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PubMed1981

Azocarcinogen-induced release of chromatin-associated RNA into liver cytoplasm: the possible role of reiterated RNA sequences in the control of RNA processing.

In the liver of rats fed the azocarcinogen 3'-methyl-4-dimethylaminoazobenzene (3'MeDAB) reiterated RNA sequence transcribed from middle repetitive DNA are released into the cytoplasm. The same repetitive nucleotide sequences can be isolated from the chromatin of the liver of control animals in the form of metabolically highly active, 13 000 daltons RNA. This small, chromatin-associated RNA originates from nuclear RNA larger than 10 S. The discontinuation of the feeding of the azocarcinogen will not stop the release of the nuclear reiterated RNA sequences into the cytoplasm. The repetitive sequences of nuclear RNA which are released into the cytoplasm in animals fed the azocarcinogen can no longer be found in the chromatin in the form of small RNA molecules. The results can be explained by the assumption that the reiterated RNA sequences are involved in the upholding of RNA processing. A cell-specific processing of RNA will be maintained by the interaction of reiterated RNA fragments from already processed RNA with the reiterated complementary sequences on RNA yet to be processed. Existence of such a feed-back circuit would make it possible to explain how a temporary interference of the azocarcinogen with RNA processing will result in the disappearance of specific reiterated RNA sequences from the chromatin. It could also explain the continuation of the release of the same repeated RNA sequences into the cytoplasm as part of larger RNA molecules even after the removal of the carcinogen.

Animals↗

PubMed2005

Functional analysis of RNA binding by the hepatitis C virus RNA-dependent RNA polymerase.

Protein-RNA interaction plays a critical role in regulating RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp). RNAs of 7 nucleotides (nt) or longer had affinities 5-fold better than an RNA of 5 nt, suggesting a minimal length required for binding. To identify RNA contact sites on the HCV RdRp, a biotinylated 7-nt RNA capable of directing de novo initiation was used in a process that coupled reversible formaldehyde cross-linking, RNA affinity chromatography, and mass spectrometry. By this process, we identified 18 peptides cross-linked to the 7-nt RNA. When these identified peptides were overlaid on the three-dimensional structures of NS5B, most mapped to the fingers subdomain, connecting loops between fingers and thumb subdomains and in the putative RNA binding channel. Two of the identified peptides resided in the active site cavity of the RdRp. Recombinant HCV RdRp with single residue changes in likely RNA contact sites were generated and characterized for effects on HCV RdRp activity. Mutant proteins had significant effects on cross-linking to 7-nt RNA and reduced RNA synthesis in vitro by 2- to 20-fold compared with wild type protein. When the mutations were tested for the replication of HCV RNA in the context of the cells transfected with the HCV subgenomic replicon, all except one prevented colony formation, indicating a defect in HCV RNA replication. These biochemical and functional analyses identified a number of residues in the HCV RdRp that are important for HCV RNA synthesis.

Amino Acid Substitution↗

PubMed1984

Interstrand duplexes in Friend erythroleukemia nuclear RNA. The interaction of non-polyadenylated nuclear RNA with polyadenylated nuclear RNA and with small nuclear RNAs.

