Multi-targeted priming for genome-wide gene expression assays

<p>Abstract</p> <p>Background</p> <p>Complementary approaches to assaying global gene expression are needed to assess gene expression in regions that are poorly assayed by current methodologies. A key component of nearly all gene expression assays is the reverse transcription of transcribed sequences that has traditionally been performed by priming the poly-A tails on many of the transcribed genes in eukaryotes with oligo-dT, or by priming RNA indiscriminately with random hexamers. We designed an algorithm to find common sequence motifs that were present within most protein-coding genes of <it>Saccharomyces cerevisiae </it>and of <it>Neurospora crassa</it>, but that were not present within their ribosomal RNA or transfer RNA genes. We then experimentally tested whether degenerately priming these motifs with multi-targeted primers improved the accuracy and completeness of transcriptomic assays.</p> <p>Results</p> <p>We discovered two multi-targeted primers that would prime a preponderance of genes in the genomes of <it>Saccharomyces cerevisiae </it>and <it>Neurospora crassa </it>while avoiding priming ribosomal RNA or transfer RNA. Examining the response of <it>Saccharomyces cerevisiae </it>to nitrogen deficiency and profiling <it>Neurospora crassa </it>early sexual development, we demonstrated that using multi-targeted primers in reverse transcription led to superior performance of microarray profiling and next-generation RNA tag sequencing. Priming with multi-targeted primers in addition to oligo-dT resulted in higher sensitivity, a larger number of well-measured genes and greater power to detect differences in gene expression.</p> <p>Conclusions</p> <p>Our results provide the most complete and detailed expression profiles of the yeast nitrogen starvation response and <it>N. crassa </it>early sexual development to date. Furthermore, our multi-targeting priming methodology for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences, facilitating a more complete and precise assay of the transcribed sequences within the genome.</p

Tasting Soil Fungal Diversity with Earth Tongues: Phylogenetic Test of SATé Alignments for Environmental ITS Data

An abundance of novel fungal lineages have been indicated by DNA sequencing of the nuclear ribosomal ITS region from environmental samples such as soil and wood. Although phylogenetic analysis of these novel lineages is a key component of unveiling the structure and diversity of complex communities, such analyses are rare for environmental ITS data due to the difficulties of aligning this locus across significantly divergent taxa. One potential approach to this issue is simultaneous alignment and tree estimation. We targeted divergent ITS sequences of the earth tongue fungi (Geoglossomycetes), a basal class in the Ascomycota, to assess the performance of SATé, recent software that combines progressive alignment and tree building. We found that SATé performed well in generating high-quality alignments and in accurately estimating the phylogeny of earth tongue fungi. Drawing from a data set of 300 sequences of earth tongues and progressively more distant fungal lineages, 30 insufficiently identified ITS sequences from the public sequence databases were assigned to the Geoglossomycetes. The association between earth tongues and plants has been hypothesized for a long time, but hard evidence is yet to be collected. The ITS phylogeny showed that four ectomycorrhizal isolates shared a clade with Geoglossum but not with Trichoglossum earth tongues, pointing to the significant potential inherent to ecological data mining of environmental samples. Environmental sampling holds the key to many focal questions in mycology, and simultaneous alignment and tree estimation, as performed by SATé, can be a highly efficient companion in that pursuit

Global gene expression and knockout analysis reveals genes associated with fungal fruiting body development in Neurospora crassa

ungi can serve as highly tractable models for understanding genetic basis of sexual development in multicellular organisms. Applying a reverse-genetic approach to advance such a model, we used random and multitargeted primers to assay gene expression across perithecial development in Neurospora crassa. We found that functionally unclassified proteins accounted for most upregulated genes, whereas downregulated genes were enriched for diverse functions. Moreover, genes associated with developmental traits exhibited stage-specific peaks of expression. Expression increased significantly across sexual development for mating type gene mat a-1 and for mat A-1 specific pheromone precursor ccg-4. In addition, expression of a gene encoding a protein similar to zinc finger, stc1, was highly upregulated early in perithecial development, and a strain with a knockout of this gene exhibited arrest at the same developmental stage. A similar expression pattern was observed for genes in RNA silencing and signaling pathways, and strains with knockouts of these genes were also arrested at stages of perithecial development that paralleled their peak in expression. The observed stage specificity allowed us to correlate expression upregulation and developmental progression and to identify regulators of sexual development. Bayesian networks inferred from our expression data revealed previously known and new putative interactions between RNA silencing genes and pathways. Overall, our analysis provides a fine-scale transcriptomic landscape and novel inferences regarding the control of the multistage development process of sexual crossing and fruiting body development in N. crassa

Metabolism and Development during Conidial Germination in Response to a Carbon-Nitrogen-Rich Synthetic or a Natural Source of Nutrition in Neurospora crassa

