Binding of Cellular Proteins to the Leader RNA of Equine Arteritis Virus

The genome of equine arteritis virus (EAV) produces a 3 coterminal-nested set of six subgenomic (sg) viral RNAs during virus replication cycle, and each set possesses a common leader sequence of 206 nucleotides (nt) in length derived from the 5 end of the viral genome. Given the presence of the leader region within both genomic and sg mRNAs, it is likely to contain cis-acting signals that may interact with cellular or viral proteins for RNA synthesis. Gel mobility shift assays indicated that proteins in Vero cell cytoplasmic extracts formed complexes with the positive (+) and negative (-) strands of the EAV leader RNA. Several cell proteins with molecular masses ranging from 74 to 31 kDa and 58 to 32 kDa were detected in UV-induced cross-linking assays with the EAV leader RNA (+) and (-) strands, respectively. In both cases, intense bands were observed at the 58–52 kDa molecular weight markers. Results from competition gel mobility shift assays using overlapping cold RNA probes spanning the leader RNA (+) strand indicated that nt 140–206 are not necessary for binding to cell proteins.     

The search for a marsupial XIC reveals a break with vertebrate synteny

X-chromosome inactivation (XCI) evolved in mammals to deal with X-chromosome dosage imbalance between the XX female and the XY male. In eutherian mammals, random XCI of the soma requires a master regulatory locus known as the ‘X-inactivation center’ (XIC/Xic), wherein lies the noncoding XIST/Xist silencer RNA and its regulatory antisense Tsix gene. By contrast, marsupial XCI is imprinted to occur on the paternal X chromosome. To determine whether marsupials and eutherians share the XIC-driven mechanism, we search for the sequence equivalents in the genome of the South American opossum, Monodelphis domestica. Positional cloning and bioinformatic analysis reveal several interesting findings. First, protein-coding genes that flank the eutherian XIC are well-conserved in M. domestica, as well as in chicken, frog, and pufferfish. However, in M. domestica we fail to identify any recognizable XIST or TSIX equivalents. Moreover, cytogenetic mapping shows a surprising break in synteny with eutherian mammals and other vertebrates. Therefore, during the evolution of the marsupial X chromosome, one or more rearrangements broke up an otherwise evolutionarily conserved block of vertebrate genes. The failure to find XIST/TSIX in M. domestica may suggest that the ancestral XIC is too divergent to allow for detection by current methods. Alternatively, the XIC may have arisen relatively late in mammalian evolution, possibly in eutherians with the emergence of random XCI. The latter argues that marsupial XCI does not require XIST and opens the search for alternative mechanisms of dosage compensation.     

The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.     

pGenesil-siHBV X 对 HepG2.2.15 细胞 HBV 表达和复制的抑制效果研究

 目的 研究针对HBV X基因区设计的小干扰RNA（siRNA）表达载体质粒pGenesil-siHBV X 对HepG2.2.15细胞HBV表达和复制的抑制效果及特异性。方法 针对HBV X区设计siRNA表达载体质粒pGenesil- siHBV X。分别用培养液（空白对照）、脂质体Metafectene、pGenesil 空载体、pGenesil-siHK（阴性对照）、pGenesil-siAFP（特异性对照）、pGenesil-siHBV X处理或转染HepG2.2.15细胞各3次。于每次转染后24 h，取各组细胞培养上清液，用时间分辨荧光免疫测定技术检测上清液中HBsAg和HBeAg含量，用化学发光法检测AFP含量，用PCR荧光定量技术检测HBV-DNA复制水平。 结果 pGenesil-siHBV X转染能抑制HepG2.2.15细胞对HBV标志物的表达，且抑制作用随转染次数增加而增强。第3次转染后，pGenesil-siHBV X组细胞上清液中 HBsAg、 HBeAg和HBV-DNA检测结果分别为（6.26 ± 1.07）ng/ml 、（0.13 ± 0.05）Ncu/ml和（3.01 ± 0.40）×107拷贝/ml，与空白对照组的（22.50 ± 1.39）ng/ml、（1.12 ± 0.11）Ncu/ml和（12.33 ± 1.28）×107拷贝/ml比较，差异有统计学意义（t值分别为12.80、12.21、9.71，P < 0.05）；pGenesil-siHBV X 转染不影响细胞对AFP的表达（t = 0.18，P = 0.86）。结论 pGenesil-siHBV X可以有效和特异地抑制HepG 2.2. 15细胞HBV-DNA的复制及HBsAg和HBeAg表达。     