Intermolecular duplexes among large nuclear RNAs, and between small nuclear RNA and heterogeneous nuclear RNA, were studied after isolation by a procedure that yielded protein-free RNA without the use of phenol or high salt. The bulk of the pulse-labeled RNA had a sedimentation coefficient greater than 45 S. After heating in 50% (v/v) formamide, it sedimented between the 18 S and 28 S regions of the sucrose gradient. Proof of the existence of interstrand duplexes prior to deproteinization was obtained by the introduction of interstrand cross-links using 4'-aminomethyl-4,5',8-trimethylpsoralen and u.v. irradiation. Thermal denaturation did not reduce the sedimentation coefficient of pulse-labeled RNA obtained from nuclei treated with this reagent and u.v. irradiated. Interstrand duplexes were observed among the non-polyadenylated RNA species as well as between polyadenylated and non-polyadenylated RNAs. beta-Globin mRNA but not beta-globin pre-mRNA also contained interstrand duplex regions. In this study, we were able to identify two distinct classes of polyadenylated nuclear RNA, which were differentiated with respect to whether or not they were associated with other RNA molecules. The first class was composed of poly(A)+ molecules that were free of interactions with other RNAs. beta-Globin pre-mRNA belongs to this class. The second class included poly(A)+ molecules that contained interstrand duplexes. beta-Globin mRNA is involved in this kind of interaction. In addition, hybrids between small nuclear RNAs and heterogeneous nuclear RNA were isolated. These hybrids were formed with all the U-rich species, 4.5 S, 4.5 SI and a novel species designated W. Approximately equal numbers of hybrids were formed by species U1a, U1b, U2, U6 and W; however, species U4 and U5 were significantly under-represented. Most of these hybrids were found to be associated stably with non-polyadenylated RNA. These observations demonstrated for the first time that small nuclear RNA-heterogeneous nuclear RNA hybrids can be isolated without crosslinking, and that proteins are not necessary to stabilize the complexes. However, not all molecules of a given small nuclear RNA species are involved in the formation of these hybrids. The distribution of a given small nuclear RNA species between the free and bound state does not reflect the stability of the complex in vitro but rather the abundance of complementary sequences in the heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2005

Evaluation of the clinical sensitivity for the quantification of human immunodeficiency virus type 1 RNA in plasma: Comparison of the new COBAS TaqMan HIV-1 with three current HIV-RNA assays--LCx HIV RNA quantitative, VERSANT HIV-1 RNA 3.0 (bDNA) and COBAS AMPLICOR HIV-1 Monitor v1.5.

The COBAS TaqMan HIV-1 test (Roche Diagnostics) was compared with the LCx HIV RNA quantitative assay (Abbott Laboratories), the Versant HIV-1 RNA 3.0 (bDNA) assay (Bayer) and the COBAS Amplicor HIV-1 Monitor v1.5 test (Roche Diagnostics), using plasma samples of various viral load levels from HIV-1-infected individuals. In the comparison of TaqMan with LCx, TaqMan identified as positive 77.5% of the 240 samples versus 72.1% identified by LCx assay, while their overall agreement was 94.6% and the quantitative results of samples that were positive by both methods were strongly correlated (r=0.91). Similarly, in the comparison of TaqMan with bDNA 3.0, both methods identified 76.3% of the 177 samples as positive, while their overall agreement was 95.5% and the quantitative results of samples that were positive by both methods were strongly correlated (r=0.95). Finally, in the comparison of TaqMan with Monitor v1.5, TaqMan identified 79.5% of the 156 samples as positive versus 80.1% identified by Monitor v1.5, while their overall agreement was 95.5% and the quantitative results of samples that were positive by both methods were strongly correlated (r=0.96). In conclusion, the new COBAS TaqMan HIV-1 test showed excellent agreement with other widely used commercially available tests for the quantitation of HIV-1 viral load.

HIV Infections↗

PubMed1992

T double-stranded RNA (dsRNA) sequence reveals that T and W dsRNAs form a new RNA family in Saccharomyces cerevisiae. Identification of 23 S RNA as the single-stranded form of T dsRNA.

Some strains of the yeast Saccharomyces cerevisiae harbor a double-stranded RNA (dsRNA) molecule, called T. We obtained T cDNA clones by random priming of denatured T dsRNA followed by reverse transcription. Sequence data of T show that only one strand ((+)-strand) has coding capacity for a protein with 940 amino acids which spans almost the entire length of the molecule (2.9 kilobases). Within this protein we found a sequence pattern characteristic of RNA-dependent RNA polymerases of (+)-strand and double-stranded RNA viruses. Although T has no homology with other dsRNAs found in S. cerevisiae, such as L-A, L-BC, M1, or W, the T-encoded protein shows a high degree of conservation with the W-encoded protein. This conservation extends beyond a region that contains the consensus sequences for RNA-dependent RNA polymerases, suggesting that both T and W are evolutionarily related. With a (+)-strand-specific probe for T we identified 23 S RNA, a new single-stranded RNA (ssRNA) species with a sedimentation coefficient of 23 S. T and 23 S RNA have the same mobility under denaturing conditions with glyoxal, suggesting that 23 S RNA is, in fact, the (+)-single-stranded RNA form of T dsRNA. 23 S RNA synthesis is induced under stress conditions such as heat shock and starvation. The relationship between T and 23 S RNA clearly resembles the one between W and its single-stranded derivative form, 20 S RNA. Thus T and W dsRNAs (and their respective single-stranded species) constitute a new RNA family in S. cerevisiae.