One of the most remarkable successes of life is its ability to flourish in response to temporally and spatially varying environments. Fungi occupy diverse ecosystems, and their sensitivity to these environmental changes often drives major fungal life history decisions, including the major switch from vegetative growth to asexual or sexual reproduction. Spore germination comprises the first and simplest stage of vegetative growth. We examined the dependence of this early life history on the nutritional environment using genome-wide transcriptomics. We demonstrated that for developmental regulatory genes, expression was generally conserved across nutritional environments, whereas metabolic gene expression was highly labile. The level of activation of developmental genes did depend on current nutrient conditions, as did the modularity of metabolic and developmental response network interactions. This knowledge is critical to the development of future technologies that could manipulate fungal growth for medical, agricultural, or industrial purposes.Fungal spores germinate and undergo vegetative growth, leading to either asexual or sexual reproductive dispersal. Previous research has indicated that among developmental regulatory genes, expression is conserved across nutritional environments, whereas pathways for carbon and nitrogen metabolism appear highly responsive—perhaps to accommodate differential nutritive processing. To comprehensively investigate conidial germination and the adaptive life history decision-making underlying these two modes of reproduction, we profiled transcription of Neurospora crassa germinating on two media: synthetic Bird medium, designed to promote asexual reproduction; and a natural maple sap medium, on which both asexual reproduction and sexual reproduction manifest. A later start to germination but faster development was observed on synthetic medium. Metabolic genes exhibited altered expression in response to nutrients—at least 34% of the genes in the genome were significantly downregulated during the first two stages of conidial germination on synthetic medium. Knockouts of genes exhibiting differential expression across development altered germination and growth rates, as well as in one case causing abnormal germination. A consensus Bayesian network of these genes indicated especially tight integration of environmental sensing, asexual and sexual development, and nitrogen metabolism on a natural medium, suggesting that in natural environments, a more dynamic and tentative balance of asexual and sexual development may be typical of N. crassa colonies

A Locus Encompassing the Epstein-Barr Virus <i>bglf4</i> Kinase Regulates Expression of Genes Encoding Viral Structural Proteins

<div><p>The mechanism regulating expression of late genes, encoding viral structural components, is an unresolved problem in the biology of DNA tumor viruses. Here we show that BGLF4, the only protein kinase encoded by Epstein-Barr virus (EBV), controls expression of late genes independent of its effect on viral DNA replication. Ectopic expression of BGLF4 in cells lacking the kinase gene stimulated the transcript levels of six late genes by 8- to 10-fold. Introduction of a BGLF4 mutant that eliminated its kinase activity did not stimulate late gene expression. In cells infected with wild-type EBV, siRNA to BGLF4 (siG4) markedly reduced late gene expression without compromising viral DNA replication. Synthesis of late products was restored upon expression of a form of BGLF4 resistant to the siRNA. Studying the EBV transcriptome using mRNA-seq during the late phase of the lytic cycle in the absence and presence of siG4 showed that BGLF4 controlled expression of 31 late genes. Analysis of the EBV transcriptome identified BGLF3 as a gene whose expression was reduced as a result of silencing BGLF4. Knockdown of BGLF3 markedly reduced late gene expression but had no effect on viral DNA replication or expression of BGLF4. Our findings reveal the presence of a late control locus encompassing BGLF3 and BGLF4 in the EBV genome, and provide evidence for the importance of both proteins in post-replication events that are necessary for expression of late genes.</p></div

Impact of knockdown of BGLF4 on the EBV transcriptome.

<p>RNA-seq analysis compared EBV transcripts expressed in 2089 cells transfected with ZEBRA versus ZEBRA plus siG4. RNA was prepared at 48 h after transfection. The plotted graph was generated by dividing the number of normalized reads of a particular transcript in the presence of siG4 by the number of reads obtained in the absence of siG4. This ratio represents the effect of BGLF4 on the abundance of a particular transcript and is presented as a log base 2-fold. A negative fold-change indicated dependency on BGLF4 for expression. Late genes illustrated in red bars while latent and other lytic genes were illustrated in blue bars.</p

Silent mutations in BGLF4 override the effect of siG4 on late gene expression.

<p>(A) Western blot analysis of 2089 cell lysates transfected with the indicated expression vectors. Two concentrations of siRNA specific to BGLF4 (siG4), 10 and 20 nM, were co-transfected with expression vectors encoding ZEBRA and wild-type BGLF4 or kinase dead BGLF4(K102I) (lanes 4, 5, 10 and 11). To demonstrate the specificity of siG4, silent mutations were introduced into the kinase active (rG4) and kinase dead (rG4(K102I)) <i>bglf4</i> genes that rendered the BGLF4 transcript resistant to siG4. Lysates of cells harvested 48 h after transfection were analyzed by immunoblotting using antibodies specific to BGLF4, ZEBRA and the late BFRF3 protein (FR3). (B) Quantitative PCR to measure the extent of EBV genome amplification in each condition.</p

Expression of siG4-resistant BGLF4 restores synthesis of EBV late transcripts.

<p>The effect of siG4 on the level of two early and six late viral transcripts was analyzed by RT-qPCR. The early transcripts are BMRF1 (polymerase processivity factor) and BRLF1 (transcription factor). The late transcripts are BLLF1 (gp350/gp220 major glycoprotein), BFRF3 (minor capsid protein), BLRF2 (tegument protein), BDLF1 (triplex capsid protein), BdRF1 (scaffold protein), and BcLF1 (major capsid protein). The relative abundance of each transcript was calculated using the ΔΔ<i>C_T</i> method.</p

Expression of BGLF4 significantly up-regulates the level of late transcripts.

<p>The abundance of five EBV lytic transcripts was assessed, using quantitative RT-PCR, in delta G4/G5 cells transfected with the following plasmids: empty vector (CMV), ZEBRA (Z), BGLF4, ZEBRA plus BGLF4, mutant BGLF4(K102I), and ZEBRA plus mutant BGLF4(K102I). BMRF1 encodes an early transcript encoding the DNA polymerase processivity factor. BFRF3, BcLF1, BLLF1 and BLRF2 are late transcripts that encode the minor viral capsid, the major capsid protein, gp350/220 major glycoprotein, and a tegument protein, respectively. The level of each of the five transcripts was normalized to empty vector control.</p