Profile of small interfering RNAs from cotton plants infected with the polerovirus Cotton leafroll dwarf virus

<p>Abstract</p> <p>Background</p> <p>In response to infection, viral genomes are processed by Dicer-like (DCL) ribonuclease proteins into viral small RNAs (vsRNAs) of discrete sizes. vsRNAs are then used as guides for silencing the viral genome. The profile of vsRNAs produced during the infection process has been extensively studied for some groups of viruses. However, nothing is known about the vsRNAs produced during infections of members of the economically important family <it>Luteoviridae</it>, a group of phloem-restricted viruses. Here, we report the characterization of a population of vsRNAs from cotton plants infected with Cotton leafroll dwarf virus (CLRDV), a member of the genus <it>Polerovirus</it>, family <it>Luteoviridae</it>.</p> <p>Results</p> <p>Deep sequencing of small RNAs (sRNAs) from leaves of CLRDV-infected cotton plants revealed that the vsRNAs were 21- to 24-nucleotides (nt) long and that their sequences matched the viral genome, with higher frequencies of matches in the 3- region. There were equivalent amounts of sense and antisense vsRNAs, and the 22-nt class of small RNAs was predominant. During infection, cotton <it>Dcl </it>transcripts appeared to be up-regulated, while Dcl2 appeared to be down-regulated.</p> <p>Conclusions</p> <p>This is the first report on the profile of sRNAs in a plant infected with a virus from the family <it>Luteoviridae</it>. Our sequence data strongly suggest that virus-derived double-stranded RNA functions as one of the main precursors of vsRNAs. Judging by the profiled size classes, all cotton DCLs might be working to silence the virus. The possible causes for the unexpectedly high accumulation of 22-nt vsRNAs are discussed. CLRDV is the causal agent of Cotton blue disease, which occurs worldwide. Our results are an important contribution for understanding the molecular mechanisms involved in this and related diseases.</p

Cartography of Methicillin-Resistant S. aureus Transcripts: Detection, Orientation and Temporal Expression during Growth Phase and Stress Conditions

BACKGROUND: Staphylococcus aureus is a versatile bacterial opportunist responsible for a wide spectrum of infections. The severity of these infections is highly variable and depends on multiple parameters including the genome content of the bacterium as well as the condition of the infected host. Clinically and epidemiologically, S. aureus shows a particular capacity to survive and adapt to drastic environmental changes including the presence of numerous antimicrobial agents. Mechanisms triggering this adaptation remain largely unknown despite important research efforts. Most studies evaluating gene content have so far neglected to analyze the so-called intergenic regions as well as potential antisense RNA molecules. PRINCIPAL FINDINGS: Using high-throughput sequencing technology, we performed an inventory of the whole transcriptome of S. aureus strain N315. In addition to the annotated transcription units, we identified more than 195 small transcribed regions, in the chromosome and the plasmid of S. aureus strain N315. The coding strand of each transcript was identified and structural analysis enabled classification of all discovered transcripts. RNA purified at four time-points during the growth phase of the bacterium allowed us to define the temporal expression of such transcripts. A selection of 26 transcripts of interest dispersed along the intergenic regions was assessed for expression changes in the presence of various stress conditions including pH, temperature, oxidative shocks and growth in a stringent medium. Most of these transcripts showed expression patterns specific for the defined stress conditions that we tested. CONCLUSIONS: These RNA molecules potentially represent important effectors of S. aureus adaptation and more generally could support some of the epidemiological characteristics of the bacterium

Specific roles for the Ccr4-Not complex subunits in expression of the genome

In this work we used micro-array experiments to determine the role of each nonessential subunit of the conserved Ccr4-Not complex in the control of gene expression in the yeast Saccharomyces cerevisiae. The study was performed with cells growing exponentially in high glucose and with cells grown to glucose depletion. Specific patterns of gene deregulation were observed upon deletion of any given subunit, revealing the specificity of each subunit's function. Consistently, the purification of the Ccr4-Not complex through Caf40p by tandem affinity purification from wild-type cells or cells lacking individual subunits of the Ccr4-Not complex revealed that each subunit had a particular impact on complex integrity. Furthermore, the micro-arrays revealed that the role of each subunit was specific to the growth conditions. From the study of only two different growth conditions, revealing an impact of the Ccr4-Not complex on more than 85% of all studied genes, we can infer that the Ccr4-Not complex is important for expression of most of the yeast genome

The Ccr4-Not Complex and yTAF1 (yTaf(II)130p/yTaf(II)145p) Show Physical and Functional Interactions

The Saccharomyces cerevisiae Ccr4-Not complex is a global regulator of transcription that is thought to regulate TATA binding protein (TBP) function at certain promoters specifically. In this paper, we show interactions between the essential domain of Not1p, which interacts with Not4p and Not5p, and the N-terminal domain of yTAF1. We isolated a temperature-sensitive nonsense allele of TAF1, taf1-4, which is synthetically lethal at the permissive temperature when combined with not4 and not5 mutants and which produces high levels of a C-terminally truncated yTAF1 derivative. Overexpression of C-terminally truncated yTAF1 is toxic in not4 or not5 mutants, whereas overexpression of full-length yTAF1 suppresses not4. Furthermore, mutations in the autoinhibitory N-terminal TAND domain of yTAF1 suppress not5, and the overexpression of similar mutants does not suppress not4. We find that, like Not5p, yTAF1 acts as a repressor of stress response element-dependent transcription. Finally, we have evidence for stress-regulated occupancy of promoter DNA by Not5p and for Not5p-dependent regulation of yTAF1 association with promoter DNA. Taken together with our finding that Not1p copurifies with glutathione S-transferase-yTaf1 in large complexes, these results provide the first molecular evidence that the Ccr4-Not complex might interact with yTAF1 to regulate its association at promoters, a function that might in turn regulate the autoinhibitory N-terminal domain of yTAF1

Bacterial Infection Drives the Expression Dynamics of microRNAs and Their isomiRs

International audienceThe optimal coordination of the transcriptional response of host cells to infection is essential for establishing appropriate immunological outcomes. In this context, the role of microRNAs (miRNAs) – important epigenetic regulators of gene expression – in regulating mammalian immune systems is increasingly well recognised. However, the expression dynamics of miRNAs, and that of their isoforms, in response to infection remains largely unexplored. Here, we characterized the genome-wide miRNA transcriptional responses of human den-dritic cells, over time, to various mycobacteria differing in their virulence as well as to other bacteria outside the genus Mycobacterium, using small RNA-sequencing. We detected the presence of a core temporal response to infection, shared across bacteria, comprising 49 miRNAs, highlighting a set of miRNAs that may play an essential role in the regulation of basic cellular responses to stress. Despite such broadly shared expression dynamics, we identified specific elements of variation in the miRNA response to infection across bacteria, including a virulence-dependent induction of the miR-132/212 family in response to myco-bacterial infections. We also found that infection has a strong impact on both the relative abundance of the miRNA hairpin arms and the expression dynamics of miRNA isoforms. That we observed broadly consistent changes in relative arm expression and isomiR distribution across bacteria suggests that this additional, internal layer of variability in miRNA responses represents an additional source of subtle miRNA-mediated regulation upon infection. Collectively, this study increases our understanding of the dynamism and role of miRNAs in response to bacterial infection, revealing novel features of their internal variability and identifying candidate miRNAs that may contribute to differences in the pathogenicity of mycobacterial infections

Differential expression of miRNAs in DCs upon infection with a panel of bacteria.

<p>(A) Numbers of significantly differentially expressed miRNAs upon infection at each time point for each bacterium. As we did not have expression profiles for the 48h time point for STP infection, this point is missing from the plot. (B) Heatmap illustrating the hierarchical clustering of experimental conditions based on the mean expression levels of the 50 most variable miRNAs. (C) Overlap of differentially expressed miRNAs between bacteria using two different significance cut-offs. Left-bar shows overlap using a single cut-off of FDR-adjusted p<0.01 and |log₂ fold change|>1, while the right bar shows the overlap using a secondary cut-off where a miRNA was called as significant if the absolute log₂ fold change was less than 1, if it passed the first more stringent fold-change cut-off upon infection with at least one of the six bacteria. (D and E) Venn diagrams showing the overlap of significantly differentially expressed miRNAs between bacteria of the MTBC (D) and between MTB-Rv and all other non-mycobacterial infections (E).</p

Changes in relative miRNA arm expression upon infection.

<p>(A) Detection and relative expression of miRNA hairpin arms. Stacked bar plot shows the proportion of detected miRNA hairpins for which either one or both mature miRNA sequences were detected. Density plot shows the distribution of log₂ expression ratios for hairpins where both arms were expressed. (B) An example of one miRNA, miR-361, which showed a strong infection-dependent change in relative arm expression. Axes show the expression of the 3p and 5p arms of all sequenced individuals at each time point. In each panel, colours denote the different time points, and infection conditions are plotted in separate panels. The displacement of points towards the right of the plot in infected samples at later time points shows that the change in the expression ratio of the two arms is due to the increased expression of the 3p arm that, in some conditions, becomes dominant.</p

Shared and specific miRNA responses to bacterial infection.

<p>(A) Plots of the temporal dynamics of two core response miRNAs. As we did not have expression profiles for the 48h time point for STP infection, this bacterium was excluded from the analysis. (B) Multidimensional scaling analysis (MDS) representing the distances between the temporal miRNA responses to different bacterial infections. Distances were based on the sum of edit distances, for each miRNA, between bacteria using STEM-assigned model temporal profiles. (C) The expression of miR-132-3p increased following infection with MTB-Rv and MTB-Bj but not BCG. Transformation of BCG with RD1 from MTB resulted in a significant increase in miR-132-3p expression, not significantly different to that induced by MTB-Rv. Both MTB-Ra and MTB-Hk showed significantly lower miR-132-3p expression than virulent mycobacteria. (D) The induction of miR-212-3p was significantly higher following BCG::RD1 infection, compared to infection with control BCG, and was not significantly different to the response to MTB-Rv. Though the difference between the induction of this miRNA upon infection with virulent or avirulent MTB strains was not significant, the tendencies observed were consistent with miR-132-3p. Significance was calculated using a Mann-Whitney test (* = p < 0.05).</p