Amino Acid Sequence↗

PubMed1996

RNA-dependent RNA polymerase activity related to the 20S RNA replicon of Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains two double-stranded RNA (dsRNA) viruses (L-A and L-BC) and two different single-stranded (ssRNA) replicons (20S RNA and 23S RNA). Replicase (dsRNA synthesis on a ssRNA template) and transcriptase (ssRNA synthesis on a dsRNA template) activities have been described for L-A and L-BC viruses, but not for 20S or 23S RNA. We report the characterization of a new in vitro RNA replicase activity in S. cerevisiae. This activity is detected after partial purification of a particulate fraction in CsCl gradients where it migrates at the density of free protein. The activity does not require the presence of L-A or L-BC viruses or 23S RNA, and its presence or absence is correlated with the presence or absence of the 20S RNA replicon. Strains lacking both this RNA polymerase activity and 20S RNA acquire this activity when they acquire 20S RNA by cytoduction (cytoplasmic mixing). This polymerase activity converts added ssRNA to dsRNA by synthesis of the complementary strand, but has no specificity for the 3' end or internal template sequence. Although it replicates all tested RNA templates, it has a template size requirement, being unable to replicate templates larger than 1 kb. The replicase makes dsRNA from a ssRNA template, but many single-stranded products due to a terminal transferase activity are also formed. These results suggest that, in contrast to the L-A and L-BC RNA polymerases, dissociation of 20S RNA polymerase from its RNA (or perhaps some cellular factor) makes the enzyme change its specificity.

Nucleotides↗

PubMed2000

RNA-Dependent replication and transcription of hepatitis delta virus RNA involve distinct cellular RNA polymerases.

Cellular DNA-dependent RNA polymerase II (pol II) has been postulated to carry out RNA-dependent RNA replication and transcription of hepatitis delta virus (HDV) RNA, generating a full-length (1.7-kb) RNA genome and a subgenomic-length (0.8-kb) mRNA. However, the supporting evidence for this hypothesis was ambiguous because the previous experiments relied on DNA-templated transcription to initiate HDV RNA synthesis. Furthermore, there is no evidence that the same cellular enzyme is involved in the synthesis of both RNA species. In this study, we used a novel HDV RNA-based transfection approach, devoid of any artificial HDV cDNA intermediates, to determine the enzymatic and metabolic requirements for the synthesis of these two RNA species. We showed that HDV subgenomic mRNA transcription was inhibited by a low concentration of alpha-amanitin (<3 microgram/ml) and could be partially restored by an alpha-amanitin-resistant mutant pol II; however, surprisingly, the synthesis of the full-length (1.7-kb) antigenomic RNA was not affected by alpha-amanitin to a concentration higher than 25 microgram/ml. By several other criteria, such as the differing requirement for the de novo-synthesized hepatitis delta antigen and temperature dependence, we further showed that the metabolic requirements of subgenomic HDV mRNA synthesis are different from those for the synthesis of genomic-length HDV RNA and cellular pol II transcripts. The synthesis of the two HDV RNA species could also be uncoupled under several different conditions. These findings provide strong evidence that pol II, or proteins derived from pol II transcripts, is involved in mRNA transcription from the HDV RNA template. In contrast, the synthesis of the 1.7-kb HDV antigenomic RNA appears not to be dependent on pol II. These results reveal that there are distinct molecular mechanisms for the synthesis of these two RNA species.

