Unknown,Transcriptomics,Genomics,Proteomics

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Global signatures of protein binding on structural RNAs in Saccharomyces cerevisiae

ABSTRACT: Protein binding is essential to the transport, decay and regulation of almost all RNA molecules. However, the structural preference of protein binding on RNAs and their cellelar functions and dynamics upon changing environmental condictions are poorly understood. Here, we integrated various high-throughput data and introduced a computational framework to describe the global interactions between RNA binding proteins (RBPs) and structured RNAs in yeast at single-nucleotide resolution. We found that on average, in terms of percent total lengths, ~15% of mRNA untranslated regions (UTRs), ~37% of canonical ncRNAs and ~11% of long ncRNA (lncRNAs) are bound by proteins. The RBP binding sites, in general, tend to occur at single-stranded loops, with evolutionarily conserved signatures, and often facilitate a specific RNA structure conformation in vivo. We found that four nucleotide modifications of tRNA are significantly associated with RBP binding. We also identified various structural motifs bound by RBPs in the UTRs of mRNAs, associated with localization, degradation and stress responces. Moreover, we identified >200 novel lncRNAs bound by RBPs, and about half of them contain conserved secondary structures. We present the first ensemble pattern of RBP binding sites in the structured noncoding regions of a eukaryotic genome, emphasizing their structural context and cellular functions. Duplicate gPAR-CLIP libraries were sequenced from yeast strains for each of three conditions: log-phase growth, growth after 2 hour glucose starvation, and growth after 2 hour nitrogen starvation. polyA RNAs were isolated for all conditions. Total RNA were isolated from log phase growth conditions. Sucrose gradient fractionation was performed: some RNAs were isolated from the "light" fraction (lighter than 40S ribosome) and some from the "heavy" fraction. gPAR-CLIP libraries were used to determine regions of RNA bound by proteins.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Mallory Freeberg

PROVIDER: E-GEOD-48888 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Protein-RNA interactions are integral components of nearly every aspect of biology including regulation of gene expression, assembly of cellular architectures, and pathogenesis of human diseases. However, studies in the past few decades have only uncovered a small fraction of the vast landscape of the protein-RNA interactome in any organism, and even less is known about the dynamics of protein-RNA interactions under changing developmental and environmental conditions. Here, we describe the gPAR-CLIP (global photoactivatable-ribonucleoside-enhanced crosslinking and immunopurification) approach for capturing regions of the transcriptome bound by RNA-binding proteins (RBPs) in budding yeast. We report over 13,000 RBP crosslinking sites in untranslated regions (UTR) covering 72% of protein-coding transcripts encoded in the genome, confirming 3M-bM-^@M-^Y UTRs as major sites for RBP interaction. Comparative genomic analyses reveal that RBP crosslinking sites are highly conserved, and RNA folding predictions indicate that secondary structural elements are constrained by protein binding and may serve as generalizable modes of RNA recognition. Finally, 38% of 3M-bM-^@M-^Y UTR crosslinking sites show changes in RBP occupancy upon glucose or nitrogen deprivation, with major impacts on metabolic pathways as well as mitochondrial and ribosomal gene expression. Our study offers an unprecedented view of the pervasiveness and dynamics of protein-RNA interactions in vivo. Duplicate gPAR-CLIP and mRNA-seq libraries were sequenced from yeast strains for each of three conditions: log-phase growth, growth after 2 hour glucose starvation, and growth after 2 hour nitrogen starvation. Additional duplicate mRNA-seq libraries were sequenced from yeast strains grown in the absence of 4-thiouracil. gPAR-CLIP libraries were used to determine regions of mRNA bound by proteins. mRNA-seq libraries served as controls for mRNA abundance. A Puf3p PAR-CLIP library was sequenced to determine how well gPAR-CLIP captured the binding signatures of a single RNA-binding protein.

