Double-stranded RNA-specific adenosine deaminase is an enzyme that in humans is encoded by the ADAR gene (which stands for adenosine deaminase acting on RNA). Adenosine deaminases acting on RNA (ADAR) are enzymes responsible for binding to double stranded RNA (dsRNA) and converting adenosine (A) to inosine (I) by deamination. ADAR protein is a RNA-binding protein, which functions in RNA-editing through post-transcriptional modification of mRNA transcripts by changing the nucleotide content of the RNA. The conversion from A to I in the RNA disrupt the normal A:U pairing which makes the RNA unstable. Inosine is structurally similar to that of guanine (G) which leads to I to cytosine (C) binding. In RNA I functions the same as G in both translation and replication. Codon changes can arise from editing which may lead to changes in the coding sequences for proteins and their functions. Most editing site are found in noncoding regions of RNA such as untranslated regions (UTRs), Alu elements and long ...
论文信息:Zhuyun Bian, Yajia Ni, Jin-Rong Xu, Huiquan Liu*.A-to-I mRNA editing in fungi: occurrence, function, and evolution. Cellular and Molecular Life Sciences (2019) 76:329-340.. JCR分区Q1,中科院大类分区二区,IF=6.721. 论文摘要: A-to-I RNA editing is an important post-transcriptional modification that converts adenosine (A) to inosine (I) in RNA molecules via hydrolytic deamination. Although editing of mRNAs catalyzed by adenosine deaminases acting on RNA (ADARs) is an evolutionarily conserved mechanism in metazoans, organisms outside the animal kingdom lacking ADAR orthologs were thought to lack A-to-I mRNA editing. However, recent discoveries of genome-wide A-to-I mRNA editing during the sexual stage of the wheat scab fungus Fusarium graminearum, model filamentous fungus Neurospora crassa, Sordaria macrospora, and an early diverging filamentous ascomycete Pyronema confluens indicated that A-to-I mRNA editing is likely an evolutionarily conserved feature in ...
TY - JOUR. T1 - Hepatitis C virus RNA quantification in right and left lobes of the liver in patients with chronic hepatitis C. AU - Idrovo, V.. AU - Dailey, P. J.. AU - Jeffers, Lennox J. AU - Coelho-Little, E.. AU - Bernstein, D.. AU - Bartholomew, M.. AU - Alvarez, L.. AU - Urdea, M. S.. AU - Collins, M. L.. AU - Schiff, Eugene R. PY - 1996/9/1. Y1 - 1996/9/1. N2 - Quantification of hepatitis C virus RNA in liver tissue is likely to be useful in the study of the natural history, pathogenesis, progression and treatment of hepatitis C virus-associated liver disease. Quantitative measurements of hepatitis C virus RNA in liver biopsy samples using the branched DNA (bDNA) signal amplification assay were carried out. The aims of this study were threefold: first, to assess the level of hepatitis C virus RNA in biopsy samples from the right and left lobes of the liver; second, to evaluate the correlation between hepatitis C virus RNA levels in serum and liver; and third, to investigate the ...
BACKGROUND Hepatitis delta virus is a unique human pathogen responsible for some 20 million infections globally. This virus is dependent on hepatitis B virus for transmission and propagation. Currently, at least three genotypes of hepatitis delta virus with different geographic distribution and clinical manifestations are described. METHODS In this study, hepatitis delta virus RNA of 35 patients sera were analyzed by RT- semi-nested polymerase chain reaction. Based on genomic differences of hepatitis delta antigen coding region of hepatitis delta virus RNA among hepatitis delta virus RNA-positive sera, the polymerase chain reaction products were digested with restriction enzymes and studied by restriction fragment length polymorphism. RESULTS Out of 35 samples, 13 (38.46%) were positive for hepatitis delta virus RNA by RT- semi-nested polymerase chain reaction. All polymorphisms were shown to be genotype I. Out of 13 hepatitis delta virus RNA-positive (13/35), eight were HBeAg negative.
Group II Self-Splicing Introns. -pre-rRNA of fungal and plant mitochondria -majority of chloroplast introns -several classes -require Mg 2+ -no cofactor. Domain Structure of a Group II Intron. A. 5 exon. 3 exon. Chemistry of Group II Self-Splicing. 1st step. 2nd step. lariat. Slideshow 3387240 by guang
TY - JOUR. T1 - Removal of divalent cations induces structural transitions in Red clover necrotic mosaic virus, revealing a potential mechanism for RNA release. AU - Sherman, Michael B.. AU - Guenther, Richard H.. AU - Tama, Florence. AU - Sit, Tim L.. AU - Brooks, Charles L.. AU - Mikhailov, Albert M.. AU - Orlova, Elena V.. AU - Baker, Timothy S.. AU - Lommel, Steven A.. PY - 2006/11/1. Y1 - 2006/11/1. N2 - The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 Å by cryoelectron microscopy. The virion capsid has prominent surface protrusions and subunits with a clearly defined shell and protruding domains. The structures of both the individual capsid protein (CP) subunits and the entire virion capsid are consistent with other species in the Tombusviridae family. Within the RCNMV capsid, there is a clearly defined inner cage formed by complexes of genomic RNA and the amino termini of CP subunits. An RCNMV virion has approximately 390 ± 30 ...