Animals↗

PubMed1986

Cytoplasmic synthesis of globin RNA in differentiated murine erythroleukemia cells: possible involvement of RNA-dependent RNA polymerase.

Three lines of evidence indicate that RNA-dependent RNA synthesis occurs in mouse erythroleukemia cells. The first involves labeling studies with [3H]uridine and shows a greater initial labeling rate of globin RNA in the cytoplasm than in the nucleus. Labeled globin RNA found in the cytoplasm after a very short pulse with tritiated uridine is of the "mature" 9S size while labeled globin RNA in the nuclei is exclusively in the form of 15S precursor molecules, suggesting that cytoplasmic globin RNA is not of nuclear origin. A high concentration of actinomycin D has no effect on the initial rate of labeling of cytoplasmic globin RNA, supporting this conclusion. Other experiments showed that the labeling of cytoplasmic globin RNA does not involve end addition to preexisting globin RNA. The second line of evidence is the identification of globin RNA minus strand in the cytoplasm of differentiated murine erythroleukemia cells by hybridization with single-stranded DNA probes containing the strand of the same sense as globin mRNA. This material has the same electrophoretic mobility as globin RNA and hybridizes with probes containing only the 5' part or only the 3' part of the gene suggesting that it is a full size copy of globin RNA. Finally, in murine erythroleukemia cells an RNA-dependent RNA polymerase activity is detected by using poly(A) . oligo(U) as a template-primer combination. This activity increases significantly after induction, suggesting that it is differentiation specific.

Acetamides↗

PubMed2002

Anti-TS1-RNA: characterization of novel antibodies against sequence-specific RNA by random RNA selection in patients with Sjögren's syndrome.

OBJECTIVE: To define a novel RNA epitope recognized by serum from a patient with Sjögren's syndrome (SS) from a randomized RNA epitope library and investigate the epitope reactivity of the anti-RNA antibodies in patients with various connective tissue diseases. METHODS: Serum from a patient with SS was used to select ligands from a library of RNA oligomers with a central region of 25 degenerate nucleotides. Bound RNA was recovered by reverse transcription, PCR amplification, and subcloning. The relationship between the antibodies to the selected RNA and disease specificity was studied using immunoprecipitation. RESULTS: From the random RNA library, several unique RNA sequences were obtained. Sera from 32 of 61 patients with SS (52.5%) precipitated with one of the selected RNA (TS1-RNA), whereas sera from 8 of 41 patients with systemic lupus erythematosus (19.5%) and 3 of 25 patients with rheumatoid arthritis (12.0%) precipitated. Although the frequency of reactivity to the TS1-RNA was higher in anti-SSA/Ro positive sera, the presence of either native or recombinant SSA/Ro antigen showed no detectable competition, and no apparent sequence homology was found between the TS1-RNA and hY RNA. CONCLUSION: These data suggest that anti-TS1-RNA is a novel antibody against sequence-specific RNA in many patients with SS.

Antibodies, Antinuclear↗

PubMed1996

Metabolism of pre-messenger RNA splicing cofactors: modification of U6 RNA is dependent on its interaction with U4 RNA.

The requirements for the formation of pseudouridine (psi) in U4 and U6 RNAs, cofactors in the splicing of pre-messenger RNA, were investigated in vitro using HeLa nuclear (NE) and cytoplasmic (S100) extracts. Maximal psi formation for both RNAs was extract order-dependent. Maximal psi formation in U4 RNA required incubation in S100 followed by the addition of NE, paralleling the in vivo maturation pathway of U4 RNA. In contrast, maximal formation of psi in U6 RNA required incubation in NE followed by the addition of S100 extract. Since U6 RNA does not exit the nucleus in vivo the contribution of S100 was investigated. In experiments where the extracts were treated with micrococcal nuclease to digest endogenous snRNAs, the efficient formation of psi in U6 RNA was dependent on the presence of U4 RNA, but not in U5 RNA or tRNA. When mutant U4 RNAs that inhibit or strengthen the interaction between U4 RNA, and U6 RNA were substituted for wild-type U4 RNA, the results confirmed the need for the interaction between these two RNAs for psi formation in U6 RNA. U6 RNA isolated from glycerol gradients after incubation in extracts had four times as much psi when associated with U4 RNA.

Base Sequence↗

PubMed1994

Association of yeast viral 23 S RNA with its putative RNA-dependent RNA polymerase.

T double-stranded RNA (dsRNA) and its single-stranded RNA (ssRNA) counterpart, 23 S RNA, are present in some strains of the yeast Saccharomyces cerevisiae. 23 S RNA is identical with the T (+)-strand, and both are cytoplasmically inherited. The T (+)-strand encodes a protein of 104 kilodaltons (kDa) (p104) that has sequences diagnostic of RNA-dependent RNA polymerases from (+)-strand and double-stranded RNA viruses. Here we show that p104 is expressed only in yeast strains carrying T dsRNA (and 23 S RNA) and that the ability to produce p104 is co-transmitted from one strain to another by cytoplasmic mixing (cytoduction) along with T dsRNA. Neither T nor 23 S RNA is encapsidated into viral particles; however, the latter was found to be non-covalently associated with p104. This finding was based on the evidence that: (i) both 23 S RNA and p104 cosedimented in sucrose gradients and that (ii) 23 S RNA was immunoprecipitated by a p104-specific polyclonal antiserum. Consistent with this association, p104 shows single-stranded RNA-binding activity in Northwestern blots. We have localized the RNA-binding domain of p104 within its C terminus 234 amino acids.

Fungal Proteins↗

PubMed2002

The ATPase, RNA unwinding, and RNA binding activities of recombinant p68 RNA helicase.

p68 RNA helicase, a nuclear RNA helicase, was identified 2 decades ago. The protein plays very important roles in cell development and organ maturation. However, the biological functions and enzymology of p68 RNA helicase are not well characterized. We report the expression and purification of recombinant p68 RNA helicase in a bacterial system. The recombinant p68 is an ATP-dependent RNA helicase. ATPase assays demonstrated that double-stranded RNA (dsRNA) is much more effective than single-stranded RNA in stimulating ATP hydrolysis by the recombinant protein. Consistently, RNA-binding assays showed that p68 RNA helicase binds single-stranded RNA weakly in an ATP-dependent manner. On the other hand, the recombinant protein has very high affinity for dsRNA. Binding of the protein to dsRNA is ATP-independent. The data indicate that p68 may directly target dsRNA as its natural substrate. Interestingly, the recombinant p68 RNA helicase unwinds dsRNA in both 3' --> 5' and 5' --> 3' directions. This is the second example of a Asp-Glu-Ala-Asp (DEAD) box RNA helicase that unwinds RNA duplexes in a bi-directional manner.

Adenosine Triphosphatases↗

PubMed2006

Characterization of norovirus 3Dpol RNA-dependent RNA polymerase activity and initiation of RNA synthesis.

Norovirus (NV) 3D(pol) is a non-structural protein predicted to play an essential role in the replication of the NV genome. In this study, the characteristics of NV 3D(pol) activity and initiation of RNA synthesis have been examined in vitro. Recombinant NV 3D(pol), as well as a 3D(pol) active-site mutant were expressed in Escherichia coli and purified. NV 3D(pol) was able to synthesize RNA in vitro and displayed flexibility with respect to the use of Mg(2+) or Mn(2+) as a cofactor. NV 3D(pol) yielded two different products when incubated with synthetic RNA in vitro: (i) a double-stranded RNA consisting of two single strands of opposite polarity or (ii) the single-stranded RNA template labelled at its 3' terminus by terminal transferase activity. Initiation of RNA synthesis occurred de novo rather than by back-priming, as evidenced by the fact that the two strands of the double-stranded RNA product could be separated, and by dissociation in time-course analysis of terminal transferase and RNA synthesis activities. In addition, RNA synthesis was not affected by blocking of the 3' terminus of the RNA template by a chain terminator, sustaining de novo initiation of RNA synthesis. NV 3D(pol) displays in vitro properties characteristic of RNA-dependent RNA polymerases, allowing the implementation of this in vitro enzymic assay for the development and validation of antiviral drugs against NV, a so far non-cultivated virus and an important human pathogen.

