Preparation of soluble extracts from adenovirus-infected cells for studies of RNA splicing

Here we describe a collection of methods that have been adapted to produce highly efficient nuclear and cytoplasmic extracts from adenovirus-infected HeLa cells. We describe how to produce extracts from virus-infected cells and how to analyze RNA splicing in vitro using T7 RNA polymerase-derived splicing substrate RNAs.

Comparison of amplification methods for transcriptomic analyses of low abundance prokaryotic RNA sources

Microarrays have established as instrumental for bacterial detection, identification, and genotyping as well as for transcriptomic studies. For gene expression analyses using limited numbers of bacteria (derived from in vivo or ex vivo origin, for example), RNA amplification is often required prior to labeling and hybridization onto microarrays. Evaluation of the fidelity of the amplification methods is crucial for the robustness and reproducibility of microarray results. We report here the first utilization of random primers and the highly processive Phi29 phage polymerase to amplify material for transcription profiling analyses. We compared two commercial amplification methods (GenomiPhi and MessageAmp kits) with direct reverse-transcription as the reference method, focusing on the robustness of mRNA quantification using either microarrays or quantitative RT-PCR. Both amplification methods using either poly-A tailing followed by in vitro transcription, or direct strand displacement polymerase, showed appreciable linearity. Strand displacement technique was particularly affordable compared to in vitro transcription-based (IVT) amplification methods and consisted in a single tube reaction leading to high amplification yields. Real-time measurements using low-, medium-, and highly expressed genes revealed that this simple method provided linear amplification with equivalent results in terms of relative messenger abundance as those obtained by conventional direct reverse-transcription.

Expression profiling with RNA from formalin-fixed, paraffin-embeeded material

Background Molecular characterization of breast and other cancers by gene expression profiling has corroborated existing classifications and revealed novel subtypes. Most profiling studies are based on fresh frozen (FF) tumor material which is available only for a limited number of samples while thousands of tumor samples exist as formalin-fixed, paraffin-embedded (FFPE) blocks. Unfortunately, RNA derived of FFPE material is fragmented and chemically modified impairing expression measurements by standard procedures. Robust protocols for isolation of RNA from FFPE material suitable for stable and reproducible measurement of gene expression (e.g. by quantitative reverse transcriptase PCR, QPCR) remain a major challenge. Results We present a simple procedure for RNA isolation from FFPE material of diagnostic samples. The RNA is suitable for expression measurement by QPCR when used in combination with an optimized cDNA synthesis protocol and TaqMan assays specific for short amplicons. The FFPE derived RNA was compared to intact RNA isolated from the same tumors. Preliminary scores were computed from genes related to the ER response, HER2 signaling and proliferation. Correlation coefficients between intact and partially fragmented RNA from FFPE material were 0.83 to 0.97. Conclusion We developed a simple and robust method for isolating RNA from FFPE material. The RNA can be used for gene expression profiling. Expression measurements from several genes can be combined to robust scores representing the hormonal or the proliferation status of the tumor.

Crystal structure of the yeast eIF4A-eIF4G complex: an RNA-helicase controlled by protein-protein interactions

Translation initiation factors eIF4A and eIF4G form, together with the cap-binding factor eIF4E, the eIF4F complex, which is crucial for recruiting the small ribosomal subunit to the mRNA 5' end and for subsequent scanning and searching for the start codon. eIF4A is an ATP-dependent RNA helicase whose activity is stimulated by binding to eIF4G. We report here the structure of the complex formed by yeast eIF4G's middle domain and full-length eIF4A at 2.6-A resolution. eIF4A shows an extended conformation where eIF4G holds its crucial DEAD-box sequence motifs in a productive conformation, thus explaining the stimulation of eIF4A's activity. A hitherto undescribed interaction involves the amino acid Trp-579 of eIF4G. Mutation to alanine results in decreased binding to eIF4A and a temperature-sensitive phenotype of yeast cells that carry a Trp579Ala mutation as its sole source for eIF4G. Conformational changes between eIF4A's closed and open state provide a model for its RNA-helicase activity.

Selective pharmacological targeting of a DEAD box RNA helicase

RNA helicases represent a large family of proteins implicated in many biological processes including ribosome biogenesis, splicing, translation and mRNA degradation. However, these proteins have little substrate specificity, making inhibition of selected helicases a challenging problem. The prototypical DEAD box RNA helicase, eIF4A, works in conjunction with other translation factors to prepare mRNA templates for ribosome recruitment during translation initiation. Herein, we provide insight into the selectivity of a small molecule inhibitor of eIF4A, hippuristanol. This coral-derived natural product binds to amino acids adjacent to, and overlapping with, two conserved motifs present in the carboxy-terminal domain of eIF4A. Mutagenesis of amino acids within this region allowed us to alter the hippuristanol-sensitivity of eIF4A and undertake structure/function studies. Our results provide an understanding into how selective targeting of RNA helicases for pharmacological intervention can be achieved.

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.

