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PubMed1997

U14 small nucleolar RNA makes multiple contacts with the pre-ribosomal RNA.

The small nucleolar RNA (snoRNA) U14 has two regions of extended primary sequence complementarity to the 18S rRNA. The 3' region (domain B) shows the consensus structure for the methylation guide class of snoRNAs, whereas base-pairing between the 5' region (domain A) and the 18S rRNA sequence is required for the formation of functional ribosomes. Between domains A and B lies another essential region (domain Y). Here we report that yeast U14 can be cross-linked in vivo to the pre-rRNA; cross-linking is detected exclusively with the 35S primary transcript. Many nucleotides in U14 that lie outside of domains A and B are cross-linked to the pre-rRNA; in particular the essential domain Y region is cross-linked at several sites. U14 is, therefore, in far more extensive contact with the pre-rRNA than predicted from simple base-pairing models. Moreover, U14 can be cross-linked to other small RNA species. The functional interactions made by U14 during ribosome synthesis are likely to be very complex.

Base Composition↗

PubMed2005

Uncharged tRNA and sensing of amino acid deficiency in mammalian piriform cortex.

Recognizing a deficiency of indispensable amino acids (IAAs) for protein synthesis is vital for dietary selection in metazoans, including humans. Cells in the brain's anterior piriform cortex (APC) are sensitive to IAA deficiency, signaling diet rejection and foraging for complementary IAA sources, but the mechanism is unknown. Here we report that the mechanism for recognizing IAA-deficient foods follows the conserved general control (GC) system, wherein uncharged transfer RNA induces phosphorylation of eukaryotic initiation factor 2 (eIF2) via the GC nonderepressing 2 (GCN2) kinase. Thus, a basic mechanism of nutritional stress management functions in mammalian brain to guide food selection for survival.

Acylation↗

PubMed2006

Conversion of pre-RISC to holo-RISC by Ago2 during assembly of RNAi complexes.

In the Drosophila RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) direct Argonaute2 (Ago2), an endonuclease, within the RNA-induced silencing complex (RISC) to cleave complementary mRNA targets. In vitro studies have shown that, for each siRNA duplex, RISC retains only one strand, the guide, and releases the other, the passenger, to form a holo-RISC complex. Here, we have isolated a new Ago2 mutant allele and provide, for the first time, in vivo evidence that endogenous Ago2 slicer activity is important to mount an RNAi response in Drosophila. We demonstrate in vivo that efficient removal of the passenger strand from RISC requires the cleavage activity of Ago2. We have also identified a new intermediate complex in the RISC assembly pathway, pre-RISC, in which Ago2 is stably bound to double-stranded siRNA.

Alleles↗

PubMed2006

Molecular probes of DNA bulges: functional assay and spectroscopic analysis.

Bulged structures in DNA and RNA have been linked to biomolecular processes involved in numerous diseases, thus probes with affinity for these nucleic acid targets would be of considerable utility to chemical biologists. Herein, we report guided chemical synthesis of small molecules capable of binding to DNA bulges by virtue of their unique (spirocyclic) geometry. The agents, modeled on a natural product congener, show pronounced selectivity for specific bulged motifs and are able to enhance slipped DNA synthesis, a hallmark functional assay of bulge binding. Significantly, bulge-agent complexes demonstrate characteristic fluorescent signatures depending on bulge and flanking sequence in the oligo. It is anticipated that these signature patterns can be harnessed as molecular probes of bulged hotspots in DNA and RNA.

DNA↗

PubMed2005

Methylation as a crucial step in plant microRNA biogenesis.

Methylation on the base or the ribose is prevalent in eukaryotic ribosomal RNAs (rRNAs) and is thought to be crucial for ribosome biogenesis and function. Artificially introduced 2'-O-methyl groups in small interfering RNAs (siRNAs) can stabilize siRNAs in serum without affecting their activities in RNA interference in mammalian cells. Here, we show that plant microRNAs (miRNAs) have a naturally occurring methyl group on the ribose of the last nucleotide. Whereas methylation of rRNAs depends on guide RNAs, the methyltransferase protein HEN1 is sufficient to methylate miRNA/miRNA* duplexes. Our studies uncover a new and crucial step in plant miRNA biogenesis and have profound implications in the function of miRNAs.

Arabidopsis↗

PubMed1987

Structure of the rat L-type pyruvate kinase gene.

