Genome-wide RNA polymerase II profiles and RNA accumulation reveal kinetics of transcription and associated epigenetic changes during diurnal cycles.

Interactions of cell-autonomous circadian oscillators with diurnal cycles govern the temporal compartmentalization of cell physiology in mammals. To understand the transcriptional and epigenetic basis of diurnal rhythms in mouse liver genome-wide, we generated temporal DNA occupancy profiles by RNA polymerase II (Pol II) as well as profiles of the histone modifications H3K4me3 and H3K36me3. We used these data to quantify the relationships of phases and amplitudes between different marks. We found that rhythmic Pol II recruitment at promoters rather than rhythmic transition from paused to productive elongation underlies diurnal gene transcription, a conclusion further supported by modeling. Moreover, Pol II occupancy preceded mRNA accumulation by 3 hours, consistent with mRNA half-lives. Both methylation marks showed that the epigenetic landscape is highly dynamic and globally remodeled during the 24-hour cycle. While promoters of transcribed genes had tri-methylated H3K4 even at their trough activity times, tri-methylation levels reached their peak, on average, 1 hour after Pol II. Meanwhile, rhythms in tri-methylation of H3K36 lagged transcription by 3 hours. Finally, modeling profiles of Pol II occupancy and mRNA accumulation identified three classes of genes: one showing rhythmicity both in transcriptional and mRNA accumulation, a second class with rhythmic transcription but flat mRNA levels, and a third with constant transcription but rhythmic mRNAs. The latter class emphasizes widespread temporally gated posttranscriptional regulation in the mouse liver.

T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension.

This article introduces a new interface for T-Coffee, a consistency-based multiple sequence alignment program. This interface provides an easy and intuitive access to the most popular functionality of the package. These include the default T-Coffee mode for protein and nucleic acid sequences, the M-Coffee mode that allows combining the output of any other aligners, and template-based modes of T-Coffee that deliver high accuracy alignments while using structural or homology derived templates. These three available template modes are Expresso for the alignment of protein with a known 3D-Structure, R-Coffee to align RNA sequences with conserved secondary structures and PSI-Coffee to accurately align distantly related sequences using homology extension. The new server benefits from recent improvements of the T-Coffee algorithm and can align up to 150 sequences as long as 10,000 residues and is available from both http://www.tcoffee.org and its main mirror http://tcoffee.crg.cat.

Phosphorylation by casein kinase 2 facilitates rRNA gene transcription by promoting dissociation of TIF-IA from elongating RNA polymerase I.

The protein kinase casein kinase 2 (CK2) phosphorylates different components of the RNA polymerase I (Pol I) transcription machinery and exerts a positive effect on rRNA gene (rDNA) transcription. Here we show that CK2 phosphorylates the transcription initiation factor TIF-IA at serines 170 and 172 (Ser170/172), and this phosphorylation triggers the release of TIF-IA from Pol I after transcription initiation. Inhibition of Ser170/172 phosphorylation or covalent tethering of TIF-IA to the RPA43 subunit of Pol I inhibits rDNA transcription, leading to perturbation of nucleolar structure and cell cycle arrest. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrate that dissociation of TIF-IA from Pol I is a prerequisite for proper transcription elongation. In support of phosphorylation of TIF-IA switching from the initiation into the elongation phase, dephosphorylation of Ser170/172 by FCP1 facilitates the reassociation of TIF-IA with Pol I, allowing a new round of rDNA transcription. The results reveal a mechanism by which the functional interplay between CK2 and FCP1 sustains multiple rounds of Pol I transcription.

RDM1, a novel RNA recognition motif (RRM)-containing protein involved in the cell response to cisplatin in vertebrates.

A variety of cellular proteins has the ability to recognize DNA lesions induced by the anti-cancer drug cisplatin, with diverse consequences on their repair and on the therapeutic effectiveness of this drug. We report a novel gene involved in the cell response to cisplatin in vertebrates. The RDM1 gene (for RAD52 Motif 1) was identified while searching databases for sequences showing similarities to RAD52, a protein involved in homologous recombination and DNA double-strand break repair. Ablation of RDM1 in the chicken B cell line DT40 led to a more than 3-fold increase in sensitivity to cisplatin. However, RDM1-/- cells were not hypersensitive to DNA damages caused by ionizing radiation, UV irradiation, or the alkylating agent methylmethane sulfonate. The RDM1 protein displays a nucleic acid binding domain of the RNA recognition motif (RRM) type. By using gel-shift assays and electron microscopy, we show that purified, recombinant chicken RDM1 protein interacts with single-stranded DNA as well as double-stranded DNA, on which it assembles filament-like structures. Notably, RDM1 recognizes DNA distortions induced by cisplatin-DNA adducts in vitro. Finally, human RDM1 transcripts are abundant in the testis, suggesting a possible role during spermatogenesis.

