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At least 325 records · Page 18Linked to original sources
PubMed2003

Order and disorder in crystals of hexameric NTPases from dsRNA bacteriophages.

The packaging of genomic RNA in members of the Cystoviridae is performed by P4, a hexameric protein with NTPase activity. Across family members such as Phi6, Phi8 and Phi13, the P4 proteins show low levels of sequence identity, but presumably have similar atomic structures. Initial structure-determination efforts for P4 from Phi6 and Phi8 were hampered by difficulties in obtaining crystals that gave ordered diffraction. Diffraction from crystals of full-length P4 showed a variety of disorder and anisotropy. Subsequently, crystals of Phi13 P4 were obtained which yielded well ordered diffraction to 1.7 A. Comparison of the packing arrangements of P4 hexamers in different crystal forms and analysis of the disorder provides insights into the flexibility of this family of proteins, which might be an integral part of their biological function.

Anisotropy↗
PubMed1998

Isolation of dsRNA-associated VLPs from the strain Cryptococcus hungaricus CBS 6569.

Double-stranded RNAs (dsRNAs) with molecular masses 1.7 and 5.0 kbp, respectively, were isolated from the strain Cryptococcus hungaricus CBS 6569. The dsRNAs were copurified with eicosahedric virus-like particles, 29 nm in diameter. This strain produced a protease-sensitive 'toxin' which inhibited the growth of strain C. hungaricus CBS 4214. The toxin had maximum activity at pH 3.7. The highest toxin amount was attained after a culture period of four days.

Cryptococcus↗
PubMed2005

dsRNA enhances eotaxin-3 production through interleukin-4 receptor upregulation in airway epithelial cells.

The exacerbation of asthma during viral infections is mainly explained by neutrophils infiltrating into the airways. However, enhanced functions of eosinophils are also observed. The aim of this study was to reveal the mechanism of how eosinophils are activated during and after viral infection of the airways, using a model of viral infection. A synthetic double-stranded RNA, poly inosinic-cytidyric acid (poly(IC)), was transfected to a human airway epithelial cell line (BEAS-2B) and the primary bronchial epithelial cells, to mimic a viral infection. The production of chemokines from the cells was investigated. The transfection of poly(IC), alone, marginally affected the eotaxin-3 production of the cells. However, the transfection of poly(IC) prior to interleukin (IL)-4 stimulation enhanced eotaxin-3 production. Poly(IC) transfection increased mRNA and protein expressions of IL-4 receptor (R)alpha and IL-2Rgamma, components of the IL-4R. In BEAS-2B cells, IL-4-mediated phosphorylation of signal transducer and activator of transcription six was enhanced in poly(IC) transfected cells. This was reversed by the addition of anti-IL-4Ralpha antibody, suggesting the role of an increased number of IL-4 receptors in enhanced IL-4-induced eotaxin-3 production. Poly(IC)-induced upregulation of IL-4Ralpha was inhibited by treatment with cycloheximide or dexamethasone. In conclusion, these results suggest that viral airway infection may enhance interleukin-4-induced eotaxin-3 production through upregulation of the interleukin-4 receptor in airway epithelial cells.

Cells, Cultured↗
PubMed2005

In vivo studies of genomic packaging in the dsRNA bacteriophage Phi8.

BACKGROUND: Phi8 is a bacteriophage containing a genome of three segments of double-stranded RNA inside a polyhedral capsid enveloped in a lipid-containing membrane. Plus strand RNA binds and is packaged by empty procapsids. Whereas Phi6, another member of the Cystoviridae, shows high stringency, serial dependence and precision in its genomic packaging in vitro and in vivo, Phi8 packaging is more flexible. Unique sequences (pac) near the 5' ends of plus strands are necessary and sufficient for Phi6 genomic packaging and the RNA binding sites are located on P1, the major structural protein of the procapsid. RESULTS: In this paper the boundaries of the Phi8 pac sequences have been explored by testing the in vivo packaging efficacy of transcripts containing deletions or changes in the RNA sequences. The pac sequences have been localized to the 5' untranslated regions of the viral transcripts. Major changes in the pac sequences are either tolerated or ameliorated by suppressor mutations in the RNA sequence. Changes in the genomic packaging program can be established as a result of mutations in P1, the major structural protein of the procapsid and the determinant of RNA binding specificity. CONCLUSION: Although Phi8 is distantly related to bacteriophage Phi6, and does not show sequence similarity, it has a similar genomic packaging program. This program, however, is less stringent than that of Phi6.