Summary of Facts and Submissions. I. European patent No. 0 846 181 with the title cDNA corresponding to the antigenome of nonsegmented negative strand RNA viruses, and process for the production of such viruses encoding additional antigenically active proteins was granted on European patent application No. 96928446.2 (published as WO 97/06270). The patent was granted with 21 claims.. II. Claim 1 of the patent as granted read as follows:. 1. A method for the production of an infectious non-segmented negative-strand RNA virus of the family Paramyxoviridae comprising. (a) introducing a cDNA molecule contained in a plasmid, wherein said cDNA molecule comprises the entire (+)-strand sequence of said negative- strand RNA virus operatively linked to an expression control sequence, which allows the synthesis of anti-genomic RNA transcripts bearing the authentic 3 -termini, and wherein said cDNA molecule consists of an integral multiple of six nucleotides, into a helper cell expressing an ...
The adenovirus major late transcription unit (MLTU) is an example of a complex alternatively spliced gene, in which more than 15 different 3 splice sites can be joined to a common 5 splice site. Maturation of the full repertoire of possible mRNAs requires late viral protein synthesis and occurs only at late stages of the infectious cycle (16-24 hpi). We are trying to decipher the mechanisms regulating alternative 3 splice site choice during the infectious cycle. Therefore, we examined the splicing activity of several 3 splice sites from the MLTU in vitro in nuclear extracts prepared from adenovirus infected cells (Ad NE) and from uninfected cells. The results suggest that pre-mRNAs with "weak" 3 splice sites (short, atypical polypyrimidine tracts) are activated and pre-mRNAs with long, prototypical polypyrimidine tracts are repressed in Ad NE. In fact, our data show a reciprocal correlation between the strength of a polypyrimidine tract, defined by its affinity for U2AF65K in vitro, and the ...
article{5993109, author = {De Backer, Lynn and Braeckmans, Kevin and Stuart, Marc CA and Demeester, Jo and De Smedt, Stefaan and Raemdonck, Koen}, issn = {0168-3659}, journal = {JOURNAL OF CONTROLLED RELEASE}, keyword = {DRUG-DELIVERY,EXOGENOUS SURFACTANT,LUNG,THERAPEUTICS,THERAPIES,SMALL-INTERFERING RNA,LOADED DEXTRAN NANOGELS,POLYMER HYBRID NANOPARTICLES,Targeted delivery,Pulmonary surfactant,siRNA delivery,Lung,Hybrid nanoparticles,Dextran nanogels,PLATFORM,CARRIERS}, language = {eng}, pages = {177--186}, title = {Bio-inspired pulmonary surfactant-modified nanogels: a promising siRNA delivery system}, url = {http://dx.doi.org/10.1016/j.jconrel.2015.03.015}, volume = {206}, year = {2015 ...
Hepatitis C virus RNA replication requires not only the viral replicase but also the host cytoskeletons, membrane structures, and cellular factors. Several hnRNPs, such as polypyrimidin-tract-binding protein (PTB) and La autoantigen were found to regulate both HCV RNA replication and translation. Another hnRNP, SYNCRIP (synaptotamin-Binding, Cytoplasmic RNA-Interacting Protein, or NSAP-1), was found to regulate HCV-IRES dependent translation. In my first part of dissertation, we identified SYNCRIP as a positive regulator of HCV RNA replication.; With a growing number of "dual-function" proteins which are able to regulate HCV RNA translation and replication, it is interesting to investigate how the two important steps in HCV life cycle are temporally and spatially regulated. Therefore, we initiated a series of experiments to examine if HCV RNA translation is dependent on its replication. The colocalization of newly-synthesized viral RNA and peptide were detected in live cell and under ...
The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and ...
We have investigated the usefulness of serum hepatitis delta virus (HDV) RNA detection using a slot hybridization analysis of serum samples from ten patients with acute hepatitis and delta markers (group I), from 28 patients with chronic delta hepatitis (group II) and from seven liver graft recipients with hepatitis B virus (HBV) and HDV related cirrhosis or fulminant hepatitis (group III). The slot-blots were hybridized with both HDV-complementary DNA and single-stranded RNA probes. With the single-stranded RNA probe, HDV RNA was detected in the first serum sample available in 9/10 of the patients with acute hepatitis (group I). In addition, HDV RNA was detected in 8/9 and 7/8 of the samples obtained within and after 1 month of the onset of hepatitis. Five of the ten patients scored positive for HDV RNA and negative for hepatitis delta antigen (HDAg) while one was negative for HDV RNA and positive for HDAg. The same RNA probe enabled the detection of serum HDV RNA in 21/28 chronic hepatitis ...