Results of An International Study of Quantitative BCR/ABL Assays Using Defined RNA Samples

Molecular monitoring of BCR/ABL transcripts by real time quantitative reverse transcription PCR (qRT-PCR) is an essential technique for clinical management of patients with BCR/ABL-positive CML and ALL. Though quantitative BCR/ABL assays are performed in hundreds of laboratories worldwide, results among these laboratories cannot be reliably compared due to heterogeneity in test methods, data analysis, reporting, and lack of quantitative standards. Recent efforts towards standardization have been limited in scope. Aliquots of RNA were sent to clinical test centers worldwide in order to evaluate methods and reporting for e1a2, b2a2, and b3a2 transcript levels using their own qRT-PCR assays. Total RNA was isolated from tissue culture cells that expressed each of the different BCR/ABL transcripts. Serial log dilutions were prepared, ranging from 100 to 10-5, in RNA isolated from HL60 cells. Laboratories performed 5 independent qRT-PCR reactions for each sample type at each dilution. In addition, 15 qRT-PCR reactions of the 10-3 b3a2 RNA dilution were run to assess reproducibility within and between laboratories. Participants were asked to run the samples following their standard protocols and to report cycle threshold (Ct), quantitative values for BCR/ABL and housekeeping genes, and ratios of BCR/ABL to housekeeping genes for each sample RNA. Thirty-seven (n=37) participants have submitted qRT-PCR results for analysis (36, 37, and 34 labs generated data for b2a2, b3a2, and e1a2, respectively). The limit of detection for this study was defined as the lowest dilution that a Ct value could be detected for all 5 replicates. For b2a2, 15, 16, 4, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. For b3a2, 20, 13, and 4 labs showed a limit of detection at the 10-5, 10-4, and 10-3 dilutions, respectively. For e1a2, 10, 21, 2, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. Log %BCR/ABL ratio values provided a method for comparing results between the different laboratories for each BCR/ABL dilution series. Linear regression analysis revealed concordance among the majority of participant data over the 10-1 to 10-4 dilutions. The overall slope values showed comparable results among the majority of b2a2 (mean=0.939; median=0.9627; range (0.399 - 1.1872)), b3a2 (mean=0.925; median=0.922; range (0.625 - 1.140)), and e1a2 (mean=0.897; median=0.909; range (0.5174 - 1.138)) laboratory results (Fig. 1-3)). Thirty-four (n=34) out of the 37 laboratories reported Ct values for all 15 replicates and only those with a complete data set were included in the inter-lab calculations. Eleven laboratories either did not report their copy number data or used other reporting units such as nanograms or cell numbers; therefore, only 26 laboratories were included in the overall analysis of copy numbers. The median copy number was 348.4, with a range from 15.6 to 547,000 copies (approximately a 4.5 log difference); the median intra-lab %CV was 19.2% with a range from 4.2% to 82.6%. While our international performance evaluation using serially diluted RNA samples has reinforced the fact that heterogeneity exists among clinical laboratories, it has also demonstrated that performance within a laboratory is overall very consistent. Accordingly, the availability of defined BCR/ABL RNAs may facilitate the validation of all phases of quantitative BCR/ABL analysis and may be extremely useful as a tool for monitoring assay performance. Ongoing analyses of these materials, along with the development of additional control materials, may solidify consensus around their application in routine laboratory testing and possible integration in worldwide efforts to standardize quantitative BCR/ABL testing.

Exploration of the visual system: Part 1: Dissection of the mouse eye for RNA, protein, and histological analyses

Due to the power of genetics, the mouse has become a widely used animal model in vision research. However, its eyeball has an axial length of only about 2 mm. The present protocol describes how to easily dissect the small rodent eye post mortem. This allows collecting different tissues of the eye, i.e., cornea, lens, iris, retina, optic nerve, retinal pigment epithelium (RPE), and sclera. We further describe in detail how to process these eye samples in order to obtain high‐quality RNA for RNA expression profiling studies. Depending on the eye tissue to be analyzed, we present appropriate lysis buffers to prepare total protein lysates for immunoblot and immuno‐precipitation analyses. Fixation, inclusion, embedding, and cryosectioning of the globe for routine histological analyses (HE staining, DAPI staining, immunohistochemistry, in situ hybridization) is further presented. These basic protocols should allow novice investigators to obtain eye tissue samples rapidly for their experiments.

