimens were DNA-PCR-positive and HIV RNA-negative. All specimens from 8 uninfected infants were HIV RNA-negative. These results suggest that plasma HIV RNA was detectable earlier and more reliably than HIV DNA in perinatal infection.

Female↗
PubMed2004

Shape-specific nucleotide binding of single-stranded RNA by the GLD-1 STAR domain.

Proteins containing the STAR RNA-binding domain fulfill vital roles in RNA biogenesis, yet a detailed understanding of STAR domain RNA binding specificity is lacking. In Caenorhabditis elegans, the STAR protein GLD-1 directly binds the 28 nucleotide recognition element TGE within the 3' untranslated region of tra-2 mRNA. The GLD-1:TGE interaction promotes translational silencing of tra-2 mRNA, marking a pivotal event in the spermatogenesis to oogenesis switch in C.elegans hermaphrodites. By measuring the binding affinities of both GLD-1 and TGE mutants, we have explored the molecular determinants of STAR domain specificity. Site-directed GLD-1 mutants were guided by sequence homology with human splicing factor 1 (SF1), for which an RNA:protein complex structure is available in the work done by Liu et al. The RNA binding affinity of 11 mutant GLD-1 proteins was measured, and their binding specificity was assessed with a series of TGE RNAs containing natural or modified nucleotides. This combinatorial analysis of both RNA and protein mutants revealed a diverse array of specificities of individual nucleotide-binding pockets along the interface. At nucleotide position 18, adenosine appears to be specified by the overall shape of a pocket lined with aliphatic side-chains. At position 19, the high preference for cytidine is dependent on both the length of an amino acid side-chain and the identity of terminal functional groups. The nucleotide 21 binding pocket exhibits low discrimination for cytidine, and accommodates most nucleobases. The highly hydrophobic binding interface and apparent small number of hydrogen bonding read-out interactions at these positions is consistent with our finding that few amino acids seem to function individually in establishing binding specificity. Rather, specificity is conferred by the shape of the nucleotide-binding pocket. Our data provide the first detailed, quantitative analysis of the STAR domain, and highlight features of STAR:RNA recognition that are distinct among single-stranded RNA-binding proteins.

Alanine↗
PubMed2024

Construction of an immunogenic cell death-related LncRNA signature to predict the prognosis of patients with lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is one of the most common malignant diseases worldwide. This study aimed to construct an immunogenic cell death (ICD)-related long non-coding RNA (lncRNA) signature to effectively predict the prognosis of LUAD. METHODS: The RNA-sequencing and clinical data of LUAD were downloaded from The Cancer Genome Atlas (TCGA). Least absolute shrinkage and selection operator (LASSO) and stepwise multivariate Cox proportional hazard regression analysis were utilized to construct lncRNA signature. Then, the reliability of the signature was evaluated in the training, validation and whole cohorts. The differences in the immune landscape and drug sensitivity between the low- and high-risk groups were analyzed. Finally, the expression level of the selected ICD-related lncRNAs in LUAD cell lines via reverse transcription quantitative PCR (RT-qPCR). CCK-8 and transwell assays were performed to study biological function of AC245014.3. RESULTS: A signature consisting of 5 ICD-related lncRNAs was constructed. Kaplan Meier (K-M) survival analysis showed shorter overall survival (OS) in high-risk group. The receiver operating characteristic (ROC) curves and Multivariate Cox regression analysis showed the signature was good predictive and independent prognostic factor in LUAD. Moreover, the high-risk group had a lower level of antitumor immunity and was less sensitive to some chemotherapeutics and targeted drugs. Finally, the expression level of selected ICD-related lncRNAs was validated in LUAD cell lines by RT-qPCR. Knockdown of AC245014.3 significantly suppressed LUAD proliferation, migration and invasion. CONCLUSIONS: In this study, an ICD-related lncRNA signature was constructed, which could accurately predict the prognosis of LUAD patients and guide clinical treatment.

Humans↗
PubMed1992

Mutational evidence for competition between the P1 and the P10 helices of a mitochondrial group I intron.

A guanosine to cytosine transversion at position 2 of the fifth intron of the mitochondrial gene COB blocks the ligation step of splicing. This mutation prevents the formation of a base pair within the P1 helix of this group I intron--the RNA duplex formed between the 3' end of the upstream exon and the internal guide sequence. The mutation also reduces the rate of the first step of splicing (guanosine addition at the 5' splice junction) while stimulating hydrolysis at the 3' intron-exon boundary. Consequently, the ligation of exons is blocked because the 3' exon is removed prior to cleavage at the 5' splice junction. The lesion can be suppressed by second-site mutations that preserve the potential for base-pairing at this position. Because the P1 duplex and the P10 duplex (between the guide sequence and the 3' exon) overlap at the affected pairings represent alternative structures that do not, indeed cannot, form simultaneously.

Base Composition↗
PubMed2004

The archaeal sRNA binding protein L7Ae has a 3D structure very similar to that of its eukaryal counterpart while having a broader RNA-binding specificity.