The genetic continuity of the potato spindle tuber viroid (PSTVd) genome was analysed after infection of tomato plants with cloned cDNAs of parental strains. During the six weeks of the experiment, several new sequence variants appeared. The sequence variants detected in the progeny population induced sequence-specific disease symptoms. The PSTVd genome therefore follows the pattern expected for typical pseudo-strains propagating in plants as a population of similar sequences. Assessing further the replicon continuity, a PSTVd cDNA mutant with a deletion in the central conserved region was constructed and proven to be non-infectious. Surprisingly, in a sub-population of potato transformants expressing the same deleted PSTVd RNA an infectious viroid was detected. This suggests specific transcript conversion followed by recovery of the full-length pathogen genome ...
Nature. 2011 Dec 1;480(7375):113-7. doi: 10.1038/nature10546. Research Support, N.I.H., Extramural; Research Support, N.I.H., Intramural; Research Support, Non-U.S. Govt; Research Support, U.S. Govt, Non-P.H.S.
In this study, we developed anionic polymer-coated liposome/siRNA complexes (lipoplexes) with chondroitin sulfate C (CS), poly-l-glutamic acid (PGA) and poly-aspartic acid (PAA) for siRNA delivery by intravenous injection, and evaluated the biodistribution and gene silencing effect in mice. The sizes of CS-, PGA- and PAA-coated lipoplexes were about 200?nm and their ?-potentials were negative. CS-, PGA- and PAA-coated lipoplexes did not induce agglutination after mixing with erythrocytes. In terms of biodistribution, siRNAs after intravenous administration of cationic lipoplexes were largely observed in the lungs, but those of CS-, PGA- and PAA-coated lipoplexes were in both the liver and the kidneys, indicating that siRNA might be partially released from the anionic polymer-coated lipoplexes in the blood circulation and accumulate in the kidney, although the lipoplexes can prevent the agglutination with blood components. To increase the association between siRNA and cationic liposome, we used ...
Telomerase, the ribonucleoprotein complex involved in telomere maintenance, is composed of two main components: hTERT and hTERC. hTERT seems to be the rate-limiting factor for telomerase activity, although hTERC expression was also shown to correlate to a certain extent with telomerase reactivation. To determine whether the absence of hTERC expression could be the consequence of DNA methylation, we quantified hTERC RNA in 60 human samples (19 telomerase-negative normal tissues, nine telomerase-positive and 22 telomerase-negative tumor tissues, eight telomerase-positive and two telomerase-negative cell lines) using a quantitative dot blot on RT-PCR products. Most of the normal tissues did not express hTERC whereas, in telomerase-positive cell lines and in telomerase-positive tumor tissues, a strong up-regulation was observed, suggesting that hTERC transcription is up-regulated during tumorigenesis. The two telomerase-negative cell lines did not express hTERC. In a series of 22 telomerase-negative ...
This invention relates to the use of tumor-derived or associated extracellular ribonucleic acid (RNA) found circulating in the plasma or serum fraction of blood for the detection, monitoring, or evaluation of cancer or premalignant conditions. Extracellular RNA may circulate as non-bound RNA, protein-bound RNA, lipid-RNA complexes, lipoprotein (proteolipid)-RNA complexes, protein-RNA complexes including within or in association with ribonucleoprotein complexes, nucleosomes, or within apoptotic bodies. Any intracellular RNA found in plasma or serum can additionally be detected by this invention. Specifically, this invention enables the extraction of circulating RNA from plasma or serum and utilizes nucleic acid amplification assays for the identification, detection, inference, monitoring, or evaluation of any neoplasm, benign, premalignant, or malignant, in humans or other animals, which might be associated with that RNA. Further, this invention allows the qualitative or quantitative detection of tumor
First, we sought to demonstrate a selective knockdown of the NaV1.8 protein. To this end, we created a stable cell line expressing the NaV1.8 protein by transfection followed by G418 selection. Figure 2is an immunocytochemical (A1) and a Western blot (A2) confirmation of the proper expression of NaV1.8 immunoreactive protein of the expected molecular mass (, 260 kd) in the NaV1.8-HEK293 cell line. Infection of this cell line with the shRNA/EGFP-expressing lentivirus resulted in a decrease in the anti-NaV1.8 antibody immunoreactive band detected by a Western blot. As expected, lentivirus only expressing the EGFP reporter or the pan-tetrodotoxin-sensitive shRNA designed to knock down the tetrodotoxin-sensitive α subunits did not inhibit the NaV1.8 protein expression. Of the five shRNA/EGFP constructs targeting the NaV1.8, all demonstrated significant reduction in the immunoreactive protein band except for the NaV1.8 (1103) construct (fig. 2B). The GFP immunoreactivity (fig. 2B, bottom) served as ...
Reactome is pathway database which provides intuitive bioinformatics tools for the visualisation, interpretation and analysis of pathway knowledge.
0041]This invention relates to methods for detecting tumor-associated RNA in plasma, serum and other bodily fluids. The methods thereby provide for the detecting, diagnosing, inferring, evaluating or monitoring of cancer or neoplastic disease in a human or animal. The inventive methods comprise the steps of extracting RNA from plasma, serum, or bodily fluid of a human or animal, and thereafter assessing the amount or concentration of mammalian extracellular RNA in said plasma, serum, or other bodily fluid of the human or animal, or cDNA derived therefrom. Particularly preferred embodiments of the inventive steps include an amplification or signal amplification step, followed by detection of the amplified product or signal. In particularly preferred embodiments this is performed by comparing the amount or concentration of one or more RNA species in said plasma, serum, or bodily fluid obtained from the human or animal with another, or with the amount or concentration of RNA found in bodily fluid ...