Project description:Carbon dioxide (CO2) sensing, transport, and metabolism play a pivotal role in survival and proliferation of pathogenic microbes infecting human host from natural environments due to the drastic difference of CO2 levels. Carbonic anhydrases (CAs) are not only key CO2-metabolic enzymes catalyzing reversible interconversion between CO2 and bicarbonate (HCO3-), but also important CO2-signaling modulators. Cryptococcus neoformans that causes fatal fungal meningitis contains two functional CAs, Can1 and Can2, among which Can2 plays essential roles for growth and sexual differentiation of the pathogen. However, downstream genes and signaling network regulated by CAs have not been studied thus far. In this study, we constructed a C. neoformans strain where CAN2 expression is controlled by the CTR4 (copper transporter) promoter and elucidated its transcriptome patterns by using DNA microarray analysis to elucidate downstream target genes of Can2. Expectedly, the CAN2 promoter replacement strain showed growth defects in a CO2-dependent manner when CAN2 is repressed. The CA-transcriptome analysis discovered a number of Can2-dependent genes, including those involved in fatty acid biosynthesis (FAS1), sexual differentiation (GPB1), capsule structure organization (CAS3), iron-metabolism (CFO2), and a number of stress-regulated genes, including an oxidative stress-responsive Atf1 transcription factor, although a majority of them do not have any orthologs in other fungi. The present study revealed other roles of Atf1 in capsule and melanin production and diverse stress responses, including thermotolerance and antifungal drug resistance, of C. neoformans. This study provides further insights into the signaling network of CA/CO2-sensing pathway in pathogenic fungi. 17 slides are used in this analysis, 3 or 2 biological replicate experiments are performed, total RNAs are extracted from 2 strains (H99 Wild type strain, CTR4::CAN2) at 2 time points (0hr time, 12hr time) in 2 conditions (BCS, CuSO4). We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye. Several experiment samples are dye swaped.

Project description:BACKGROUND: Lithospermum erythrorhizon, popularly known as Gromwell, is a perennial herb belonging to the family of Boraginaceae and its extract is known to have diverse pharmacological, cosmetic, and nutritional values for human. Nevertheless the biological influence of Gromwell extract on general physiology of eukaryotic cells remains unknown. In this study, we for the first time performed a global transcriptome analysis by using DNA microarray analysis to identify genes affected by the addition of Gromwell extract (GE) by using a eukaryotic microorganism, basidiomycete fungus Cryptococcus neoformans, as a model system. RESULTS: The transcriptome analysis revealed that a total of 1932 genes are differentially regulated in a statistically significant manner and expressions of 715 genes are up- (315 genes) or down-regulated (457 genes) more than twofold upon GE treatment. In response to GE treatment, genes involved in signal transduction genes are immediately regulated and the evolutionarily conserved set of genes involved in the core cellular functions, including DNA replication, RNA transcription/processing, and protein translation/processing, are generally upregulated. In contrast, a number of genes involved in carbohydrate metabolism and transport, inorganic ion transport and metabolism, post-translational modification/protein turnover/chaperone functions, and signal transduction are downregulated by the GE treatment. Among the GE-responsive genes that are also evolutionarily conserved in the human genome, we confirmed expression patterns of YSA1, TPO2, FET5 (CFO1), and PZF1 by Northern blot analysis. Based on the functional characterization of some GE-responsive genes through phenotypic analysis of gene knockout mutants, we found that GE treatment may promote cellular tolerance against a variety of environmental stresses in eukaryotes. CONCLUSIONS: A significant portion of the Cryptococcus genome is affected in expression levels by treatment of GE, implying that the general physiology of eukaryotic cells is significantly affected by the GE treatment. There are more than 95% of genome homology between JEC21 and H99. Therefore 6 slides of JEC21 (Cryptococcus neoformans var. neoformans serotype D) 70-mer oligo are used in this analysis, 3 biological replicate experiments are performed, total RNAs are extracted from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A)) with 6.4 mg/ml Gromwell extract in 0, 10, 30, 60 min. We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy3 as Sample dye and Cy5 as a control dye.

Project description:WD40 motif-containing Msi1-like (MSIL) proteins play pleiotropic cellular functions as a negative regulator of the Ras/cAMP-pathways and a component of chromatin assembly factor-I (CAF-I), and yet have not been studied in fungal pathogens. Here we identified and characterized an MSIL protein, Msl1, in Cryptococcus neoformans, which can cause fatal meningoencephalitis in humans. Notably, Msl1 was not a functional ortholog for the yeast Msi1 but played pleiotropic roles in C. neoformans in both cAMP-dependent and -independent manners but mainly Ras-independently. Msl1 negatively controlled antioxidant melanin production and sexual differentiation, which can be repressed by inhibiting the cAMP-signaling pathways. In contrast, Msl1 controlled thermotolerance, diverse stress responses, and antifungal drugs resistance in Ras/cAMP-independent manners. Cac2, which is the second CAF-I component, appeared to play both redundant and distinct function with Msl1. Msl1 is required for full virulence of C. neoformans. Transcriptome and proteomic analysis identified a group of Msl1-regulated genes or -interacting proteins, respectively, which mostly include stress-related genes, including HSP12, HSP78, SSA1, SSA4, and STM1. Furthermore, we identified the third putative component of CAF-1, Rlf2, in C. neoformans. In conclusion, this study demonstrated the pleiotropic roles of Msl1 in human fungal pathogen C. neoformans, providing a novel antifungal therapeutic target. There is more than 95% genome homology between JEC21 and H99. Therefore, 6 slides of JEC21 (Cryptococcus neoformans var. neoformans serotype D) 70-mer oligos are used in this analysis. Total RNAs are extracted from 2 strains from H99 (H99 wild-type strain (Cryptococcus neoformans var. grubii serotype A), msl1M-NM-^T). 3 biological replicate experiments are performed for each strain. We use the mix of all total RNAs from this experiment as the control RNA. We use Cy3 as the test sample dye and Cy5 as the control dye.

Project description:The high osmolarity glycerol response (HOG) pathway plays a pivotal role in the stress response, virulence regulation, and differentiation of fungi, including Cryptococcus neoformans that causes fatal meningoencephalitis. Core signaling components of in the HOG pathway, including the Tco-Ypd1-Ssk1 phosphorelay system and the Ssk2-Pbs2-Hog1 MAPK module, have been elucidated but its downstream transcription factors remain unclear. Here we demonstrated that Atf1 with a basic leucine zipper domain is the transcription factor downstream of Hog1 in C. neoformans. We found that ATF1 expression was differentially regulated by oxidative damaging agents, mainly in a Hog1-dependent, but Mpk1-independent, manner. Interestingly, Atf1 not only promoted oxidative stress response and adaptation, but also played an opposing role to Hog1 in the process. Atf1 primarily localized to the nucleus under both unstressed and oxidative stress conditions in a Hog1-independent manner. Our data demonstrated that Atf1 promoted pheromone production and sexual differentiation under negative control by Hog1. Finally, a DNA microarray-based transcriptome analysis of the atf1M-bM-^HM-^F mutant under unstressed and oxidative stress conditions revealed that Atf1 regulated oxidative stress response genes, including a sulfiredoxin gene (SRX1). Intriguing, the array data further demonstrated that Atf1 modulated basal expression of genes involved in DNA repair and genotoxic stress response. Supporting this, we found that the atf1M-bM-^HM-^F mutant was highly sensitive to genotoxic agents. In conclusion, this study provided a further insight into the Hog1-dependent oxidative and genotoxic stress response and differentiation mechanism of C. neoformans. There are more than 95% of genome homology between JEC21 and H99. Therefore 6 slides of JEC21 (Cryptococcus neoformans var. neoformans serotype D) 70-mer oligo are used in this analysis, 3 biological replicate experiments are performed, total RNAs are extracted 2 conditions (with or without treatment of Hydroten peroxide) from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), and atf1M-NM-^T). We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy3 as Sample dye and Cy5 as a control dye.

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Global signatures of protein binding on structural RNAs in Saccharomyces cerevisiae

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