The ribosomal L7Ae protein of archaea has the peculiarity to be a component of the C/D and H/ACA snRNPs, that guide rRNA post-transcriptional modifications. Its yeast (Snu13p) and human (15.5kDa protein) homologs are only found in C/D snoRNPs and the (U4/U6, U5) spliceosomal tri-snRNP. By using a large variety of RNAs, we compared the RNA-binding specificities of the recombinant Pyrococcus abyssi L7Ae and Saccharomyces cerevisiae Snu13 proteins. Unlike Snu13p, protein L7Ae binds terminal loops closed by two A:G and G:A pairs and canonical K-turn structures with similar efficiencies, provided that the terminal loop contains at least 5nt. In contrast to Snu13p, binding of protein L7Ae to canonical K-turn structures is not dependent on the identity of the residue at position 2 in the bulge. The peculiar KT-15 motif of P. abyssi 23S rRNA, that is recognized by L7Ae, does not associate with Snu13p. To get more information on the P. abyssi L7Ae protein, we solved its X-ray structure at 1.9A resolution. In spite of their sequence divergence, the free P. abyssi and bound H. marismortui proteins were found to have highly similar structures. Only a limited number of side-chain conformational changes occur at the protein-RNA interface upon RNA binding. In particular, one ion pair that is formed by residues Glu43 and Lys46 in the free protein is disrupted in the ribosomal 50S subunit, so that, residue Glu43 can interact with the RNA residue G264. The Glu43-Lys46 ion pair of protein L7Ae belongs to a complex network of ion pairs that may participate to protein thermostability.

Amino Acid Sequence↗
PubMed2005

[Construction of a trans-splicing ribozyme for restoring EGFP truncation mutation].

Special designed group I intron ribozymes can specifically splice objective RNA, repair the mutant gene in RNA level. The specificity of ribozyme is determined by nucleotides specific internal guide sequence (IGS) introduced to the enzyme. In this study, fragment sequence containing Tetrahymena thermophilia intron I of 26S rRNA gene was cloned and cis-splicing activity of this ribozyme was confirmed by in vitro transcription. For evaluating the trans-splicing activity of this ribozyme, a truncated mutant Green Fluorescence Protein (GFP) vector, XYQ5/XYQ10- pEGFP-C2, was constructed. This vector deleted the 3' end 564bp fragment of EGFP coding sequence, led to the lost the activity of emitting green fluorescence. Trans-splicing ribozyme plasmids ptrans-rib-CMV2 for remedy of the truncated mutant EGFP was constructed by PCR and molecular cloning techniques. This vector utilizing cloned 26S rRNA intron 1 as core enzyme; selecting T-G site at 194bp of EGFP coding sequence as splicing receptor, designed an IGS which is inversely complement to the 188-193nt of EGFP mRNA; the 195-890bp fragment of EGFP coding sequence was ligated to the 3'-end of ribozyme core. The fragment containing these components was inserted to a eukayotic expression vector pRC-CMV2. Using linearized XYQ5/XYQ10- pEGFP-C2 and ptrans-rib-CMV2 as templates, truncated EGFP mRNA and the constructed ribozyme vector were transcribed and mixed to evaluate the trans-splicing activity. Analysis of in vitro transcription products mix by RT-PCR verified the existence of wild type EGFP mRNA molecule. Co-transfection of XYQ5/XYQ10- pEGFP-C2 with ptrans-rib-CMV2 to Hela cells proved this ribozyme restored green fluorescence within cell, but the efficiency was low.

Animals↗
PubMed2003

Interactions of yeast ribosomal protein rpS14 with RNA.

Yeast ribosomal protein S14 (rpS14) binds to two different RNA molecules: (1). helix 23 of 18S rRNA during its assembly into 40S ribosomal subunits and (2). a stem-loop structure in RPS14B pre-mRNA to repress expression of the RPS14B gene. We used the three-dimensional structure of Thermus thermophilus ribosomal protein S11, a bacterial homologue of rpS14, as a guide to identify conserved, surface-exposed amino acid residues that are likely to contact RNA. Eight residues that met these criteria were mutated to alanine. Most of these mutations affected interaction of rpS14 with either helix 23 or the RPS14B stem-loop RNA or both. Assembly of 40S ribosomal subunits and repression of RPS14B were also affected. S11 contains an extended carboxy-terminal domain rich in basic amino acids, which interacts with rRNA. We systematically evaluated the importance of each of the last ten amino acid residues in the basic, carboxy-terminal tail of yeast rpS14 for binding to RNA, by mutating each to alanine. Mutations in nine of these residues decreased binding of rpS14 to one or both of its RNA ligands. In addition, we examined the importance of four structural motifs in helix 23 of 18S rRNA for binding to rpS14. Mutations that altered either the terminal loop, the G-U base-pair closing the terminal loop, or the internal loop affected binding of rpS14 to helix 23.

Amino Acid Sequence↗
PubMed1996

Niruriside, a new HIV REV/RRE binding inhibitor from Phyllanthus niruri.

During the screening of natural products for their ability to inhibit the binding of HIV-REV protein to [33P]-labeled RRE RNA, one novel compound, niruriside (1), was isolated from the MeOH extract of the dried leaf of Phyllanthus niruri L. by bioassay-guided fractionation. The structure of niruriside was determined by spectroscopic methods. Niruriside showed specific inhibitory activity against the binding of REV protein to RRE RNA with an IC50 value of 3.3 microM; however, niruriside did not protect CEM-SS cells from acute HIV infection at concentrations up to 260 microM using an XTT dye reduction assay.

Antiviral Agents↗