DExD/H-box proteins are a diverse class of proteins that are implicated in RNA and RNP remodeling. They have sequence homology to DNA helicases and share conserved ATPase domains, suggesting that they use the energy of ATP binding and hydrolysis to mediate conformational rearrangements in RNAs. In the past, the action of DExD/H-box proteins has been characterized primarily on simple model substrates such as small RNA duplexes. It is not known how DExD/H-box proteins manipulate structured RNA, what determines target specificity and what molecular events follow their action. Here, using the well-characterized Tetrahymena group I intron ribozyme, I performed kinetic and thermodynamic studies to understand the mechanism of CYT-19 and related DExD/Hbox proteins. CYT-19 has been shown previously to facilitate the folding of several group I and group II introns. I demonstrated that CYT-19 acts as a chaperone, accelerating the re-folding of a long-lived misfolded species of the Tetrahymena group I ...
COMPOSITIONS AND METHODS FOR CANCER AND CANCER STEM CELL DETECTION AND ELIMINATION - In alternative embodiments, the invention provides compositions and methods for inhibiting or ablating cancer stem cells. In alternative embodiments, the invention provides compositions and methods for inhibiting the action of double-stranded RNA-specific adenosine deaminases, or ADAR, enzymes. In alternative embodiments, the invention provides compositions and methods for treating, ameliorating or preventing diseases and conditions responsive to the inhibition of cell differentiation and/or self-renewal of dysfunctional cells, cancer cells, leukemia cells, hematopoietic stem cells or cancer stem cells, e.g., leukemia or Chronic Myeloid Leukemia (CML). In alternative embodiments, the invention provides compositions and methods for inhibiting a Sonic Hedgehog (Shh) pathway, e.g., by using a Smoothened (SMO) protein inhibitor. In alternative embodiments, the invention provides compositions and methods for ...
Maximiliano DAngelo, PhD, Research Associate, Salk Institute for Biological Studies: Nuclear Pore Deterioration during Aging and Its Consequences for Cellular Function. Kristian Doyle, PhD, Post-Doctoral Scholar, Stanford University: Does Increased TGF-Beta Signaling in the Elderly Increase Astrogliosis and Impair Functional Recovery Following Stroke?. Jeremy Herskowitz, PhD, Postdoctoral Fellow, Emory University School of Medicine: The Role of LR11 in Alzheimers Disease Pathogenesis. Maria Lehtinen, PhD, Postdoctoral Fellow, HHMI/Childrens Hospital Boston: Regulation of the Neural Stem Cell Niche by the Aging Cerebrospinal Fluid (CSF) Proteome. Lingbo Li, PhD, Stanford University: Understanding the Role of RNA-editing Enzyme ADAR in Aging Neurons. Daijun Ling, PhD, Research Fellow, Beckman Research Institute of City of Hope: Autophagic-Lysosomal Storage of Amyloid Beta and Its Connection to Development of Senile Plaques. Brian Onken, PhD, Postdoctoral Fellow, Rutgers, The State University of ...
Avian encephalomyelitis virus (AEV) is a picornavirus that affects young chickens, quails, pheasants and turkeys. Translation initiation on picornavirus mRNA is cap independent and occurs through a mechanism known as internal initiation, which depends on Internal Ribosome Entry Site (IRES) element within the 5 untranslated region (UTR) of the viral RNA. AEV has been assigned within the Hepatovirus genus and shares protein sequence similarity with hepatitis A virus (HAV). I have demonstrated that the 494 nucleotide 5 UTR of the AEV genome contains an internal ribosome entry site (IRES) element. However, in contrast to the HAV IRES, the AEV IRES functions efficiently in the presence of cleaved eEF4G, suggesting functional differences exist. Characterization of the AEV IRES element revealed that there are remarkable structural and functional similarities between the AEV, flavivirus [especially hepatitis C virus (HCV)] and newly discovered type 4 picornaviras IRES elements, including porcine ...