Low current and nadir CD4+ T-cell counts are associated with higher hepatitis C virus RNA levels in the Swiss HIV cohort study.

BACKGROUND: The aim of this study was to evaluate the effect of CD4+ T-cell counts and other characteristics of HIV-infected individuals on hepatitis C virus (HCV) RNA levels. METHODS: All HIV-HCV-coinfected Swiss HIV Cohort Study participants with available HCV RNA levels and concurrent CD4+ T-cell counts before starting HCV therapy were included. Potential predictors of HCV RNA levels were assessed by multivariate censored linear regression models that adjust for censored values. RESULTS: The study included 1,031 individuals. Low current and nadir CD4+ T-cell counts were significantly associated with higher HCV RNA levels (P = 0.004 and 0.001, respectively). In individuals with current CD4+ T-cell counts < 200/microl, median HCV RNA levels (6.22 log10 IU/ml) were +0.14 and +0.24 log10 IU/ml higher than those with CD4+ T-cell counts of 200-500/microl and > 500/microl. Based on nadir CD4+ T-cell counts, median HCV RNA levels (6.12 log10 IU/ml) in individuals with < 200/microl CD4+ T-cells were +0.06 and +0.44 log10 IU/ml higher than those with nadir T-cell counts of 200-500/microl and > 500/microl. Median HCV RNA levels were also significantly associated with HCV genotype: lower values were associated with genotype 4 and higher values with genotype 2, as compared with genotype 1. Additional significant predictors of lower HCV RNA levels were female gender and HIV transmission through male homosexual contacts. In multivariate analyses, only CD4+ T-cell counts and HCV genotype remained significant predictors of HCV RNA levels. Conclusions: Higher HCV RNA levels were associated with CD4+ T-cell depletion. This finding is in line with the crucial role of CD4+ T-cells in the control of HCV infection.

Targeting the proteolytic processing of the viral glycoprotein precursor is a promising novel antiviral strategy against arenaviruses.

A crucial step in the arenavirus life cycle is the biosynthesis of the viral envelope glycoprotein (GP) responsible for virus attachment and entry. Processing of the GP precursor (GPC) by the cellular proprotein convertase site 1 protease (S1P), also known as subtilisin-kexin-isozyme 1 (SKI-1), is crucial for cell-to-cell propagation of infection and production of infectious virus. Here, we sought to evaluate arenavirus GPC processing by S1P as a target for antiviral therapy using a recently developed peptide-based S1P inhibitor, decanoyl (dec)-RRLL-chloromethylketone (CMK), and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). To control for off-target effects of dec-RRLL-CMK, we employed arenavirus reverse genetics to introduce a furin recognition site into the GPC of LCMV. The rescued mutant virus grew to normal titers, and the processing of its GPC critically depended on cellular furin, but not S1P. Treatment with the S1P inhibitor dec-RRLL-CMK resulted in specific blocking of viral spread and virus production of LCMV. Combination of the protease inhibitor with ribavirin, currently used clinically for treatment of human arenavirus infections, resulted in additive drug effects. In cells deficient in S1P, the furin-dependent LCMV variant established persistent infection, whereas wild-type LCMV underwent extinction without the emergence of S1P-independent escape variants. Together, the potent antiviral activity of an inhibitor of S1P-dependent GPC cleavage, the additive antiviral effect with ribavirin, and the low probability of emergence of S1P-independent viral escape variants make S1P-mediated GPC processing by peptide-derived inhibitors a promising strategy for the development of novel antiarenaviral drugs.

Maf1, a new player in the regulation of human RNA polymerase III transcription.

BACKGROUND: Human RNA polymerase III (pol III) transcription is regulated by several factors, including the tumor suppressors P53 and Rb, and the proto-oncogene c-Myc. In yeast, which lacks these proteins, a central regulator of pol III transcription, called Maf1, has been described. Maf1 is required for repression of pol III transcription in response to several signal transduction pathways and is broadly conserved in eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We show that human endogenous Maf1 can be co-immunoprecipitated with pol III and associates in vitro with two pol III subunits, the largest subunit RPC1 and the alpha-like subunit RPAC2. Maf1 represses pol III transcription in vitro and in vivo and is required for maximal pol III repression after exposure to MMS or rapamycin, treatments that both lead to Maf1 dephosphorylation. CONCLUSIONS/SIGNIFICANCE: These data suggest that Maf1 is a major regulator of pol III transcription in human cells.