TP73-AS1在肿瘤中的表达及调控作用

<正>随着高通量测序技术的发展,成千上万种长链非编码RNA(long non-coding RNA,lncRNA)进入人们的视野。lncRNA是一类长度超过200个核苷酸并缺少开放阅读框的不具备蛋白质编码功能的RNA~([1-2])。研究发现lncRNA参与诸多生物学进程的关键步骤,包括染色质重塑、基因转录、转录后调节和蛋白质翻译等~([3-5])。LncRNA机制的不断被阐明,也为肿瘤的研究提供了新的可能。LncRNA在多种肿瘤的增殖、迁移侵袭和抗凋亡     

Three mitochondrial unassigned open reading frames of Podospora anserina represent remnants of a viral-type RNA polymerase gene

The mitochondrial DNA of Podospora anserina is complex, consisting of a characteristic set of genes with a large number of introns and a substantial amount of sequence of unknown function and origin. In addition, as indicated by various types of reorganization, this genome is highly flexible. Here we report the identification of three unassigned mitochondrial open reading frames (ORF P', ORF Q', ORF 11) as remnants of a rearranged viral-type RNA polymerase gene. These ORFs are not transcribed and may be derived from the integration of a linear plasmid of the type recently identified in a mutant of P. anserina.     

Loss-of-function mutations in caspase recruitment domain-containing protein 14 (CARD14) are associated with a severe variant of atopic dermatitis

1. Download high-res image (356KB)
2. Download full-size image

     

Intraaxonaler transport von Ethidium-Bromid-sensitiven RNS- und niedermolekularen 3H-Uridin-Verbindungen im tractus opticus von teleosteern

Zusammenfassung Am Sehtrakt von Carassius carassius und Scardinius erythrophthalmus wurde die axonale Ausbreitungsweise hoch- und niedermolekularer 3H-Uridin-Verbindungen untersucht. Dabei wurde nach intraocularer Injektion des Tracers für TCE-resistente Verbindungen eine intraaxonale Transportgeschwindigkeit von 2–4 mm/d bestimmt, für TCE-lösliche Verbindungen eine von ca. 30–50 mm/d. Durch Applikation des spezifisch mitochondrialen RNS-Synthese-Hemmers Ethidium-Bromid konnte die Einbaurate von 3H-Uridin in hochmolekulare RNS um 70–80% erniedrigt werden, was dafür spricht, daß die langsam im Axoplasma wandernden Mitochondrien einen Großteil der axonalen RNS synthetisieren.In der TCE-löslichen Fraktion konnten durch dünnschichtchromatographische Analyse noch nach 8d p. i. 3H-Uridin und 3H-UDPG nachgewiesen werden. Dieser Befund wird hinsichtlich eines transneuronalen Stoffübertritts von Uridin und der möglichen Bedeutung des UDPG-Transportes im Nervengewebe diskutiert.

---

Intraaxonal transport of ethidium-bromide-sensitive RNA- and lowmolecular 3H-uridine-compounds in the optic tract of teleost

Summary In the optic system of teleosts (Carassius carassius and Scardinius erythrophthalmus) the axonal flow of high and low molecular 3H-uridine-compounds was investigated. After injection of the tracer into one eyeball and TCA-extraction of the samples a transport-rate of 2–4 mm/d was demonstrated. By the specific inhibitor of mitochondrial RNA-synthesis, Ethidium-Bromide, the amount of axonal radioactivity could be reduced to 20–30% of the control. This indicates the mitochondria as being the site of synthesis most of axonal RNA. Considering the TCA-soluble 3H-uridine-compounds, an intraaxonal flow also could be demonstrated, with a transport-rate of 30–50 mm/d, 16 times higher as the one of RNA. The analysis by thin layer chromatography indicated the existence of 3H-uridine and 3H-UDPG in the axonal fraction of TCA-soluble compounds still after an incorporation time of 8d. The possibility of a transneuronal convection of uridine and the function of UDPG-transport in the axon are discussed.

Frau Prof. Dr. H. Kersten (Erlangen) danke ich für die Überlassung einer Probe Ethidium-Bromid.