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At least 595 records · Page 33Linked to original sources
PubMed2003

Induction of an interferon response by RNAi vectors in mammalian cells.

DNA vectors that express short hairpin RNAs (shRNAs) from RNA polymerase III (Pol III) promoters are a promising new tool to reduce gene expression in mammalian cells. shRNAs are processed to small interfering RNAs (siRNAs) of 21 nucleotides (nt) that guide the cleavage of the cognate mRNA by the RNA-induced silencing complex. Although siRNAs are thought to be too short to induce interferon expression, we report here that a substantial number of shRNA vectors can trigger an interferon response.

2',5'-Oligoadenylate Synthetase↗
PubMed2006

An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana.

ARGONAUTE4 (AGO4) and RNA polymerase IV (Pol IV) are required for DNA methylation guided by 24 nucleotide small interfering RNAs (siRNAs) in Arabidopsis thaliana. Here we show that AGO4 localizes to nucleolus-associated bodies along with the Pol IV subunit NRPD1b; the small nuclear RNA (snRNA) binding protein SmD3; and two markers of Cajal bodies, trimethylguanosine-capped snRNAs and the U2 snRNA binding protein U2B''. AGO4 interacts with the C-terminal domain of NRPD1b, and AGO4 protein stability depends on upstream factors that synthesize siRNAs. AGO4 is also found, along with the DNA methyltransferase DRM2, throughout the nucleus at presumed DNA methylation target sites. Cajal bodies are conserved sites for the maturation of ribonucleoprotein complexes. Our results suggest a function for Cajal bodies as a center for the assembly of an AGO4/NRPD1b/siRNA complex, facilitating its function in RNA-directed gene silencing at target loci.

Arabidopsis↗
PubMed2003

Modification of Sm small nuclear RNAs occurs in the nucleoplasmic Cajal body following import from the cytoplasm.

Biogenesis of functional spliceosomal small nuclear RNAs (snRNAs) includes the post-transcriptional covalent modification of numerous internal nucleotides. We have recently demonstrated that synthesis of 2'-O-methylated nucleotides and pseudouridines in the RNA polymerase II-synthesized Sm snRNAs is directed by sequence-specific guide RNAs. Here, we provide evidence supporting the notion that modification of Sm snRNAs occurs in nucleoplasmic Cajal bodies (CBs), where modification guide RNAs accumulate. We show that short fragments of Sm snRNAs are correctly and efficiently modified when targeted to CBs, but not when these same fragments are targeted to the nucleolus. We also demonstrate that internal modification of the U2 snRNA occurs exclusively after nuclear import of the newly assembled Sm snRNP from the cytoplasm. Finally, we show that p80 coilin, the CB marker protein, is not required for snRNA modification. In coilin knockout cells, Sm snRNAs and their modification guide RNAs colocalize in residual CBs, which do not stockpile fibrillarin and fail to recruit the U3 small nucleolar RNA.

Active Transport, Cell Nucleus↗
PubMed2006

A general RNA-capping ribozyme retains stereochemistry during cap exchange.

Numerous natural and artificial ribozymes have been shown to facilitate reactions that invert stereochemistry. Here, we demonstrate that an RNA-capping ribozyme retains stereochemistry at a phosphorus reaction center. The ribozyme synthesizes a broad range of 5'-5' RNA caps by exchanging phosphate groups around the alpha-phosphate found at the 5' terminus of the ribozyme. A ribozyme prepared with an Rp adenosine(5')alpha-thiotetraphosphate cap was found to exchange this cap for an Rp 4-thiouridine(5')alpha-thiotetraphosphate cap when incubated with 4-thiouridine triphosphate. The same Rp capped construct, when incubated with [gamma-(32)P]-ATP, exchanged the unlabeled ATP for a radiolabeled one while maintaining the same stereoconfiguration. In contrast, ribozymes prepared with an Sp cap failed to react even in the presence of thiophilic metal ions such as manganese. The kinetics of capping was also unusual as compared to inverting ribozymes. When the ribozyme was prepared with a triphosphate, capping was found to follow Michaelis-Menten-type kinetics even though the rate of pyrophosphate release was completely independent of nucleotide substrate concentration. Interestingly, the rate of capping and hydrolysis, when summed, was found to be indistinguishable from the rate of pyrophosphate release, indicating that an early rate-limiting step precedes both capping and hydrolysis. Together the retention of stereochemistry and kinetics imply that capping utilizes two inverting chemical steps that are separated by the transient formation of a rate-limiting covalent intermediate. As all protein enzymes that mediate similar capping reactions utilize a covalent intermediate, chemical necessity may have strongly guided the evolution of both protein and RNA-capping catalysts.