Effects of cognitive behavioral stress management on HIV-1 RNA, CD4 cell counts and psychosocial parameters of HIV-infected persons

OBJECTIVE: To determine the effects of cognitive-behavioral stress management (CBSM) training on clinical and psychosocial markers in HIV-infected persons. METHODS: A randomized controlled trial in four HIV outpatient clinics of 104 HIV-infected persons taking combination antiretroviral therapy (cART), measuring HIV-1 surrogate markers, adherence to therapy and well-being 12 months after 12 group sessions of 2 h CBSM training. RESULTS: Intent-to-treat analyses showed no effects on HIV-1 surrogate markers in the CBSM group compared with the control group: HIV-1 RNA < 50 copies/ml in 81.1% [95% confidence interval (CI), 68.0-90.6] and 74.5% (95% CI, 60.4-85.7), respectively (P = 0.34), and mean CD4 cell change from baseline of 53.0 cells/microl (95% CI, 4.1-101.8) and 15.5 cells/microl (95% CI, -34.3 to 65.4), respectively (P = 0.29). Self-reported adherence to therapy did not differ between groups at baseline (P = 0.53) or at 12 month's post-intervention (P = 0.47). Significant benefits of CBSM over no intervention were observed in mean change of quality of life scores: physical health 2.9 (95% CI, 0.7-5.1) and -0.2 (95% CI, -2.1 to 1.8), respectively (P = 0.05); mental health 4.8 (95% CI, 1.8-7.3) and -0.5 (95% CI, -3.3 to 2.2) (P = 0.02); anxiety -2.1 (95% CI, -3.6 to -1.0) and 0.3 (95% CI, -0.7 to 1.4), respectively (P = 0.002); and depression -2.1 (95% CI, -3.2 to -0.9) and 0.02 (95% CI, -1.0 to 1.1), respectively (P = 0.001). Alleviation of depression and anxiety symptoms were most pronounced among participants with high psychological distress at baseline. CONCLUSION: CBSM training of HIV-infected persons taking on cART does not improve clinical outcome but has lasting effects on quality of life and psychological well-being.

Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization

Quantitative reverse transcriptase real-time PCR (QRT-PCR) is a robust method to quantitate RNA abundance. The procedure is highly sensitive and reproducible as long as the initial RNA is intact. However, breaks in the RNA due to chemical or enzymatic cleavage may reduce the number of RNA molecules that contain intact amplicons. As a consequence, the number of molecules available for amplification decreases. We determined the relation between RNA fragmentation and threshold values (Ct values) in subsequent QRT-PCR for four genes in an experimental model of intact and partially hydrolyzed RNA derived from a cell line and we describe the relation between RNA integrity, amplicon size and Ct values in this biologically homogenous system. We demonstrate that degradation-related shifts of Ct values can be compensated by calculating delta Ct values between test genes and the mean values of several control genes. These delta Ct values are less sensitive to fragmentation of the RNA and are unaffected by varying amounts of input RNA. The feasibility of the procedure was demonstrated by comparing Ct values from a larger panel of genes in intact and in partially degraded RNA. We compared Ct values from intact RNA derived from well-preserved tumor material and from fragmented RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples of the same tumors. We demonstrate that the relative abundance of gene expression can be based on FFPE material even when the amount of RNA in the sample and the extent of fragmentation are not known.

RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells

BACKGROUND: With current treatment strategies, nearly half of all medulloblastoma (MB) patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to cause anaplasia and correlate with unfavorable prognosis. METHODS: To study the role of c-MYC in MB biology, we down-regulated c-MYC expression by using small interfering RNA (siRNA) and investigated changes in cellular proliferation, cell cycle analysis, apoptosis, telomere maintenance, and response to ionizing radiation (IR) and chemotherapeutics in a representative panel of human MB cell lines expressing different levels of c-MYC (DAOY wild-type, DAOY transfected with the empty vector, DAOY transfected with c-MYC, D341, and D425). RESULTS: siRNA-mediated c-MYC down-regulation resulted in an inhibition of cellular proliferation and clonogenic growth, inhibition of G1-S phase cell cycle progression, and a decrease in human telomerase reverse transcriptase (hTERT) expression and telomerase activity. On the other hand, down-regulation of c-MYC reduced apoptosis and decreased the sensitivity of human MB cells to IR, cisplatin, and etoposide. This effect was more pronounced in DAOY cells expressing high levels of c-MYC when compared with DAOY wild-type or DAOY cells transfected with the empty vector. CONCLUSION: In human MB cells, in addition to its roles in growth and proliferation, c-MYC is also a potent inducer of apoptosis. Therefore, targeting c-MYC might be of therapeutic benefit when used sequentially with chemo- and radiotherapy rather than concomitantly.