Base Sequence↗

PubMed1996

Initiation of (-)-strand RNA synthesis catalyzed by the BMV RNA-dependent RNA polymerase: synthesis of oligonucleotides.

RNA replication, a process of fundamental importance for pathogenesis by many viruses, remains poorly understood at the mechanistic level because relatively few of the responsible enzymes have been purified and characterized biochemically. Partially purified RNA-dependent RNA polymerase (RdRp) from brome mosaic virus (BMV)-infected barley leaves is able to synthesize (-)-strand RNAs from input (+)-strand templates. In resolving RdRp products generated during (-)-strand BMV RNA synthesis, we found that an RNA of eight nucleotides was generated at approximately 10-fold molar excess to the full-length (-)-strand RNA. Production of the 8-mer was dependent upon and specific to BMV RNA templates. Furthermore, inhibitors of full-length (-)-strand RNA synthesis by RdRp also affected the production of the 8-mer. Analysis of the sequence of the 8-mer suggests that it is initiated at the authentic initiation site on the BMV RNA template, consistent with the possibility that the 8-mer is an abortive initiation product of RNA synthesis by RdRp in vitro. Addition of heparin or Mn2+ differentially affected production of the 8-mer and full-length (-)-strand RNA, as did some nucleotide changes near the site of RNA initiation. These studies further define the initiation process of (-)-strand RNA synthesis.

Bromovirus↗

PubMed1999

RNA-Stimulated ATPase and RNA helicase activities and RNA binding domain of hepatitis G virus nonstructural protein 3.

Hepatitis G virus (HGV) nonstructural protein 3 (NS3) contains amino acid sequence motifs typical of ATPase and RNA helicase proteins. In order to examine the RNA helicase activity of the HGV NS3 protein, the NS3 region (amino acids 904 to 1580) was fused with maltose-binding protein (MBP), and the fusion protein was expressed in Escherichia coli and purified with amylose resin and anion-exchange chromatography. The purified MBP-HGV/NS3 protein possessed RNA-stimulated ATPase and RNA helicase activities. Characterization of the ATPase and RNA helicase activities of MBP-HGV/NS3 showed that the optimal reaction conditions were similar to those of other Flaviviridae viral NS3 proteins. However, the kinetic analysis of NTPase activity showed that the MBP-HGV/NS3 protein had several unique properties compared to the other Flaviviridae NS3 proteins. The HGV NS3 helicase unwinds RNA-RNA duplexes in a 3'-to-5' direction and can unwind RNA-DNA heteroduplexes and DNA-DNA duplexes as well. In a gel retardation assay, the MBP-HGV/NS3 helicase bound to RNA, RNA/DNA, and DNA duplexes with 5' and 3' overhangs but not to blunt-ended RNA duplexes. We also found that the conserved motif VI was important for RNA binding. Further deletion mapping showed that the RNA binding domain was located between residues 1383 and 1395, QRRGRTGRGRSGR. Our data showed that the MBP-HCV/NS3 protein also contains the RNA binding domain in the similar domain.

Adenosine Triphosphatases↗

PubMed2003

Mechanism of RNA recombination in carmo- and tombusviruses: evidence for template switching by the RNA-dependent RNA polymerase in vitro.