RNALogo: a new approach to display structural RNA alignment - Regulatory RNAs play essential roles in many essential biological processes, ranging from gene regulation to protein synthesis. This work presents a web-based tool, RNALogo, to create a new graphical representation of the patterns in a multiple RNA sequence alignment with a consensus structure. The RNALogo graph can indicate significant features within an RNA sequence alignment and its consensus RNA secondary structure. RNALogo extends Sequence logos, and specifically incorporates RNA secondary structures and mutual information of base-paired regions into the graphical representation. Each RNALogo graph is composed of stacks of letters, with one stack for each position in the consensus RNA secondary structure. RNALogo provides a convenient and high configurable logo generator. An RNALogo graph is generated for each RNA family in Rfam, and these generated logos are accumulated into a gallery of RNALogo. Users can search or browse RNALogo
SR proteins are a conserved family of proteins involved in RNA splicing. SR proteins are named because they contain a protein domain with long repeats of serine and arginine amino acid residues, whose standard abbreviations are "S" and "R" respectively. SR proteins are ~200-600 amino acids in length and composed of two domains, the RNA recognition motif (RRM) region and the RS domain. SR proteins are more commonly found in the nucleus than the cytoplasm, but several SR proteins are known to shuttle between the nucleus and the cytoplasm. SR proteins were discovered in the 1990s in Drosophila and in amphibian oocytes, and later in humans. In general, metazoans appear to have SR proteins and unicellular organisms lack SR proteins. SR proteins are important in constitutive and alternative pre-mRNA splicing, mRNA export, genome stabilization, nonsense-mediated decay, and translation. SR proteins alternatively splice pre-mRNA by preferentially selecting different splice sites on the pre-mRNA strands ...
The molecular basis of a significant number of cases of isolated growth hormone deficiency remains unknown. We describe three sisters affected with severe isolated growth hormone deficiency and pituitary hypoplasia caused by biallelic mutations in the RNPC3 gene, which codes for a minor spliceosome protein required for U11/U12 small nuclear ribonucleoprotein (snRNP) formation and splicing of U12‐type introns. We found anomalies in U11/U12 di‐snRNP formation and in splicing of multiple U12‐type introns in patient cells. Defective transcripts include preprohormone convertases SPCS2 and SPCS3 and actin‐related ARPC5L genes, which are candidates for the somatotroph‐restricted dysfunction. The reported novel mechanism for familial growth hormone deficiency demonstrates that general mRNA processing defects of the minor spliceosome can lead to very narrow tissue‐specific consequences ...
Mutations of an alternative splice donor site located within the gag region has previously been shown to broaden the pathogenic potential of the T-lymphomagenic gammaretrovirus Moloney murine leukemia virus, while the equivalent mutations in the erythroleukemia inducing Friend murine leukemia virus seem to have no influence on the disease-inducing potential of this virus. In the present study we investigate the splice pattern as well as the possible effects of mutating the alternative splice sites on the oncogenic properties of the B-lymphomagenic Akv murine leukemia virus. By exon-trapping procedures we have identified a novel gammaretroviral exon, resulting from usage of alternative splice acceptor (SA) and splice donor (SD) sites located in the capsid region of gag of the B-cell lymphomagenic Akv murine leukemia virus. To analyze possible effects in vivo of this novel exon, three different alternative splice site mutant viruses, mutated in either the SA, in the SD, or in both sites, respectively,
Hepatitis C is a common cause of chronic liver disease but is rarely associated with acute hepatitis. The majority of patients have no clinical symptoms and jaundice in this phase of acute viral hepatitis C. Clinical symptoms are not difference with other types of hepatitis [2]. It is necesseray to treat acute hepatitis C infection. HCV infection becomes chronic in about 85 % of individuals as demonstrated by the persistence of HCV. HCV is the major cause of cirrhosis and hepatocellular carcinoma [2]. Interferon-α is effective in improving biochemical outcomes and achieving sustained virologic clearance in patients with acute hepatitis C [3]. If acute infection is confirmed (with or without acute hepatitis), recent data suggest that early treatment of acute HCV infection with interferon-α may be highly effective in preventing chronic HCV infection [1]. These data underscore the importance of identifying persons with acute HCV infection and promptly referring them to experienced clinicians who ...
Ribosome biogenesis is a highly complex process that requires several cofactors, including DExD/H-box RNA helicases (RHs). RHs are a family of ATPases that rearrange the secondary structures of RNA and thus remodel ribonucleoprotein (RNP) complexes. DExD/H-box RHs are found in most organisms and play critical roles in a variety of RNA-involved cellular events. In human and yeast cells, many DExD/H box RHs participate in multiple steps of ribosome biogenesis and regulate cellular proliferation and stress responses. In plants, several DExD/H-box RHs have been demonstrated to be associated with plant development and abiotic and biotic stress tolerance through their functions in modulating pre-rRNA processing. In this review, we summarize the pleiotropic roles of DExD/H-box RHs in rRNA biogenesis and other biological functions. We also describe the overall function of the DExD/H-box RH family in ribosome biogenesis based on data from human and yeast.
TY - JOUR. T1 - Importance of the positive-strand RNA secondary structure of a murine coronavirus defective interfering RNA internal replication signal in positive-strand RNA synthesis. AU - Repass, John F.. AU - Makino, Shinji. PY - 1998/10. Y1 - 1998/10. N2 - The RNA elements that are required for replication of defective interfering (DI) RNA of the JHM strain of mouse hepatitis virus (MHV) consist of three discontinuous genomic regions: about 0.46 to 0.47 kb from both terminal sequences and an internal 58-nucleotide (nt)-long sequence (58-nt region) present at about 0.9 kb from the 5 end of the DI genome. The internal region is important for positive-strand DI RNA synthesis (Y. N. Kim and S. Makino, J. Virol. 69:4963-4971, 1995). We further characterized the 58-nt region in the present study and obtained the following results. (i) The positive-strand RNA structure in solution was comparable with that predicted by computer modeling. (ii) Positive-strand RNA secondary structure, but not ...