Prognosis of patients treated with cART from 36 months after initiation, according to current and previous CD4 cell count and plasma HIV-1 RNA measurements

OBJECTIVES: CD4 cell count and plasma viral load are well known predictors of AIDS and mortality in HIV-1-infected patients treated with combination antiretroviral therapy (cART). This study investigated, in patients treated for at least 3 years, the respective prognostic importance of values measured at cART initiation, and 6 and 36 months later, for AIDS and death. METHODS: Patients from 15 HIV cohorts included in the ART Cohort Collaboration, aged at least 16 years, antiretroviral-naive when they started cART and followed for at least 36 months after start of cART were eligible. RESULTS: Among 14 208 patients, the median CD4 cell counts at 0, 6 and 36 months were 210, 320 and 450 cells/microl, respectively, and 78% of patients achieved viral load less than 500 copies/ml at 6 months. In models adjusted for characteristics at cART initiation and for values at all time points, values at 36 months were the strongest predictors of subsequent rates of AIDS and death. Although CD4 cell count and viral load at cART initiation were no longer prognostic of AIDS or of death after 36 months, viral load at 6 months and change in CD4 cell count from 6 to 36 months were prognostic for rates of AIDS from 36 months. CONCLUSIONS: Although current values of CD4 cell count and HIV-1 RNA are the most important prognostic factors for subsequent AIDS and death rates in HIV-1-infected patients treated with cART, changes in CD4 cell count from 6 to 36 months and the value of 6-month HIV-1 RNA are also prognostic for AIDS.

Base pairing and miscoding properties of 1,N(6)-ethenoadenine- and 3,N(4)-ethenocytosine-containing RNA oligonucleotides

Two RNA phosphoramidites containing the bases 1,N(6)-ethenoadenine (εA) and 3,N(4)-ethenocytosine (εC) were synthesized. These building blocks were incorporated into two 12-mer oligoribonucleotides for evaluation of the base pairing properties of these base lesions by UV melting curve (Tm) and circular dichroism measurements. The Tm data of the resulting duplexes with the etheno modifications opposing all natural bases showed a substantial destabilization compared to the corresponding natural duplexes, confirming their inability to form base pairs. The coding properties of these lesions were further investigated by introducing them into 31-mer oligonucleotides and assessing their ability to serve as templates in primer extension reactions with HIV, AMV, and MMLV reverse transcriptases (RT). Primer extension reactions showed complete arrest of the incorporation process using MMLV RT and AMV RT, while HIV RT preferentially incorporates dAMP opposite εA and dAMP as well as dTMP opposite εC. The properties of these RNA lesions are discussed in the context of its putative biological role.

A 6'-Fluoro-Substituent in Bicyclo-DNA Increases Affinity to Complementary RNA Presumably by CF-HC Pseudohydrogen Bonds

The synthesis of a novel bicyclic thymidine analogue carrying a β-fluoro substituent at C6' (6'F-bcT) has been achieved. Key steps of the synthesis were an electrophilic fluorination/stereospecific hydrogenation sequence of a bicyclo sugar intermediate, followed by an N-iodo-succinimide-induced stereoselective nucleosidation. A corresponding phosphoramidite building block was then prepared and used for oligonucleotide synthesis. Tm measurements of oligonucleotides with single and double incorporations showed a remarkable stabilization of duplex formation particularly with RNA as complement without compromising pairing selectivity. Increases in Tm were in the range of +1-2 °C compared to thymidine and +1-3 °C compared to a standard bc-T residue. Structural investigations of the 6'F-bcT nucleoside by X-ray crystallography showed an in-line arrangement of the fluorine substituent with H6 of thymine, however, with a distance that is relatively long for a nonclassical CF-HC hydrogen bond. In contrast, structural investigations in solution by (1)H and (13)C NMR clearly showed scalar coupling of fluorine with H6 and C6 of the nucleobase, indicating the existence of at least weak electrostatic interactions. On the basis of these results, we put forward the hypothesis that these weak CF-HC6 electrostatic interactions increase duplex stability by orienting and partially freezing torsion angle χ of the 6'F-bcT nucleoside.

RNA-Seq-based analysis of the physiologic cold shock-induced changes in Moraxella catarrhalis gene expression

BACKGROUND Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. The prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis is greatest in winter. We investigated how M. catarrhalis uses the physiologic exposure to cold air to regulate pivotal survival systems that may contribute to M. catarrhalis virulence. RESULTS In this study we used the RNA-seq techniques to quantitatively catalogue the transcriptome of M. catarrhalis exposed to a 26 °C cold shock or to continuous growth at 37 °C. Validation of RNA-seq data using quantitative RT-PCR analysis demonstrated the RNA-seq results to be highly reliable. We observed that a 26 °C cold shock induces the expression of genes that in other bacteria have been related to virulence a strong induction was observed for genes involved in high affinity phosphate transport and iron acquisition, indicating that M. catarrhalis makes a better use of both phosphate and iron resources after exposure to cold shock. We detected the induction of genes involved in nitrogen metabolism, as well as several outer membrane proteins, including ompA, m35-like porin and multidrug efflux pump (acrAB) indicating that M. catarrhalis remodels its membrane components in response to downshift of temperature. Furthermore, we demonstrate that a 26 °C cold shock enhances the induction of genes encoding the type IV pili that are essential for natural transformation, and increases the genetic competence of M. catarrhalis, which may facilitate the rapid spread and acquisition of novel virulence-associated genes. CONCLUSION Cold shock at a physiologically relevant temperature of 26 °C induces in M. catarrhalis a complex of adaptive mechanisms that could convey novel pathogenic functions and may contribute to enhanced colonization and virulence.