Transcriptional expression level during exponential growth phase in Kluyveromyces marxianus
ABSTRACT: To understand functional roles of ncRNAs during exponential growth phase, we captured transcriptome changes by conducting strand-specific RNA-seq. Directional RNA-seq (dUTP method by TruSeq®) were generated in two biological replicates by using Illumina MiSeq
Project description:Maize RNA Polymerase D1 (RPD1), the largest subunit of RNA polymerase IV (Pol IV), is required for normal plant development, repression of transposable elements (TEs), and for the regulation of specific alleles associated with TEs. Here, we define the nascent transcriptomes of rpd1 mutant and wild-type (WT) seedlings using global run-on sequencing (GRO-seq) to identify the broader targets of RPD1-based transcriptional regulation. Surprisingly, although TE-like sequences comprise >85% of the maize genome, most TEs are not transcribed at the seedling stage, even in rpd1 mutants. Profile comparisons identify the global set of genes and TEs whose transcription is altered in the absence of RPD1, in some cases in antisense orientation. These results indicate that maize Pol IV specifies Pol II-based transcriptional regulation for certain regions of the maize genome. Nuclei isolated from 10 wild-type and 10 rpd1 mutant seedlings were pooled and used to make two global run-on sequencing libraries.
Project description:The human malaria parasite Plasmodium falciparum has a complex and multi-stage life cycle that requires extensive immune escape, invasion of human liver and blood cells, and transmission through the female Anopholes mosquito. To date, the regulatory elements orchestrating these critical parasite processes remain largely unknown. However, there is mounting evidence across a broad range of species that intergenic long non-coding RNA (lncRNA) and antisense RNA can regulate chromatin state and gene expression. To pursue such functional roles for lncRNAs in P. falciparum, we performed deep, strand-specific RNA sequencing of fifteen non-polyA-selected blood stage samples, and assembled and characterized the properties of 660 intergenic lncRNAs, 474 antisense RNAs, and 1381 circular RNAs (circRNAs). We further validated the non-canonical splice junctions of seven P. falciparum circRNAs, an emerging class of non-coding RNA with regulatory potential and unexplored functional significance in P. falciparum. Our comprehensive analysis of P. falciparum lncRNAs indicates a functional role for these transcripts; P. falciparum intergenic lncRNAs and antisense RNAs are developmentally regulated in a similar periodic fashion to annotated transcripts, and sense-antisense pair expression is significantly anti-correlated. Notable outliers include intergenic lncRNAs that strongly peak in expression during parasite invasion, such as the telomere-associated lncRNA-TARE family, antisense transcripts that drop in expression during parasite invasion, and a highly correlated, multi-exonic, antisense counterpart to P. falciparum Gametocyte Developmental Protein 1 (PfGDV1). Taken together, our results present over two thousand P. falciparum intergenic lncRNA, antisense, and circRNA candidates and highlight promising P. falciparum lncRNAs for future investigation. We harvested fifteen blood stage samples from two biological replicate time-courses. The first time-course comprised of eleven samples that finely map temporal changes during P. falciparum blood stage development. We harvested samples over 56 hours, at roughly 4-hour time intervals, from a tightly synchronized P. falciparum 3D7 parasite population. As the asexual blood stage is an approximately 48-hour cycle, this time-course allowed us to profile gene expression during RBC rupture and parasite invasion. The second time-course comprised of four samples harvested in synchronous P. falciparum 3D7 parasites approximately four hours before and after the ring to trophozoite and trophozoite to schizont morphological stage transitions, which occur during the blood stage at 24 hours post invasion (hpi) and 36 hpi, respectively.
Project description:Functional characterization of the Xap5 protein using epistatic genetic interaction analysis (E-MAP), RNA-seq and ChIP-chip along with other growth experiments. wild type and mutant cells were grown at 37C in EMM50 and RNA was extracted from exponentially growing cells. Strand specific libraries were constructed with random primers. Ribo zero was used to remove r-RNA. For ChIP-chip Agilent 60mer array was used to analyze DNA recovered by chromatin immunoprecipitation of Xap5 from fission yeast culture.
Project description:We use ChIP-seq to identify the targets of Efg1 in Candida parapsilosis. We show that Efg1 binds to 502 promoter regions, including 70 potential transcription factors or regulatory proteins. Several of the transcription factors belong to networks that regulate biofilm development and white-opaque switching in C. albicans. Efg1 also binds to its own promoter. The binding site for C. parapsilosis Efg1 resembles that of orthologs in other fungi. Many Efg1 targets are probably also regulated by the Ndt80 transcription factor. Efg1 in C. parapsilosis was tagged in situ using a myc epitope. Examination of Efg1-myc DNA binding sites by ChIP-seq. Three immunoprecipitated samples were sequenced and three input samples (pre-immunoprecipitation) were sequenced as controls.
Project description:In oligotrophic ocean waters where bacteria are often subjected to chronic nutrient limitation, community transcriptome sequencing has pointed to the presence of highly abundant small RNAs (sRNAs). The role of sRNAs in regulating response to nutrient stress was investigated in a model heterotrophic marine bacterium Ruegeria pomeroyi grown in continuous culture under carbon and nitrogen limitation. RNAseq analysis identified 98 sRNAs, of which 69 were cis-encoded and located antisense to their target genes, and 30 were trans-encoded and linked to predicted target genes through complementarity analysis. The most prevalent functional roles of target genes were transport, cell-cell interactions, signal transduction, and transcriptional regulation. Thirty-two percent of the sRNAs had been identified in a previous study of R. pomeroyi growth on organic sulfur compounds, and may be constitutively expressed, while 69% were not identified in previous studies. Eighty-six percent and were transcribed equally under both carbon and nutrient limitation, and may be involved in a general stress response; 14% were differentially regulated under carbon versus nitrogen stress, and may respond to specific nutrient limitations. A network analysis of the predicted target genes of the R. pomeroyi sRNAs indicated that they average fewer connections than typical protein-encoding genes, and appear to be more important in peripheral or niche-defining functions encoded in the pan genome rather than central metabolism encoded in the core genome.
Project description:We describe a refined approach to identify new human RNA-protein interactions. In vitro transcribed labeled RNA is bound to ~9,400 human recombinant proteins spotted on protein microarrays. This approach identified 137 RNA-protein interactions for 10 human coding and non-coding RNAs, including an interaction between Staufen 1 protein and TP53 mRNA that promoted the latter’s stability. RNA hybridization to protein microarrays allows rapid identification of human RNA-protein interactions on a large scale. Sense and antisense strands for 10 RNA transcripts representing protein coding RNAs TP53, HRAS, MYC, BCL2 and non-coding sequences PWRN1, SOX2OT, OCC1, IGF2RNC, lncRBM26 and DLEU1 were in vitro transcribed, labeled with Cy5 and independently hybridized on human protein microarrays. The labeling process was optimized in order to achieve ~ 3 pmol dye per every microgram RNA with average efficacy of 1 dye molecule for approximately every 850 bp RNA to minimally influence RNA native structure and at the same time yield in signal intensities that were readily visualized.
Project description:Transcript leaders (TLs) can have profound effects on mRNA translation and stability. To map TL boundaries genome-wide, we developed TL-Sequencing (TL-Seq), a technique combining enzymatic capture of m7G-capped mRNA 5'-ends with high-throughput sequencing. TL-Seq identified mRNA start sites for the majority of yeast genes and revealed many examples of intragenic TL heterogeneity. Transcription initiation sites occur in at least 5% of protein-coding regions and are concentrated near the 5'ends of ORFs. These truncated mRNAs are translated, based on ribosome density analysis. Translation Associated TL-Seq (TATL-Seq), which combines TL-Seq with polysome fractionation, revealed substantial differences in translation of alternative TL isoforms. Globally, while some TL features are associated with poor translation and nonsense-mediated mRNA decay (uAUGs, very short length), others (secondary structure, long length) have much less impact than predicted from analyses of individual genes. TL-Seq and TATL-Seq can be applied to any eukaryote to investigate TL-mediated regulation of gene expression. TL-Seq (+/- pyrophosphatase), TATL-Seq (TL-Seq libraries from polysome gradient)
Project description:Background: Mycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular pathogen that can persist inside host macrophages during infection via a diverse range of mechanisms that subvert the host immune response. In the current study, we have analysed and compared the transcriptomes of M. bovis-infected monocyte-derived macrophages (MDM) purified from six Holstein-Friesian females with the transcriptomes of non-infected control MDM from the same animals over a 24 h period using strand-specific RNA sequencing (RNA-seq). In addition, we compare gene expression profiles generated using RNA-seq with those previously generated by us using the high-density Affymetrix® GeneChip® Bovine Genome Array platform from the same MDM-extracted RNA. Results: A mean of 7.2 million reads from each MDM sample mapped uniquely and unambiguously to single Bos taurus reference genome locations. Analysis of these mapped reads showed 2,584 genes (1,392 upregulated; 1,192 downregulated) and 757 putative natural antisense transcripts (558 upregulated; 119 downregulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). Of the differentially expressed genes, 694 were common to both the sense and antisense data sets, with the direction of expression (i.e. up- or downregulation) positively correlated for 693 genes and negatively correlated for the remaining gene. Gene ontology analysis of the differentially expressed genes revealed an enrichment of immune, apoptotic and cell signalling genes. Notably, the number of differentially expressed genes identified from RNA-seq sense strand analysis was greater than the number of differentially expressed genes detected from microarray analysis (2,584 genes versus 2,015 genes). Furthermore, our data reveal a greater dynamic range in the detection and quantification of gene transcripts for RNA-seq compared to microarray technology. Conclusions: This study highlights the value of RNA-seq in identifying novel immunomodulatory mechanisms that underlie host-mycobacterial pathogen interactions during infection, including possible complex post-transcriptional regulation of host gene expression involving antisense RNA. Description of the transcriptomic host response after infection of bovine monocyte-derived macrophages with Mycobacterium bovis
Project description:We performed a pooled GWAS and individual genotyping in 269 children with allergic respiratory diseases comparing allergic children with and without asthma. We used a modular approach to identify the most significant loci associated with asthma by combining silhouette statistics and physical distance method with cluster-adapted thresholding. We found 97% concordance between pooled GWAS and individual genotyping, with 36 out of 37 top-scoring SNPs significant at individual genotyping level. The most significant SNP is located inside the coding sequence of C5, an already identified asthma susceptibility gene, while the other loci regulate functions that are relevant to bronchial physiopathology, as immune- or inflammation-mediated mechanisms and airway smooth muscle contraction. Integration with gene expression data (from mouse experimental asthma model taken from GSE6858 and GSE1301) showed that almost half of the putative susceptibility genes are differentially expressed in experimental asthma mouse models. Affymetrix SNP arrays (Mapping 250K NspI and StyI) were performed according to the manufacturer's directions on pooled DNA extracted from peripheral blood samples.The design is a pooled-GWAS. DNA samples were assigned to the Asthma group if displaying symptoms of asthma, alone or associated to other allergic phenotypes, including rhinoconjunctivitis (RC), and assigned to the RC group if displaying rhinitis or rhinoconjunctivits alone or associated to other allergic phenotypes, excluding asthma. Each of the two groups was subdiveded into 4 independent groups of samples, each containing 31-36 individuals. Individual DNA samples were then added to their respective pools in equivalent molar amounts. Each pool was labeled and hybridized independently on three different arrays (3 technical replicates for each pool).
Project description:RNA helicases are important regulators of gene expression that act by remodeling RNA secondary structures and as RNA-protein interactions. Here, we demonstrate that MOV10 has an ATP-dependent 5' to 3' in vitro RNA unwinding activity and determine the RNA-binding sites of MOV10 and its helicase mutants using PAR-CLIP. We find that MOV10 predominantly binds to 3' UTRs upstream of regions predicted to form local secondary structures and provide evidence that MOV10 helicase mutants are impaired in their ability to translocate 5' to 3' on their mRNA targets. MOV10 interacts with UPF1, the key component of the nonsense-mediated mRNA decay pathway. PAR-CLIP of UPF1 reveals that MOV10 and UPF1 bind to RNA in close proximity. Knockdown of MOV10 resulted in increased mRNA half-lives of MOV10-bound as well as UPF1-regulated transcripts, suggesting that MOV10 functions in UPF1-mediated mRNA degradation as an RNA clearance factor to resolve structures and displace proteins from 3' UTRs. Flp-In T-REx HEK293 cells expressing FLAG/HA-tagged MOV10 WT, MOV10 K530A, MOV10 D645N and UPF1 were sequenced. mRNA half-life data under GSE56751.