RNA recombination occurs frequently during replication of tombusviruses and carmoviruses, which are related small plus-sense RNA viruses of plants. The most common recombinants generated by these viruses are either defective interfering (DI) RNAs or chimeric satellite RNAs, which are thought to be generated by template switching of the viral RNA-dependent RNA polymerase (RdRp) during the viral replication process. To test if RNA recombination is mediated by the viral RdRp, we used either a purified recombinant RdRp of Turnip crinkle carmovirus or a partially purified RdRp preparation of Cucumber necrosis tombusvirus. We demonstrated that these RdRp preparations generated RNA recombinants in vitro. The RdRp-driven template switching events occurred between either identical templates or two different RNA templates. The template containing a replication enhancer recombined more efficiently than templates containing artificial sequences. We also observed that AU-rich sequences promote recombination more efficiently than GC-rich sequences. Cloning and sequencing of the generated recombinants revealed that the junction sites were located frequently at the ends of the templates (end-to-end template switching). We also found several recombinants that were generated by template switching involving internal positions in the RNA templates. In contrast, RNA ligation-based RNA recombination was not detected in vitro. Demonstration of the ability of carmo- and tombusvirus RdRps to switch RNA templates in vitro supports the copy-choice models of RNA recombination and DI RNA formation for these viruses.

Base Sequence↗

PubMed1990

RNA dependent RNA polymerase activity associated with the double-stranded RNA virus of Giardia lamblia.

Giardia lamblia, a parasitic protozoan, can contain a double-stranded RNA (dsRNA) virus, GLV (1). We have identified an RNA polymerase activity present specifically in cultures of GLV infected cells. This RNA polymerase activity is present in crude whole cell lysates as well as in lysates from GLV particles purified from the culture medium. The RNA polymerase has many characteristics common to other RNA polymerases (e.g. it requires divalent cations and all four ribonucleoside triphosphates), yet it is not inhibited by RNA polymerase inhibitors such as alpha-amanitin or rifampicin. The RNA polymerase activity synthesizes RNAs corresponding to one strand of the GLV genome, although under the present experimental conditions, the RNA products of the reaction are not full length viral RNAs. The in vitro products of the RNA polymerase reaction co-sediment through sucrose gradients with viral particles; and purified GLV viral particles have RNA polymerase activity. The RNA polymerase activities within and outside of infected cells closely parallel the amount of virus present during the course of viral infection. The similarities between the RNA polymerase of GLV and the polymerase associated with the dsRNA virus system of yeast are discussed.

Animals↗

PubMed2025

Dual RNA isolation from blood: an optimized protocol for host and bacterial RNA purification for dual RNA-sequencing analysis in whole blood sepsis samples.

Dual RNA-sequencing (dual RNA-seq) holds significant promise for deciphering bacterial virulence mechanisms during systemic infections. However, its application in sepsis research is hindered by technical challenges, including a low bacterial burden in blood and limited sample volumes and RNA yield from vulnerable populations, such as neonates. We developed an optimized protocol [dual RNA isolation from blood (DRIB)] for simultaneous stabilization, isolation and purification of high-quality host leukocyte and bacterial RNA from low-volume whole blood samples (0.5 ml). This protocol is compatible with clinical sample collection workflows and high-throughput RNA sequencing. The feasibility of DRIB for dual RNA-seq was validated using a pilot cohort of clinical adult sepsis samples, enabling the investigation of host-bacterial gene expression during sepsis. The DRIB protocol yielded 2.10-6.91 µg of total RNA per clinical sample in our pilot cohort. Dual-species ribosomal RNA (rRNA) depletion and RNA-seq generated 16.6-24.8 million filtered reads per sample, with 63±7% of reads uniquely mapped to host or bacterial sequences. Host genes accounted for 51-68% (8.4-10.9 million) reads, while 0.5-6.7% (79,496-789,808 reads) mapped to bacterial genomes. Bioinformatic analysis revealed that both shared and individual transcriptional patterns were identified in host and bacterial responses, including pathways related to immune metabolism and metal-ion binding. Our optimized DRIB protocol and RNA-seq pipeline effectively captured both host and bacterial RNA transcription in clinical sepsis samples. Expanding this approach to larger cohorts and varying disease timepoints will provide crucial new insights into host-bacterial gene co-expression dynamics in sepsis progression and outcomes.

Humans↗