Bacteriophages↗
PubMed2004

Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs.

microRNAs (miRNAs) are widespread among eukaryotes, and studies in several systems have revealed that miRNAs can regulate expression of specific genes. Primary miRNA transcripts are initially processed to approximately 70-nucleotide (nt) stem-loop structures (pre-miRNAs), exported to the cytoplasm, further processed to yield approximately 22-nt dsRNAs, and finally incorporated into ribonucleoprotein particles, which are thought to be the active species. Here we study nuclear export of pre-miRNAs and show that the process is saturable and thus carrier-mediated. Export is sensitive to depletion of nuclear RanGTP and, according to this criterion, mediated by a RanGTP-dependent exportin. An unbiased affinity chromatography approach with immobilized pre-miRNAs identified exportin 5 as the pre-miRNA-specific export carrier. We have cloned exportin 5 from Xenopus and demonstrate that antibodies raised against the Xenopus receptor specifically block pre-miRNA export from nuclei of Xenopus oocytes. We further show that exportin 5 interacts with double-stranded RNA in a sequence-independent manner.

Active Transport, Cell Nucleus↗
PubMed2005

Molecular dynamics simulations and free energy calculations of base flipping in dsRNA.

The family of adenosine deaminases acting on RNA (ADARs) targets adenosines in RNA that is mainly double stranded. Some substrates are promiscuously deaminated whereas others, such as the mammalian glutamate receptor B (gluR-B) pre-mRNA, are more selectively deaminated. Many DNA/RNA-base modification enzymes use a base flipping mechanism to be able to reach their target base and it is believed that ADARs function in a similar way. In this study we used molecular dynamics (MD) simulations to describe two sites on the gluR-B pre-mRNA, the selectively targeted R/G site and the nontargeted 46 site, in an attempt to explain the substrate specificity. We used regular MD and also a forced base flipping method with umbrella sampling to calculate the free energy of base opening. Spontaneous opening of the mismatched adenosine was observed for the R/G site but not for the 46 site.

Adenosine↗
PubMed2004

Evidence for auto-inhibition by the N terminus of hADAR2 and activation by dsRNA binding.

Adenosine deaminases that act on RNA (ADARs) catalyze adenosine to inosine conversion in RNA that is largely double stranded. Human ADAR2 (hADAR2) contains two double-stranded RNA binding motifs (dsRBMs), separated by a 90-amino acid linker, and these are followed by the C-terminal catalytic domain. We assayed enzymatic activity of N-terminal deletion constructs of hADAR2 to determine the role of the dsRBMs and the intervening linker peptide. We found that a truncated protein consisting of one dsRBM and the deaminase domain was capable of deaminating a short 15-bp substrate. In contrast, full-length hADAR2 was inactive on this short substrate. In addition, we observed that the N terminus, which was deleted from the truncated protein, inhibits editing activity when added in trans. We propose that the N-terminal domain of hADAR2 contains sequences that cause auto-inhibition of the enzyme. Our results suggest activation requires binding to an RNA substrate long enough to accommodate interactions with both dsRBMs.

Adenosine Deaminase↗
PubMedDate not supplied

Use of polymerase chain reaction for efficient cloning of dsRNA segments of infectious bursal disease virus.

A method is described for efficient cloning of the large RNA segment of infectious bursal disease virus (IBDV) after reverse transcription and cDNA amplification. Complementary DNA segments of IBDV were prepared using reverse transcriptase and specific primers homologous to the conserved region at the 3' end of the IBDV sequence. The resulting cDNA segments were amplified using Taq DNA polymerase and a pair of specific primers. Three separate primer pairs were used for amplification, each yielding a cDNA fragment of the predicted size. The amplified products were directly used for cloning into a cloning vector, pCR1000. Three overlapping cDNA clones, containing the entire coding region of the large RNA segment of IBDV, were obtained. The identity of these clones was confirmed by hybridization with IBDV-specific probe, as well as by sequence analysis. By this method, approximately 3.2 kilobases of the large genome segments of two different strains of IBDV were cloned, and their complete nucleotide sequence was determined.

Amino Acid Sequence↗