Adenosine Triphosphate↗
PubMed1997

Association of a group I intron with its splice junction in 50S ribosomes: implications for intron toxicity.

The effect of genetic context on splicing of group I introns is not well understood at present. The influence of ribosomal RNA conformation on splicing of rDNA introns in vivo was investigated using a heterologous system in which the Tetrahymena group I intron is inserted into the homologous position of the Escherichia coli 23S rRNA. Mutations that block splicing in E. coli result in accumulation of unspliced 23S rRNA that is assembled into 50S complexes, but not 70S ribosomes. The data indicate that accommodation of the intron structure on the surface of the 50S subunit inhibits interactions with the small ribosomal subunit. Spliced intron RNA also remains noncovalently bound to 50S subunits on sucrose gradients. This interaction appears to be mediated by base pairing between the intron guide sequence and the 23S rRNA, because the fraction of bound intron RNA is reduced by point mutations in the IGS or deletion of the P1 helix. Association of the intron with 50S subunits correlates with slow cell growth. The results suggest that group I introns have the potential to inhibit protein synthesis in prokaryotes by direct interactions with ribosomes.

Animals↗
PubMed2000

RNA-binding properties of the mitochondrial Y-box protein RBP16.

We have previously identified a mitochondrial Y-box protein in Trypanosoma brucei that we designated RBP16. The predicted RBP16 amino acid sequence revealed the presence of a cold-shock domain at its N-terminus and a glycine- and arginine-rich C-terminus reminiscent of an RGG RNA-binding motif. Since RBP16 is capable of interacting with different guide RNAs (gRNAs) in vitro and in vivo primarily via the oligo(U) tail, as well as with ribosomal RNAs, possible functions of RBP16 may be in kinetoplastid RNA editing and/or translation. Herein, we report experiments that further define the RNA-binding properties of RBP16. RBP16 forms a single stable complex with the gRNA gA6[14] at low protein concentration, while at higher protein concentration two stable complexes that possibly represent two different conformations are observed. Both complexes are stable at relatively high salt and moderate heparin concentrations indicating that the binding of RBP16 to gA6[14] does not rely primarily on ionic interactions. Phenylglyoxal treatment of the protein indicates that arginine residues are important in RNA binding. The minimal length of RNA sequence necessary for the binding of RBP16 was assessed by gel retardation and UV cross-linking competition assays using oligo(U) ribonucleotides of varying lengths (4-40 nt). Although RBP16 can bind to oligonucleotides as small as U(4), its affinity increases with the length of the oligo(U) ribonucleotide, with a dramatic increase in binding efficiency observed when the length is increased to 10 nt. Gel retardation assays employing T.brucei mRNAs demonstrated that, although it acts as a major binding determinant, a 3' U tail is not an absolute requirement for efficient RBP16-RNA binding. Experiments with oligonucleotides containing U stretches embedded at different positions in oligo(dC) indicated that high-affinity binding requires both a uridine stretch, as well as 5' and 3' non-specific sequences. These results suggest a model for the molecular interactions involved in RBP16-RNA binding.

Animals↗
PubMed1997

Design of short external guide sequences (EGSs) for cleavage of target molecules with RNase P.

The minimal substrate for human RNase P consists of the 5' leader sequence, aminoacyl acceptor stem, T-stem and T-loop of tRNA. The sequences corresponding to the D-stem, anticodon stem and loop and variable loop are replaced by a bulge which can be as small as 1 nt, but requires > 4 nt for optimal cleavage by RNase P. We found that a trans construct in which the T loop is opened between G57 and A58 (tRNA numbering system) is still processed by RNase P. The strand that is cleaved can be considered the target RNA while the other strand serves as an External Guide Sequence (EGS). We were also able to delete the nucleotides corresponding to nt 58 to 60 in the T-loop without affecting cleavage of the substrate. We propose that the sequence UUCG or UUCA (nucleotide 55 to 57 in the T-loop) positioned 3' to a double helical region of 12 to 13 basepairs containing a bulge of > 4 nt can form a structure that is recognized by human RNase P. The four nucleotides UUCR probably form a structure that resembles the uridine turn in the Tloop of tRNA. Since recognition by RNase P seems to be independent of the helical sequence, we suggest that this motif can be used for targeting RNA molecules for EGS-directed cleavage by RNase P. Based on these results, several 13-mer EGSs targeted to the 2.1 Kb surface antigen mRNA of hepatitis B virus (HBV) were designed and tested using a co-transcriptional cleavage assay with a 2.1 Kb HBV transcript. Some of these were capable of inducing cleavage of the HBV RNA by RNase P. The use of such small EGSs for the inactivation of various genes will be discussed.

Base Sequence↗
PubMed2003

Small nucleolar RNA interference induced by antisense or double-stranded RNA in trypanosomatids.

In trypanosomes the C/D- and H/ACA-like small nucleolar RNAs (snoRNAs) are clustered and repeated in the genome. The snoRNAs studied to date are transcribed as polycistronic transcripts by RNA polymerase II and then processed, resulting in mature snoRNAs. In this study we demonstrated that snoRNA genes can be silenced in three trypanosomatid species: Leptomonas collosoma, Leishmania major, and Trypanosoma brucei. Silencing was achieved in L. collosoma and L. major by the expressing of an antisense transcript complementary to the snoRNA gene and was accompanied by the accumulation of small interfering RNA. Silencing eliminated the mature snoRNA but not its precursor and abolished the specific 2'-O-methylation guided by the snoRNA. In T. brucei, silencing was achieved by using the inducible synthesis of double-stranded RNA from T7 opposing promoters. Silencing varied between the different snoRNA genes, which may reflect the accessibility of small interfering RNA to the target RNAs. This study suggests that RNA interference can degrade snoRNAs. This study has further implications in elucidating the function of nucleolar RNAs and specific modifications guided by these RNAs in trypanosomatids and perhaps in other eukaryotes as well.

Animals↗
PubMed1993

Ligation-based DNA diagnostics.

A number of novel gene detection techniques all revolve around the ligation of synthetic nucleic acid probes. In such ligase-assisted gene detection reactions, specific DNA or RNA sequences are investigated by using them as guides for the covalent joining of pairs of probe molecules. The probes are designed to hybridize immediately next to each other on the target nucleic acid strand. Demonstration of ligated probes results in highly specific detection of and efficient distinction between similar sequence variants under standard reaction conditions. Accordingly, the principle has been applied in automated genetic screening procedures. Ligation reactions are also integral to a number of amplification procedures and they will be of value in an expanding range of genetic analyses.

DNA↗
PubMed1987

A three-dimensional model of domain III of the Escherichia coli small ribosomal subunit.

A three-dimensional model of domain III (nucleotides 920 to 1395) of the 30S ribosomal subunit of E. coli is proposed. The data used as a guide in folding the secondary structure of the RNA into a tertiary structure are four long range RNA-RNA interactions proposed by us on the basis of experiments performed in this laboratory plus two sets of data from other laboratories: protein-RNA cross-linking sites for proteins S1, S3, S7, S10 and S12, and the interprotein distances determined by neutron scattering. The model is consistent with nearly all of the published experimental findings on the structure of domain III.

Base Sequence↗
PubMed2006

Short-term treatment duration for HCV-2 and HCV-3 infected patients.

BACKGROUND: We have shown that 12-14 weeks treatment is effective in HCV-2 or -3 patients with undetectable HCV-RNA after 4 weeks of therapy (rapid virologic response). PATIENTS: To identify predictors of sustained virologic response, rapid virologic response and relapse following short treatment, we pooled data from the original Italian and Norwegian studies. Four hundreds and three patients were treated with PegIFN alpha-2b (1.0, n=281 or 1.5 microg/kg, n=122) and ribavirin (800-1200 mg) for 12-14 or 24 weeks, depending on negative or positive HCV-RNA at week 4. RESULTS: Sustained virologic response differed between cases with and without rapid virologic response (85% versus 62%, P<0.0001), mild and severe fibrosis (83% versus 67%, P=0.004), and HCV-2 and -3 (81% versus 73%, P=0.05). In a regression model, RVR (odds ratio 3.49, confidence interval 1.73-5.36) and mild fibrosis (odds ratio 2.91, confidence interval 1.57-5.38) independently predicted sustained virologic response. Rapid virologic response was obtained in 274 (68%) patients, 163/242 (67%) HCV-2, and 111/161 (69%) HCV-3. Patients with RVR had more frequently mild fibrosis (70% versus 54%, P=0.03), and high PegIFN dose (78% versus 64%, P=0.005). In a regression model, mild fibrosis independently predicted rapid virologic response (odds ratio 1.87, confidence interval 1.10-3.16). In rapid virologic response patients, sustained virologic response was achieved in 85% of both HCV-2 and -3. Virologic relapse was observed in 10.6% rapid virologic response patients and was more frequent among those with low ALT (14% versus 2%, P=0.04). CONCLUSION: In HCV-2 or -3, the HCV-RNA status after 4 weeks of therapy may guide treatment duration. HCV-2 and HCV-3 patients with severe fibrosis are less likely to experience both rapid virologic response and sustained virologic response, and more frequently relapse after a 12 or 14 weeks duration of antiviral therapy.

Adult↗
PubMed2003

Analysis of NCp7-dependent activation of HIV-1 cDNA integration and its conservation among retroviral nucleocapsid proteins.

HIV-1 nucleocapsid protein NCp7 is a small basic protein with two zinc fingers, found in the virion core where several hundred molecules coat the genomic RNA. NCp7 has nucleic acid chaperone properties that guide reverse transcriptase (RT) to synthesize the proviral DNA flanked by the long terminal repeats (LTR). In vitro, NCp7 can strongly activate magnesium-dependent LTR-DNA strand transfer by integrase (IN). Here we show that IN activation relies on both the basic residues and the zinc fingers of NCp7. NCp7 lacking the zinc fingers binds DNA but moderately stimulates strand transfer by IN. The NCp7 zinc-finger domain binds DNA poorly and does not efficiently stimulate IN activity. However, the NC zinc-finger domain can complement DNA binding to restore full activation of strand transfer by IN. We propose that the basic residues and the zinc fingers function together to stabilize IN at the LTR ends and promote the formation of a nucleoprotein complex competent for integration. We also show that these properties of HIV-1 NCp7 are remarkably conserved among nucleocapsid proteins of retrotransposon and retrovirus origins.

Amino Acid Sequence↗
PubMed2001

Kinetics of the hepatitis C virus during interferon therapy as a marker of therapeutic response.

BACKGROUND: The viral load and subtype of hepatitis C virus (HCV) are predictors of the efficacy of interferon (IFN) therapy. The kinetics of HCV during IFN therapy have been described recently, suggesting that HCV infection is highly dynamic. These observations have raised the issue as to whether early monitoring of the viral load can help guide IFN therapy. METHODS: We measured HCV-RNA levels at 0, 24 and 48 h after the start of IFN-alpha treatment (10 MU daily for 2 weeks and then three times weekly for 22 weeks) or IFN-beta treatment (6 MU daily for 6 weeks). Then we analyzed the relationship between HCV kinetics and therapeutic response using stepwise multivariate logistic regression analysis. RESULTS: The exponential decay slope of the viral load during the first 24 h, not the first 48 h or the next 24 h, was a predictor of viral eradication at 6 months after completion of the treatment (sustained response; P = 0.0023). This decay slope was not affected by the HCV serotype or the type of IFN used. Initial viral load and HCV serotype were also predictors, as reported previously (P < 0.0001 and P = 0.0347, respectively). We also proposed a model using a prognostic index that predicted a sustained response with more than 80% sensitivity, specificity and efficacy in an independent and external group of patients. CONCLUSION: This study demonstrated that the exponential decay slope of the viral load during the first 24 h was an important predictor of the response to IFN therapy as well as the initial viral load and HCV serotype. The model may also be useful for the clinical management of IFN therapy.

Antiviral Agents↗
PubMed2001

Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate.

Duplexes of 21-23 nucleotide (nt) RNAs are the sequence-specific mediators of RNA interference (RNAi) and post-transcriptional gene silencing (PTGS). Synthetic, short interfering RNAs (siRNAs) were examined in Drosophila melanogaster embryo lysate for their requirements regarding length, structure, chemical composition and sequence in order to mediate efficient RNAi. Duplexes of 21 nt siRNAs with 2 nt 3' overhangs were the most efficient triggers of sequence-specific mRNA degradation. Substitution of one or both siRNA strands by 2'-deoxy or 2'-O-methyl oligonucleotides abolished RNAi, although multiple 2'-deoxynucleotide substitutions at the 3' end of siRNAs were tolerated. The target recognition process is highly sequence specific, but not all positions of a siRNA contribute equally to target recognition; mismatches in the centre of the siRNA duplex prevent target RNA cleavage. The position of the cleavage site in the target RNA is defined by the 5' end of the guide siRNA rather than its 3' end. These results provide a rational basis for the design of siRNAs in future gene targeting experiments.

Animals↗
PubMed1996

Age- and time-related changes in extracellular viral load in children vertically infected by human immunodeficiency virus.

BACKGROUND: It is known that plasma or serum viral load is high in vertically HIV-infected children during the first year of life, but the changes in these titers after the first birthday have not been described. Information on the natural history of circulating extracellular virus will be useful in elucidating the pathogenesis of pediatric HIV infection and in using viral load measurement to guide prognosis and therapy. METHODS: We measured serum RNA by reverse transcriptase-polymerase chain reaction and immune complex-dissociated p24 antigen enzyme-linked immunosorbent assay over time in 48 unselected children followed in our clinics and analyzed the findings in relation to age and clinical outcome. RESULTS: In first-available samples from the 48 children there was a gradual reduction in HIV RNA values with increasing age, with a slope of -0.21 log copy/ml/year (P < 0.001, R2 = 0.6022). This downward trend was seen in subsets of children with all degrees of immunodeficiency. The mean slope of repeated HIV RNA measurements in individual children was similarly in a downward direction (slope -0.11 (P = 0.007 for difference from zero)). The slope was more negative in children who were younger at baseline. Immune complex-dissociated p24 antigen values were much less predictable and predictive. CONCLUSIONS: Viral load in vertically infected children, measured by reverse transcriptase-polymerase chain reaction, falls very gradually over time, descending from very high titers at the end of the first year, and reaching values seen in horizontally infected adults at approximately 5 years of age.

Adult↗
PubMed2006

Identification and characterization of the nuclear localization/retention signal in the EWS proto-oncoprotein.

Ewing sarcoma (EWS) protein, a member of a large family of RNA-binding proteins, contains an N-terminal transcriptional activation domain (EAD) and a C-terminal RNA-binding domain (RBD). Due to its multifunctional properties EWS protein is involved in processes such as gene expression, RNA processing and transport, and cell signaling. Chimeric EWS proteins generated by chromosomal translocations cause malignant tumors. EWS protein is located predominantly in the nucleus, but was found also in the cytosol and associated with the cell membrane. The determinants responsible for the nuclear localization of the protein were as yet unknown. We identified the nuclear localization signal of EWS protein at its C terminus (C-NLS), which is required for the nuclear import and retention of the protein. The C-NLS sequence is conserved in related proto-oncoproteins suggesting an NLS function also in these proteins. Two arginine residues, due to their positive charge, a proline residue and a tyrosine residue are essential for C-NLS function. The nuclear localization of EWS protein is independent of the regions in RBD containing numerous arginine methylation sites, RNA-recognition and zinc finger motifs. Regions in EAD guide the subnuclear partition of EWS protein and contain another but different NLS that allows nucleocytoplasmic shuttling of the N-terminal domain.

Amino Acid Motifs↗
PubMed2005

The Putative RNA Helicase Dbp4p Is Required for Release of the U14 snoRNA from Preribosomes in Saccharomyces cerevisiae.

Around 70 yeast snoRNAs guide rRNA modification, frequently forming base-paired interactions predicted to be very stable at physiological temperatures. Eighteen putative RNA helicases are required for ribosome synthesis, but their actual substrates were not known. We report that depletion of the DEAD box helicase Dbp4p dramatically increased cosedimentation of the snoRNAs U14 and snR41 with preribosomes. Cosedimentation was maintained after deproteinization by proteinase K, indicating that the snoRNAs remained base paired to the pre-rRNA. Affinity purification showed that U14 was strongly accumulated in early 90S preribosomes and depleted from later pre-40S complexes. U14 is required for pre-rRNA processing, and depletion of Dbp4p caused a very similar pre-rRNA processing defect, perhaps due to the reduced pool of free U14. Point mutations in helicase motifs I and III of Dbp4p blocked release of U14 from preribosomes. We conclude that the helicase activity of Dbp4p is required to unwind U14 and snR41 from the pre-rRNA.

Amino Acid Motifs↗
PubMed2006

Encoding folding paths of RNA switches.

RNA co-transcriptional folding has long been suspected to play an active role in helping proper native folding of ribozymes and structured regulatory motifs in mRNA untranslated regions (UTRs). Yet, the underlying mechanisms and coding requirements for efficient co-transcriptional folding remain unclear. Traditional approaches have intrinsic limitations to dissect RNA folding paths, as they rely on sequence mutations or circular permutations that typically perturb both RNA folding paths and equilibrium structures. Here, we show that exploiting sequence symmetries instead of mutations can circumvent this problem by essentially decoupling folding paths from equilibrium structures of designed RNA sequences. Using bistable RNA switches with symmetrical helices conserved under sequence reversal, we demonstrate experimentally that native and transiently formed helices can guide efficient co-transcriptional folding into either long-lived structure of these RNA switches. Their folding path is controlled by the order of helix nucleations and subsequent exchanges during transcription, and may also be redirected by transient antisense interactions. Hence, transient intra- and inter-molecular base pair interactions can effectively regulate the folding of nascent RNA molecules into different native structures, provided limited coding requirements, as discussed from an information theory perspective. This constitutive coupling between RNA synthesis and RNA folding regulation may have enabled the early emergence of autonomous RNA-based regulation networks.

Base Sequence↗