The total sequence of a 13,021 base-pair (bp) genomic fragment containing the rat L-type pyruvate kinase (L-PK) gene was determined by "shot gun" sequencing. This fragment includes 8360 bp of the L-PK gene, plus 3193 bp of the 5'-flanking and 1468 bp of the 3'-flanking regions. Like the chicken PK-M1 gene, the rat L-PK gene exhibits a fully conserved exon-intron structure, with 11 exons and 10 introns. In the chicken M1 gene, the coding sequences are well conserved (about 70%), in particular at the level of the exons implicated in the formation of PK active sites, exons that are also partially homologous to the corresponding sequences of the yeast gene. Various types of repetitive sequences exist in the L-PK gene, especially two ID (identifier) sequences located in the second intron and the 11th exon. Elements very similar to the "cyclic AMP-dependent regulatory element" recently described in the phosphoenolpyruvate carboxykinase and somatostatin genes are found in the sequenced fragment, but far upstream (-2338) and downstream (+5788) from the cap site. Various sequences homologous to described regulatory elements (glucocorticoid regulatory elements, enhancers, potential Z-DNA) are also observed 5' and 3' of the cap site. A comparison of the 5'-flanking region of the L-PK gene with the same regions of liver-specific or non-specific, cyclic-AMP-responsive or non-responsive genes was also made. It revealed various potentially interesting features that will be used to guide a further functional study. The cap site was determined by primer extension and nuclease S1 mapping using either mature mRNA or precursor RNA as templates. With both templates the start site of transcription appeared to be microheterogeneous, 19 to 14 bp before the ATG translation initiation codon.

Animals↗

PubMed2005

Identification of 20 snoRNA-like RNAs from the primitive eukaryote, Giardia lamblia.

From a specialized cDNA library of Giardia lamblia, 20 snoRNA-like RNAs, including 16 box C/D sRNAs and four box H/ACA sRNAs, were first identified. The sRNAs were predicted to guide a total of 11 2'-O-methylation and four pseudouridylation sites on the G. lamblia rRNAs, respectively. By using primer extension assay, seven methylation sites were precisely mapped in the G. lamblia 16S rRNA, despite its high GC content. All of the sRNA genes locate on the small intergenic regions of the G. lamblia genome and seem to be independently transcribed from their own promoters. Particularly, a cluster composed of GlsR17 and GlsR18 genes is transcribed as a dicistronic precursor, implying a mechanism of endonuclease cleavage for the maturation of the two sRNAs. The systematic identification of the sRNAs in G. lamblia has provided valuable information about the characteristics of the two major families of small guide RNAs in one of the most primitive eukaryotes and would contribute to the understanding of the evolution of small non-messenger RNA genes from prokaryotes to eukaryotes.

Animals↗

PubMed2003

High prevalence of M184 mutation among patients with viroimmunologic discordant responses to highly active antiretroviral therapy and outcomes after change of therapy guided by genotypic analysis.

Whether highly active antiretroviral therapy (HAART) should be modified in patients with persistent increases in CD4(+) T cells despite detectable viral loads is an unresolved question. Forty-three heavily pretreated human immunodeficiency virus (HIV)-infected patients with virologic failure during HAART were studied before a change of therapy guided by genotypic analysis and during follow-up. Patients with an increase in CD4(+) cell count (>100 cells/ml) over pre-HAART values were considered to be discordant patients (20 individuals), whereas patients with a lower increase or no increase in CD4(+) cell count were considered failing patients (23 individuals). Based on univariate analysis, a high CD4(+) cell count before antiretroviral treatment, homosexual behavior as a risk factor for HIV infection, reduced drug exposure to nonnucleoside reverse transcriptase inhibitors, low replicative capacity of HIV isolates, and more frequent detection of HIV isolates with a non-B subtype, an R5 biological phenotype, and M184V and T215Y/F mutations were factors associated with a discordant response to HAART. Based on multivariate analysis, only the M184V mutation remained significantly associated with a viroimmunologic discordant response (odds ratio, 25.48; 95% confidence interval, 1.43 to 453.93). No difference in lamivudine exposure was found between discordant (95%) and failing (91%) patients. Twelve months after the genotypic analysis-guided change of therapy, 3 discordant (15%) and 6 failing patients (26%) achieved undetectable viral loads (<50 copies/ml), whereas in patients with HIV RNA loads of >500 copies/ml, discordant responses were observed in 5 out of 15 discordant patients and in 4 out of 16 failing patients. A relationship between the M184V mutation and a viroimmunologic discordant response to HAART was found. After the genotypic analysis-driven change of therapy, similar rates of virologic suppression were detected in the two groups.

Antiretroviral Therapy, Highly Active↗

PubMed2006

Trypanosoma cruzi mitochondrial maxicircles display species- and strain-specific variation and a conserved element in the non-coding region.

BACKGROUND: The mitochondrial DNA of kinetoplastid flagellates is distinctive in the eukaryotic world due to its massive size, complex form and large sequence content. Comprised of catenated maxicircles that contain rRNA and protein-coding genes and thousands of heterogeneous minicircles encoding small guide RNAs, the kinetoplast network has evolved along with an extreme form of mRNA processing in the form of uridine insertion and deletion RNA editing. Many maxicircle-encoded mRNAs cannot be translated without this post-transcriptional sequence modification. RESULTS: We present the complete sequence and annotation of the Trypanosoma cruzi maxicircles for the CL Brener and Esmeraldo strains. Gene order is syntenic with Trypanosoma brucei and Leishmania tarentolae maxicircles. The non-coding components have strain-specific repetitive regions and a variable region that is unique for each strain with the exception of a conserved sequence element that may serve as an origin of replication, but shows no sequence identity with L. tarentolae or T. brucei. Alternative assemblies of the variable region demonstrate intra-strain heterogeneity of the maxicircle population. The extent of mRNA editing required for particular genes approximates that seen in T. brucei. Extensively edited genes were more divergent among the genera than non-edited and rRNA genes. Esmeraldo contains a unique 236-bp deletion that removes the 5'-ends of ND4 and CR4 and the intergenic region. Esmeraldo shows additional insertions and deletions outside of areas edited in other species in ND5, MURF1, and MURF2, while CL Brener has a distinct insertion in MURF2. CONCLUSION: The CL Brener and Esmeraldo maxicircles represent two of three previously defined maxicircle clades and promise utility as taxonomic markers. Restoration of the disrupted reading frames might be accomplished by strain-specific RNA editing. Elements in the non-coding region may be important for replication, transcription, and anchoring of the maxicircle within the kinetoplast network.

Amino Acid Sequence↗

PubMed1998

Nocardia, nocardiosis and mycetoma.

The recent emergence of invasive infections due to Nocardia spp., including nosocomial outbreak, is now evident. Newer molecular diagnostic and typing methods are developed. Although sulfonamide-based therapy is generally effective, optimal treatment may be guided by antimicrobial susceptibility testing of isolates. The improved classification of nocardiae and other related genera such as actinomadurae, using the 16S ribosomal RNA sequencing, provide a sound basis for improved diagnostic methods for the identification of members of clinically significant species. The commonest cause of eumycetoma in Sudan is Madurella mycetomatis, and Streptomyces somaliensis and Actinomadura madurae for actinomycetoma. The humoral immunity response in actinomycetoma patients and in experimental mice was measured and significant titre of anti-P24 antibody was demonstrated.

Actinomycetales↗

PubMed1995

Mutants of the bacteriophage MS2 coat protein that alter its cooperative binding to RNA.

An RNA binding assay measuring cooperative protein binding has been used to evaluate the effects of mutations in the MS2 phage coat protein expected to disrupt capsid assembly. By using the crystal structure of the virus as a guide, six different mutations in the FG loop structure were selected in which hydrophobic residues were replaced with charged residues. Most of these proteins form capsids in Escherichia coli, but not in an in vitro assembly assay, suggesting that interdimer interactions are weaker than wild type. These mutant proteins reduce the free energy of cooperative protein binding to a double-hairpin RNA from its wild-type value of -1.9 kcal/mol. Several of the variants that have large effects on cooperativity have no effect on RNA affinity, suggesting that protein-RNA interactions can be affected independently of dimer-dimer interactions. The V75E;A81G protein, which shows no measurable cooperativity, binds operator RNA equally well as the wild-type protein under a variety of buffer conditions. Because this protein also exhibits similar specificity for variant RNA sequences, it will be useful for studying RNA binding properties independent of capsid assembly.

Base Sequence↗

PubMed2006

Pseudouridine synthases.

Pseudouridine synthases are the enzymes responsible for the most abundant posttranscriptional modification of cellular RNAs. These enzymes catalyze the site-specific isomerization of uridine residues that are already part of an RNA chain, and appear to employ both sequence and structural information to achieve site specificity. Crystallographic analyses have demonstrated that all pseudouridine synthases share a common core fold and active site structure and that this core is modified by peripheral domains, accessory proteins, and guide RNAs to give rise to remarkable substrate versatility.

Catalysis↗

PubMed2004

Interference probing of rRNA with snoRNPs: a novel approach for functional mapping of RNA in vivo.

Synthesis of eukaryotic ribosomal RNAs (rRNAs) includes methylation of scores of nucleotides at the 2'-O-ribose position (Nm) by small nucleolar RNP complexes (snoRNPs). Sequence specificity is provided by the snoRNA component through base-pairing of a guide sequence with rRNA. Here, we report that methylation snoRNPs can be targeted to many new sites in yeast rRNA, by providing the snoRNA with a novel guide sequence, and that in some cases growth and translation activity are strongly impaired. Novel snoRNAs can be expressed individually or by a unique library strategy that yields guide sequences specific for a large target region. Interference effects were observed for sites in both the small and large subunits, including the reaction center region. Targeting guide RNAs to nucleotides flanking the sensitive sites caused little or no defect, indicating that methylation is responsible for the interference rather than a simple antisense effect or misguided chaperone function. To our knowledge, this is the only approach that has been used to mutagenize the backbone of rRNA in vivo.

Base Sequence↗

PubMed2003

Crystal structure of the 2'-specific and double-stranded RNA-activated interferon-induced antiviral protein 2'-5'-oligoadenylate synthetase.

2'-5'-oligoadenylate synthetases are interferon-induced, double-stranded RNA-activated antiviral enzymes which are the only proteins known to catalyze 2'-specific nucleotidyl transfer. This crystal structure of a 2'-5'-oligoadenylate synthetase reveals a structural conservation with the 3'-specific poly(A) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2'- and 3'-specific nucleotidyl transferases. Comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. The 2'-5'-oligoadenylate synthetases are activated by viral double-stranded RNA in infected cells and initiate a cellular response by synthesizing 2'-5'-oligoadenylates, which in turn activate RNase L. This crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsRNA activation site that is probed by mutagenesis, thus providing structural insight into cellular recognition of viral double-stranded RNA.

2',5'-Oligoadenylate Synthetase↗

PubMed2006

CD4+ count-guided interruption of antiretroviral treatment.

BACKGROUND: Despite declines in morbidity and mortality with the use of combination antiretroviral therapy, its effectiveness is limited by adverse events, problems with adherence, and resistance of the human immunodeficiency virus (HIV). METHODS: We randomly assigned persons infected with HIV who had a CD4+ cell count of more than 350 per cubic millimeter to the continuous use of antiretroviral therapy (the viral suppression group) or the episodic use of antiretroviral therapy (the drug conservation group). Episodic use involved the deferral of therapy until the CD4+ count decreased to less than 250 per cubic millimeter and then the use of therapy until the CD4+ count increased to more than 350 per cubic millimeter. The primary end point was the development of an opportunistic disease or death from any cause. An important secondary end point was major cardiovascular, renal, or hepatic disease. RESULTS: A total of 5472 participants (2720 assigned to drug conservation and 2752 to viral suppression) were followed for an average of 16 months before the protocol was modified for the drug conservation group. At baseline, the median and nadir CD4+ counts were 597 per cubic millimeter and 250 per cubic millimeter, respectively, and 71.7% of participants had plasma HIV RNA levels of 400 copies or less per milliliter. Opportunistic disease or death from any cause occurred in 120 participants (3.3 events per 100 person-years) in the drug conservation group and 47 participants (1.3 per 100 person-years) in the viral suppression group (hazard ratio for the drug conservation group vs. the viral suppression group, 2.6; 95% confidence interval [CI], 1.9 to 3.7; P<0.001). Hazard ratios for death from any cause and for major cardiovascular, renal, and hepatic disease were 1.8 (95% CI, 1.2 to 2.9; P=0.007) and 1.7 (95% CI, 1.1 to 2.5; P=0.009), respectively. Adjustment for the latest CD4+ count and HIV RNA level (as time-updated covariates) reduced the hazard ratio for the primary end point from 2.6 to 1.5 (95% CI, 1.0 to 2.1). CONCLUSIONS: Episodic antiretroviral therapy guided by the CD4+ count, as used in our study, significantly increased the risk of opportunistic disease or death from any cause, as compared with continuous antiretroviral therapy, largely as a consequence of lowering the CD4+ cell count and increasing the viral load. Episodic antiretroviral therapy does not reduce the risk of adverse events that have been associated with antiretroviral therapy. (ClinicalTrials.gov number, NCT00027352 [ClinicalTrials.gov].).

AIDS-Related Opportunistic Infections↗

PubMed2006

Ab initio determination of the ionization potentials of DNA and RNA nucleobases.

Quantum chemical high level ab initio coupled-cluster and multiconfigurational perturbation methods have been used to compute vertical and adiabatic ionization potentials of the five canonical DNA and RNA nucleobases: uracil, thymine, cytosine, adenine, and guanine. Several states of their cations have been also calculated. The present results represent a systematic compendium of these magnitudes, establishing theoretical reference values at a level not reported before, calibrating computational strategies, and guiding the assignment of the features in the experimental photoelectron spectra.

Adenine↗

PubMed2006

UTP-bound and Apo structures of a minimal RNA uridylyltransferase.

3'-Uridylylation of RNA is emerging as a phylogenetically widespread phenomenon involved in processing events as diverse as uridine insertion/deletion RNA editing in mitochondria of trypanosomes and small nuclear RNA (snRNA) maturation in humans. This reaction is catalyzed by terminal uridylyltransferases (TUTases), which are template-independent RNA nucleotidyltransferases that specifically recognize UTP and belong to a large enzyme superfamily typified by DNA polymerase beta. Multiple TUTases, recently identified in trypanosomes, as well as a U6 snRNA-specific TUTase enzyme in humans, are highly divergent at the protein sequence level. However, they all possess conserved catalytic and UTP recognition domains, often accompanied by various auxiliary modules present at the termini or between conserved domains. Here we report identification, structural and biochemical analyses of a novel trypanosomal TUTase, TbTUT4, which represents a minimal catalytically active RNA uridylyltransferase. The TbTUT4 consists of only two domains that define the catalytic center at the bottom of the nucleoside triphosphate and RNA substrate binding cleft. The 2.0 Angstroms crystal structure reveals two significantly different conformations of this TUTase: one molecule is in a relatively open apo conformation, whereas the other displays a more compact TUTase-UTP complex. A single nucleoside triphosphate is bound in the active site by a complex network of interactions between amino acid residues, a magnesium ion and highly ordered water molecules with the UTP's base, ribose and phosphate moieties. The structure-guided mutagenesis and cross-linking studies define the amino acids essential for catalysis, uracil base recognition, ribose binding and phosphate coordination by uridylyltransferases. In addition, the cluster of positively charged residues involved in RNA binding is identified. We also report a 2.4 Angstroms crystal structure of TbTUT4 with the bound 2' deoxyribonucleoside, which provides the structural basis of the enzyme's preference toward ribonucleotides.

Amino Acid Sequence↗

PubMed1971

Host-gene control of C-type tumor virus-expression and tumorigenesis: relevance of studies in inbred mice to cancer in man and other species.

C-type RNA viruses are tumorigenic in mice and other species. They are widespread and transmitted in an intimate relationship with host-cell genetic material. Tumorigenesis is no doubt a complex biological phenomenon, and involves both genetic and environmental factors. Among the genetic factors, single-locus determination is probably exceptional; yet, a number of genes are known in inbred mice and other species, including man, that can exert a major influence. Clearly, studies with inbred mice apply outside the murine model and justify generalizations on the etiology of cancer. The types of cancer observed in man are similar to those of inbred strains of mice, and their stochastic and often familial occurrences suggest that genetic factors play important roles in both man and mouse. A number of heritable disorders predipsose to tumorigenesis. Investigations of the relationship between various polymorphic phenotypes and human tumors should be intensified. For instance, a study of high-risk human-tumor groups could aid considerably not only in the recognition of etiological agents but also of underlying host-gene controlled mechanisms. Indeed epidemiological studies have already led to the identification of mutant genes that influence the incidence of human cancer. Such studies may be guided by findings in inbred strains of mice. There is good evidence in inbred mice that mutations in host chromosomes determine whether or not genes coding for RNA oncogene viral "functions" become expressed as cancer. Although an etiological role of C-type RNA virus has not yet been demonstrated in human cancer, studies of the interaction of viral and host-cell genomes in mice and other species, and the visualization of C-type virus in human tumors have provided good reasons for suspecting the presence in man of oncogenic genes (oncogenes) homologous to those of the C-type viral genomes, which are well studied in mice, hamsters, cats, rats, and chickens.

Animals↗