Using digital RNA counting and flow cytometry to compare mRNA with protein expression in acute leukemias.

BACKGROUND: The diagnosis of malignant hematologic diseases has become increasingly complex during the last decade. It is based on the interpretation of results from different laboratory analyses, which range from microscopy to gene expression profiling. Recently, a method for the analysis of RNA phenotypes has been developed, the nCounter technology (Nanostring® Technologies), which allows for simultaneous quantification of hundreds of RNA molecules in biological samples. We evaluated this technique in a Swiss multi-center study on eighty-six samples from acute leukemia patients. METHODS: mRNA and protein profiles were established for normal peripheral blood and bone marrow samples. Signal intensities of the various tested antigens with surface expression were similar to those found in previously performed Affymetrix microarray analyses. Acute leukemia samples were analyzed for a set of twenty-two validated antigens and the Pearson Correlation Coefficient for nCounter and flow cytometry results was calculated. RESULTS: Highly significant values between 0.40 and 0.97 were found for the twenty-two antigens tested. A second correlation analysis performed on a per sample basis resulted in concordant results between flow cytometry and nCounter in 44-100% of the antigens tested (mean = 76%), depending on the number of blasts present in a sample, the homogeneity of the blast population, and the type of leukemia (AML or ALL). CONCLUSIONS: The nCounter technology allows for fast and easy depiction of a mRNA profile from hematologic samples. This technology has the potential to become a valuable tool for the diagnosis of acute leukemias, in addition to multi-color flow cytometry.

Role of mutations in the phosphoprotein on RNA polymerase activity of canine distemper virus following cell culture adaptation

Abstract: The canine distemper virus A75/17 wild-type strain, which is unable to replicate in cell lines, was adapted to growth in Vero cells. Sequence comparison between the A75/17 and the Vero cell-adapted A75/17-V virus revealed 7 amino acid differences between the 2 viruses. Three of these were located in the matrix protein, three in the phosphoprotein also changing the V protein but not the C protein and one in the large protein. The phosphoprotein and the large protein constituted the viral RNA polymerase whose activity was studied by transfection experiments using a reverse genetic system with a plasmid encoding a minireplicon and expression plasmids encoding the nucleocapsid protein and the viral RNA polymerase subunits. Surprinsingly, the enzyme of A75/17 CDV was significantly more active in cell lines compared to the polymerase of A75/17-V CDV. The decrease in overall enzyme activity was found to be due to both decreased replication and transcription activity. This polymerase attenuation was confirmed in CHO cells infection stably expressing the dog SLAM receptor mainly found in dog's lymphoid organs and allowing both virus strains to enter these cells at the same efficiency. A75/17-V CDV replicated more slowly in CHODogSLAM cells than A75/17 CDV and syncytium formation was significantly decreased compared to A75/17 infected CHODogSLAM cells.. Cell culture adaptation lead to an attenuated virus strain both in vitro and in vivo with decreased polymerase activity and syncytium forming capability showing an important role of the polymerase in determining the phenoytpe of the virus. In addition, this reduced phenotype of A75/17-V CDV was shown to be due to the P mutations in the P protein only, showing an important function of the polycistronic P gene in the adaptation process. The role of the matrix protein was found not to have any effect on polymerase activity, however its participation in the adaptation process still needs to be elucidated. The accessory proteins V and C were shown to act on polymerase activity, but their functions in virus pathogenicity and in inhibiting the interferon system have not been studied in this thesis. The V proteins have an activating effect on the polymerase of both the A75/17 and the A75/17-V CDV strains. Although the C protein amino acid sequence was not changed during adaptation of wild-type canine distemper virus in Vero cells, the C protein was demonstrated to have opposite effects on polymerase activity of both virus strains suggesting a different interaction of the C protein with the proteins forming the polymerase complex, which could modulate polymeras activity. These effects were demonstrated by transfection experiments and studying recombinant viruses not expressing the C protein. Thus, the abrogation of the C protein decrease the activity of the wild-type polymerase. In contrast, the polymerase activity of the Vero cell- adapted virus is enhanced in the absence of the C protein and this has also been demonstrated with a recombinant virus, which grew faster in the first 48 hours of infection. Future studies will focus on the generation of recombinant wild-type viruses, which should be very helpful in understanding the molecular mechanisms underlying the adaptation process and the loss of pathogenicity.

The Pseudomonas aeruginosa antimetabolite L -2-amino-4-methoxy-trans-3-butenoic acid (AMB) is made from glutamate and two alanine residues via a thiotemplate-linked tripeptide precursor.