Understanding how biomolecules interact is a major task of systems biology. To model protein-nucleic acid interactions, it is important to identify the DNA or RNA-binding residues in proteins. Protein sequence features, including the biochemical property of amino acids and evolutionary information in terms of position-specific scoring matrix (PSSM), have been used for DNA or RNA-binding site prediction. However, PSSM is rather designed for PSI-BLAST searches, and it may not contain all the evolutionary information for modelling DNA or RNA-binding sites in protein sequences. In the present study, several new descriptors of evolutionary information have been developed and evaluated for sequence-based prediction of DNA and RNA-binding residues using support vector machines (SVMs). The new descriptors were shown to improve classifier performance. Interestingly, the best classifiers were obtained by combining the new descriptors and PSSM, suggesting that they captured different aspects of evolutionary
The Drosophila piRNA pathway provides an RNA-based immune system that defends the germline genome against selfish genetic elements. Two inter-related branches of the piRNA system exist: somatic cells that support oogenesis only employ Piwi, whereas germ cells utilize a more elaborated pathway centered on the three gonad-specific Argonaute proteins Piwi, Aubergine, and Argonaute3. While several key factors of each branch have been identified, our current knowledge is insufficient to explain the complex workings of the piRNA machinery. Here, we report a reverse genetic screen spanning the ovarian transcriptome in an attempt to uncover the full repertoire of genes required for piRNA-mediated transposon silencing in the female germline. Our screen reveals new key factors of piRNA-mediated transposon silencing, including the novel piRNA biogenesis factors, CG2183 (GASZ) and Deadlock. Last, our data uncovers a previously unanticipated set of factors preferentially required for repression of different
In vertebrates, a nuclear cap-binding complex (CBC) formed by two cap- binding proteins, CBP20 and CBP80, is involved in several steps of RNA metabolism, including pre-mRNA splicing and nuclear export of some RNA polymerase II-transcribed U snRNAs. The CBC is highly conserved, and antibodies against human CBP20 cross-react with the CBP20 counterpart in the dipteran Chironomus tentans. Using immunoelectron microscopy, the in situ association of CBP20 with a specific pre-mRNP particle, the Balbiani ring particle, has been analyzed at different stages of pre-mRNA synthesis, maturation, and nucleo-cytoplasmic transport. We demonstrate that CBP20 binds to the nascent pre-mRNA shortly after transcription initiation, stays in the RNP particles after splicing has been completed, and remains attached to the 5 domain during translocation of the RNP through the nuclear pore complex (NPC). The rapid association of CBP20 with nascent RNA transcripts in situ is consistent with the role of CBC in splicing, ...
A negative-sense single-stranded RNA virus (or (-)ssRNA virus) is a virus that uses negative sense, single-stranded RNA as its genetic material. Single stranded RNA viruses are classified as positive or negative depending on the sense or polarity of the RNA. The negative viral RNA is complementary to the mRNA and must be converted to a positive RNA by RNA polymerase before translation. Therefore, the purified RNA of a negative sense virus is not infectious by itself, as it needs to be converted to a positive sense RNA for replication. These viruses belong to Group V on the Baltimore classification. In addition, negative-sense single-stranded RNA viruses have complex genomic sequences, cell cycles, and replication habits that use various protein complexes to arrange in specific conformations and carry out necessary processes for survival and reproduction of their genomic sequences. The complexity of negative-sense single-stranded RNA viruses carries into its ability to suppress the innate immune ...
Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer using carbohydrates as the feedstock [1]. Therefore, it is important to develop molecular biology tools to further manipulate this microorganism. Extraction of high-quality RNA from R. toruloides is particular challenging due to high level of polysaccharides, lipids and other secondary metabolites [2]. To obtain an optimal protocol for RNA extraction from R. toruloides, four methods, including guanidine thiocyanate [3], glass beads-hot acid phenol, glass beads-TRIzol, and modified RNAiso, were evaluated. RNA quality was assessed using UV absorbance (A260/A280 and A260/A230), agarose gel electrophoresis, reverse transcription (RT)-PCR reactions, and Gel-Pro analyzer 4.5. Large differences in RNA yield and quality among protocols were found. The optimum method was modified RNAiso method, where RNA was isolated using liquid nitrogen-RNAiso with salt precipitation and the addition of PVP andβ-mercaptoethanol. This ...
Epitranscriptomic events such as adenosine-to-inosine (A-to-I) RNA editing by ADAR can recode mRNAs to translate novel proteins. Editing of the mRNA that encodes actin crosslinking protein Filamin A (FLNA) mediates a Q-to-R transition in the interactive C-terminal region. While FLNA editing is conserved among vertebrates, its physiological function remains unclear. Here, we show that cardiovascula...
Viral vectors have become the best option for the delivery of therapeutic genes in conventional and RNA interference-based gene therapies. The current viral vectors for the delivery of small regulatory RNAs are based on DNA viruses and retroviruses/lentiviruses. Cytoplasmic RNA viruses have been excluded as viral vectors for RNAi therapy because of the nuclear localization of the microprocessor complex and the potential degradation of the viral RNA genome during the excision of any virus-encoded pre-microRNAs. However, in the last few years, the presence of several species of small RNAs (e.g., virus-derived small interfering RNAs, virus-derived short RNAs, and unusually small RNAs) in animals and cell cultures that are infected with cytoplasmic RNA viruses has suggested the existence of a non-canonical mechanism of microRNA biogenesis. Several studies have been conducted on the tick-borne encephalitis virus and on the Sindbis virus in which microRNA precursors were artificially incorporated and
Tan,M.H., Li,Q., Shanmugam,R., Piskol,R., Kohler, J., et al. (2017) Dynamic landscape and regulation of RNA editing in mammals. Nature 550(7675):249-254. Ma, D, George C.X., Nomburg, J., Pfaller, C.K., Cattaneo, R., and Samuel, C.E. (2017). Upon infection the cellular WD repeat-containing protein 5 localizes to cytoplasmic inclusion bodies and enhances measles virus replication. J. Virol.: in press. George, C.X., Ramaswami, G., Li, J.B., Samuel, C.E. (2016) Editing of cellular self-RNAs by adenosine deaminase ADAR1 suppresses innate immune stress responses. J Biol Chem. 291:6158-68. Kainulainen, M., Lau, S., Samuel, C.E., Hornung, V., Weber, F. (2016) NSs virulence factor of Rift Valley Fever Virus engages the F-box proteins FBXW11 and β-TRCP1 to degrade the antiviral protein kinase PKR. J Virol. 90:6140-6147. George, C. X., Samuel, C. E. (2015) STAT2-dependent induction of RNA adenosine deaminse ADAR1 by type 1 interferon differs between mouse and human cells in the requirement for STAT1. ...
Plays role in pre-mRNA splicing as core component of the SMN-Sm complex that mediates spliceosomal snRNP assembly and as component of the spliceosomal U1, U2, U4 and U5 small nuclear ribonucleoproteins (snRNPs), the building blocks of the spliceosome. Component of both the pre-catalytic spliceosome B complex and activated spliceosome C complexes. Is also a component of the minor U12 spliceosome (By similarity). As part of the U7 snRNP it is involved in histone pre-mRNA 3-end processing (PubMed:19470752).
Purpose: The strongest genetic association of Fuchs endothelial corneal dystrophy (FECD) is with an expanded trinucleotide repeat (CTG·CAG) in an intron of the TCF4 gene. The same expanded repeat sequence in the 3UTR of the DMPK gene causes Myotonic Dystrophy type 1 (DM1) via a RNA toxicity mechanism. In DM1, poly(CUG) transcripts accumulate in foci and sequester the splicing factor MBNL1, causing missplicing of essential transcripts in skeletal muscle. Our hypothesis is that the same molecular mechanism causes RNA toxicity in the corneal endothelium of FECD patients. Because FECD patients do not present signs or symptoms of DM1, we also ask whether the molecular signature of RNA toxicity is present in muscle cells derived from FECD patients.. Methods: Corneal endothelial tissue was obtained at the time of endothelial keratoplasty, and skin fibroblasts were derived from skin biopsy specimens from patients with FECD. Using fluorescence in situ hybridization (FISH), protein-RNA aggregates can be ...
We previously reported that sialyl Lewisy, synthesized by fucosyltransferases, is involved in angiogenesis. Fucosyltransferase 1 (fut1) is an α(1,2)-fucosyltransferase responsible for synthesis of the H blood group and Lewisy antigens. However, the angiogenic involvement of fut 1 in the pathogenesis of rheumatoid arthritis synovial tissue (RA ST) has not been clearly defined. Assay of α(1,2)-linked fucosylated proteins in RA was performed by enzyme-linked lectin assay. Fut1 expression was determined in RA ST samples by immunohistological staining. We performed angiogenic Matrigel assays using a co-culture system of human dermal microvascular endothelial cells (HMVECs) and fut1 small interfering RNA (siRNA) transfected RA synovial fibroblasts. To determine if fut1 played a role in leukocyte retention and cell proliferation in the RA synovium, myeloid THP-1 cell adhesion assays and fut1 siRNA transfected RA synovial fibroblast proliferation assays were performed. Total α(1,2)-linked fucosylated
Gene expression is tightly regulated at the level of transcription through cooperation between cis-regulatory elements and trans-factors that bind to the regulatory elements. Together, these factors regulate the higher order chromatin structure which establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms. CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and ...
Activation-induced deaminase (AID) is expressed only in germinal center B cells. There, it is required for somatic hypermutation, gene conversion and class switch recombination of antibody variable region segments, three processes that diversify antibodies during immune responses. Although AID has homology to RNA-editing enzymes, three recent reports suggest it could initiate the diversification processes by deaminating cytidine residues within the antibody genes themselves.
Drug resistance is a major factor for the limited efficacy of chemotherapy in gastric cancer treatment. Hypoxia-inducible factor-1α (HIF-1α), a central transcriptional factor in hypoxia, is suggested to participate in the resistance. Here, we identified a hypoxia-mimic (cobalt chloride) sensitive gastric cell line BGC-823 to explore whether diosgenin, an aglycone of steroidal saponins, can inhibit cancer cell invasion and survival of solid tumor in a hypoxic mimic microenvironment. We have shown that diosgenin is a potent candidate for decreasing the ability of invasion and survival in cobalt chloride treated BGC-823 cells. In addition, when combined with HIF-1α specific short hairpin RNA (shRNA), diosgenin can inhibit BGC-823 cells more effectively. The anti-invasion role of diosgenin may be related to E-cadherin, integrinα5 and integrinβ6. These results suggest that diosgenin may be a useful compound in controlling gastric cancer cells in hypoxia condition, especially when combined with down
The particle-bound RNA polymerase activity of vesicular stomatitis virus (VSV) can be demonstrated in vivo. Linear synthesis of viral RNA persists for 5 to 6 hours at 34°C in infected monolayers of chick embryo cells treated with cycloheximide and actinomycin D to block synthesis of protein and cell-specific RNA. At least 55 percent of the RNA made under these conditions is complementary to virion RNA. RNA synthesis mediated by VSV polymerase activity is inhibited in cells first treated with chick-derived interferon or polyriboinosinate• polyribocytidylate, but not by mouse interferon. The RNA product of VSV polymerase activity is present throughout the cytoplasm, and its synthesis is inhibited by the interferon system, as judged by autoradiographs that show the physical distribution, in cells, of RNA produced by virion polymerase in the absence of translation-a demonstration of the transcription product of the viral genome. ...
The Y-box-binding protein 1 (YB-1), a member of the cold-shock domain RNA-and DNA-binding protein family, has pleiotropic functions such as regulation of the cell cycle. The aim of this study was to evaluate if YB-1 is a proliferative marker in breast cancer and elucidate potential downstream targets involved in YB-1-mediated cell cycle regulation using RNA interference technology. YB-1 protein expression was evaluated in tissue microarrays of 131 breast invasive ductal carcinomas by immunohistochemistry, while the YB-1 gene expression profile was evaluated in the T-47D, MDA-MB-231, ZR-75-1 and MCF7 breast cancer cell lines. Silencing of the YB-1 gene in T-47D breast cancer cells was performed using siRNA and the effects of down-regulation of YB-1 on cell growth and regulation of the cell cycle were ascertained. A focused panel of 84 genes involved in cell cycle progression was also examined. In tissue microarrays, YB-1 expression was shown to be associated with proliferating cell nuclear ...
Uncontrolled transposable element (TE) insertions and excisions can cause chromosome breaks and mutations with dramatic deleterious effects. The PIWI interacting RNA (piRNA) pathway functions as an adaptive TE silencing system during germline development. Several essential piRNA pathway proteins appear to be rapidly evolving, suggesting that TEs and the silencing machinery may be engaged in a classical evolutionary arms race. Using a variety of molecular evolutionary and population genetic approaches, we find that the piRNA pathway genes rhino, krimper, and aubergine show patterns suggestive of extensive recurrent positive selection across Drosophila species. We speculate that selection on these proteins reflects crucial roles in silencing unfamiliar elements during vertical and horizontal transmission of TEs into naive populations and species, respectively.
Abstract: Infection by human papillomavirus (HPV) alters the microenvironment of keratinocytes as a mechanism to evade the immune system. A-to-I editing by ADAR1 has been reported to regulate innate immunity in response to viral infections. Here, we evaluated the role of ADAR1 in HPV infection in vitro and in vivo. Innate immune activation was characterized in human keratinocyte cell lines constitutively infected or not with HPV. ADAR1 knockdown induced an innate immune response through enhanced expression of RIG-I-like receptors (RLR) signaling cascade, over-production of type-I IFNs and pro-inflammatory cytokines. ADAR1 knockdown enhanced expression of HPV proteins, a process dependent on innate immune function as no A-to-I editing could be identified in HPV transcripts. A genetic association study was performed in a cohort of HPV/HIV infected individuals followed for a median of 6 years (range 0.1-24). We identified the low frequency haplotype AACCAT significantly associated with recurrent ...
The RTG1591 is funded by the German Research Foundation (DFG). We are dedicated to training PhD students in research projects concerning the post-transcriptional control of gene expression in directing cell fate, human diseases and plant-pathogen interactions. Successful candidates will characterize, on the molecular level, how RNA-binding proteins, enzymes acting on RNA, and various regulatory RNAs, including microRNAs, long non-coding RNAs and bacterial sRNAs, modulate gene expression. On the basis of animal and plant model organisms, students will explore how the post-transcriptional control of gene expression determines cell fate and diseases. The RTG1591 provides a structured post-graduate training program including various workshops and seminars supporting students in establishing a successful scientific career. We offer MD stipends (stipend, approximately 830€/month) for one year with an optional extension for an additional two years.. ...