The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a non-proteinogenic amino acid which is toxic for prokaryotes and eukaryotes. Production of AMB requires a five-gene cluster encoding a putative LysE-type transporter (AmbA), two non-ribosomal peptide synthetases (AmbB and AmbE), and two iron(II)/α-ketoglutarate-dependent oxygenases (AmbC and AmbD). Bioinformatics analysis predicts one thiolation (T) domain for AmbB and two T domains (T1 and T2) for AmbE, suggesting that AMB is generated by a processing step from a precursor tripeptide assembled on a thiotemplate. Using a combination of ATP-PPi exchange assays, aminoacylation assays, and mass spectrometry-based analysis of enzyme-bound substrates and pathway intermediates, the AmbB substrate was identified to be L-alanine (L-Ala), while the T1 and T2 domains of AmbE were loaded with L-glutamate (L-Glu) and L-Ala, respectively. Loading of L-Ala at T2 of AmbE occurred only in the presence of AmbB, indicative of a trans loading mechanism. In vitro assays performed with AmbB and AmbE revealed the dipeptide L-Glu-L-Ala at T1 and the tripeptide L-Ala-L-Glu-L-Ala attached at T2. When AmbC and AmbD were included in the assay, these peptides were no longer detected. Instead, an L-Ala-AMB-L-Ala tripeptide was found at T2. These data are in agreement with a biosynthetic model in which L-Glu is converted into AMB by the action of AmbC, AmbD, and tailoring domains of AmbE. The importance of the flanking L-Ala residues in the precursor tripeptide is discussed.

The striatal long noncoding RNA Abhd11os is neuroprotective against an N-terminal fragment of mutant huntingtin in vivo.

A large number of gene products that are enriched in the striatum have ill-defined functions, although they may have key roles in age-dependent neurodegenerative diseases affecting the striatum, especially Huntington disease (HD). In the present study, we focused on Abhd11os, (called ABHD11-AS1 in human) which is a putative long noncoding RNA (lncRNA) whose expression is enriched in the mouse striatum. We confirm that despite the presence of 2 small open reading frames (ORFs) in its sequence, Abhd11os is not translated into a detectable peptide in living cells. We demonstrate that Abhd11os levels are markedly reduced in different mouse models of HD. We performed in vivo experiments in mice using lentiviral vectors encoding either Abhd11os or a small hairpin RNA targeting Abhd11os. Results show that Abhd11os overexpression produces neuroprotection against an N-terminal fragment of mutant huntingtin, whereas Abhd11os knockdown is protoxic. These novel results indicate that the loss lncRNA Abhd11os likely contribute to striatal vulnerability in HD. Our study emphasizes that lncRNA may play crucial roles in neurodegenerative diseases.

A phase IIa randomised clinical study of GNbAC1, a humanised monoclonal antibody against the envelope protein of multiple sclerosis-associated endogenous retrovirus in multiple sclerosis patients.

BACKGROUND: GNbAC1 is an immunoglobulin (IgG4) humanised monoclonal antibody against multiple sclerosis-associated retrovirus (MSRV)-Env, a protein of endogenous retroviral origin, expressed in multiple sclerosis (MS) lesions, which is pro-inflammatory and inhibits oligodendrocyte precursor cell differentiation. OBJECTIVE: This is a randomised, double-blind placebo-controlled dose-escalation study followed by a six-month open-label phase to test GNbAC1 in MS patients. The primary objective was to assess GNbAC1 safety in MS patients, and the other objectives were pharmacokinetic and pharmacodynamic assessments. METHODS: Ten MS patients were randomised into two cohorts to receive a single intravenous infusion of GNbAC1/placebo at doses of 2 or 6 mg/kg. Then all patients received five infusions of GNbAC1 at 2 or 6 mg/kg at four-week intervals in an open-label setting. Safety, brain magnetic resonance imaging (MRI), pharmacokinetics, immunogenicity, cytokines and MSRV RNA expression were studied. RESULTS: All patients completed the study. GNbAC1 was well tolerated in all patients. GNbAC1 pharmacokinetics is dose-linear with mean elimination half-life of 27-37 d. Anti-GNbAC1 antibodies were not detected. Cytokine analysis did not indicate an adverse effect. MSRV-transcripts showed a decline after the start of treatment. Nine patients had stable brain lesions at MRI. CONCLUSION: The safety, pharmacokinetic profile, and pharmacodynamic responses to GNbAC1 are favourable in MS patients over a six-month treatment period.

RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification.