scholarly journals Programmable RNA recognition using a CRISPR-associated Argonaute

2017 ◽  
Author(s):  
Audrone Lapinaite ◽  
Jennifer A. Doudna ◽  
Jamie H. D. Cate
Keyword(s):  
Gene Expression ◽  
Gene Expression Regulation ◽  
Complex Mixture ◽  
Seed Region ◽  
Single Nucleotide ◽  
Guide Rna ◽  
Argonaute Proteins ◽  
Wide Range ◽  
Domains Of Life

ABSTRACTArgonaute proteins (Agos) are present in all domains of life. While the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci and use non-canonical 5’-hydroxyled guide RNAs (gRNAs) for nucleic acid targeting. Here we show that using 5-bromo-2′-deoxyuridine (BrdU) as the 5’ nucleotide of gRNAs stabilizes in vitro reconstituted CRISPR-associated Marinitoga piezophila Argonaute-gRNA complexes (MpAgo RNPs) and significantly improves their specificity and affinity for RNA targets. Using reconstituted MpAgo RNPs with 5’-BrdU modified gRNAs, we mapped the seed region of the gRNA, and identified the nucleotides of the gRNA that play the most significant role in targeting specificity. We also show that these MpAgo RNPs can be programmed to distinguish between substrates that differ by a single nucleotide, using permutations at the 6th and 7th positions in the gRNA. Using these specificity features, we employed MpAgo RNPs to detect specific Adenosine to Inosine edited RNAs in a complex mixture. These findings broaden our mechanistic understanding of the interactions of Argonautes with guide and substrate RNAs, and demonstrate that MpAgo RNPs with 5’-BrdU modified gRNAs can be used as a highly-specific RNA-targeting platform to probe RNA biology.SIGNIFICANCEArgonaute proteins are present in bacteria, archaea and eukaryotes. They play an important role in a wide range of biological processes, from transcriptional and translational gene expression regulation to defense against viruses and silencing of mobile genetic elements. Here we present mechanistic insights into the interactions of the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) with its guide RNA (gRNA) and RNA substrates. By modifying the 5’-nucleotide of the gRNA, we demonstrate that MpAgo-gRNA complexes (RNPs) are easily programmable, have high affinity to fully complementary RNA substrates, and can discriminate by over 300 fold between substrates that differ by only a single nucleotide. These MpAgo RNPs should be useful for probing endogenous RNAs in living cells.

scholarly journals Programmable RNA recognition using a CRISPR-associated Argonaute

2018 ◽  
Vol 115 (13) ◽  
pp. 3368-3373 ◽  
Author(s):  
Audrone Lapinaite ◽  
Jennifer A. Doudna ◽  
Jamie H. D. Cate
Keyword(s):  
Complex Mixture ◽  
Seed Region ◽  
Rna Recognition ◽  
Single Nucleotide ◽  
Guide Rnas ◽  
Rna Targets ◽  
Rna Biology ◽  
Rna Targeting ◽  
Domains Of Life

Argonaute proteins (Agos) are present in all domains of life. Although the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (clustered regularly interspaced short palindromic repeats) loci and use noncanonical 5′-hydroxylated guide RNAs (gRNAs) for nucleic acid targeting. Here we show that using 5-bromo-2′-deoxyuridine (BrdU) as the 5′ nucleotide of gRNAs stabilizes in vitro reconstituted CRISPR-associated Marinitoga piezophila Argonaute–gRNA complexes (MpAgo RNPs) and significantly improves their specificity and affinity for RNA targets. Using reconstituted MpAgo RNPs with 5′-BrdU-modified gRNAs, we mapped the seed region of the gRNA and identified the nucleotides of the gRNA that play the most significant role in targeting specificity. We also show that these MpAgo RNPs can be programmed to distinguish between substrates that differ by a single nucleotide, using permutations at the sixth and seventh positions in the gRNA. Using these specificity features, we employed MpAgo RNPs to detect specific adenosine-to-inosine–edited RNAs in a complex mixture. These findings broaden our mechanistic understanding of the interactions of Argonautes with guide and substrate RNAs, and demonstrate that MpAgo RNPs with 5′-BrdU-modified gRNAs can be used as a highly specific RNA-targeting platform to probe RNA biology.

scholarly journals Synonymous single nucleotide polymorphism in arsenic (+3) methyltransferase of the Western mosquitofish (Gambusia affinis) and its gene expression among field populations

Ecotoxicology ◽  
2021 ◽  
Author(s):  
Daesik Park ◽  
Catherine R. Propper ◽  
Guangning Wang ◽  
Matthew C. Salanga
Keyword(s):  
Gene Expression ◽  
Sequence Variation ◽  
Allelic Variation ◽  
Arsenic Concentration ◽  
Gambusia Affinis ◽  
Contamination Level ◽  
Water Medium ◽  
Single Nucleotide ◽  
Wide Range ◽  
High Arsenic

AbstractNaturally occurring arsenic is toxic at extremely low concentrations, yet some species persist even in high arsenic environments. We wanted to test if these species show evidence of evolution associated with arsenic exposure. To do this, we compared allelic variation across 872 coding nucleotides of arsenic (+3) methyltransferase (as3mt) and whole fish as3mt gene expression from three field populations of Gambusia affinis, from water sources containing low (1.9 ppb), medium-low (3.3 ppb), and high (15.7 ppb) levels of arsenic. The high arsenic site exceeds the US EPA’s Maximum Contamination Level for drinking water. Medium-low and high populations exhibited homozygosity, and no sequence variation across all animals sampled. Eleven of 24 fish examined (45.8%) in the low arsenic population harbored synonymous single nucleotide polymorphisms (SNPs) in exons 4 and/or 10. SNP presence in the low arsenic population was not associated with differences in as3mt transcript levels compared to fish from the medium-low site, where SNPs were noted; however, as3mt expression in fish from the high arsenic concentration site was significantly lower than the other two sites. Low sequence variation in fish populations from sites with medium-low and high arsenic concentrations suggests greater selective pressure on this allele, while higher variation in the low population suggests a relaxed selection. Our results suggest gene regulation associated with arsenic detoxification may play a more crucial role in influencing responses to arsenic than polymorphic gene sequence. Understanding microevolutionary processes to various contaminants require the evaluation of multiple populations across a wide range of pollution exposures.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

Gene Expression Analysis to Investigate Biological Networks Underlying Nasal Inflammatory Dysfunctions Induced by Diesel Exhaust Particles Using an In Vivo System

2019 ◽  
Vol 129 (3) ◽  
pp. 245-255 ◽  
Author(s):  
Hyun Soo Kim ◽  
Byeong-Gon Kim ◽  
Sohyeon Park ◽  
Nahyun Kim ◽  
An-Soo Jang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signaling Pathways ◽  
Diesel Exhaust ◽  
Expression Profiles ◽  
Complex Mixture ◽  
Diesel Exhaust Particles ◽  
The Body ◽  
Exhaust Particles

Objectives: Diesel exhaust particles (DEP)s are notorious ambient pollutants composed of a complex mixture of a carbon core and diverse chemical irritants. Several studies have demonstrated significant relationships between DEP exposure and serious nasal inflammatory response in vitro, but available information regarding underlying networks in terms of gene expression changes has not sufficiently explained potential mechanisms of DEP-induced nasal damage, especially in vivo. Methods: In the present study, we identified DEP-induced gene expression profiles under short-term and long-term exposure, and identified signaling pathways based on microarray data for understanding effects of DEP exposure in the mouse nasal cavity. Results: Alteration in gene expression due to DEP exposure provokes an imbalance of the immune system via dysregulated inflammatory markers, predicted to disrupt protective responses against harmful exogenous substances in the body. Several candidate markers were identified after validation using qRT-PCR, including S100A9, CAMP, IL20, and S100A8. Conclusions: Although further mechanistic studies are required for verifying the utility of the potential biomarkers suggested by the present study, our in vivo results may provide meaningful suggestions for understanding the complex cellular signaling pathways involved in DEP-induced nasal damages.

scholarly journals Single Nucleotide Polymorphisms That Cause Structural Changes in the Cyclic AMP Receptor Protein Transcriptional Regulator of the Tuberculosis Vaccine Strain Mycobacterium bovis BCG Alter Global Gene Expression without Attenuating Growth

2008 ◽  
Vol 76 (5) ◽  
pp. 2227-2234 ◽  
Author(s):  
Debbie M. Hunt ◽  
José W. Saldanha ◽  
John F. Brennan ◽  
Pearline Benjamin ◽  
Molly Strom ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hydrogen Bond ◽  
Cyclic Amp ◽  
Single Nucleotide Polymorphisms ◽  
Mycobacterium Bovis ◽  
Transcriptional Regulator ◽  
Receptor Protein ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide

ABSTRACT Single nucleotide polymorphisms (SNPs) are present in the global transcriptional regulator cyclic AMP (cAMP) receptor protein (CRP) of the attenuated vaccine strain Mycobacterium bovis, bacillus Calmette-Guérin (BCG). We have found that these SNPs resulted in small but significant changes in the expression of a number of genes in M. tuberculosis when a deletion of the Rv3676 CRP was complemented by the BCG allele, compared to complementation by the M. tuberculosis allele. We can explain these changes in gene expression by modeling the structure of the mycobacterial protein on the known structure of CRP from Escherichia coli. Thus, the SNP change in the DNA-binding domain, Lys178, is predicted to form a hydrogen bond with the phosphate backbone of the DNA, as does the equivalent residue in E. coli, whereas Glu178 in M. tuberculosis/M. bovis does not, thus explaining the stronger binding reported for CRP of BCG to CRP-binding sites in mycobacterial DNA. In contrast, the SNP change in the nucleotide binding domain (Leu47Pro) is predicted to result in the loss of one hydrogen bond, which is accommodated by the structure, and would not therefore be expected to cause any change in function relating to cAMP binding. The BCG allele fully complemented the growth defect caused by the deletion of the Rv3676 protein in M. tuberculosis, both in vitro and in macrophage and mouse infections, suggesting that these SNPs do not play any role in the attenuation of BCG. However, they may have allowed BCG to grow better under the in vitro-selective conditions used in its derivation from the M. bovis wild type.

scholarly journals Variability in porcine microRNA genes and its association with mRNA expression phenotypes

2020 ◽  
Author(s):  
Emilio Mármol-Sánchez ◽  
Dailu Guan ◽  
Raquel Quintanilla ◽  
Raul Tonda ◽  
Marcel Amills
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Binding Sites ◽  
Expression Patterns ◽  
Critical Role ◽  
Apical Region ◽  
Seed Region ◽  
Association Analyses ◽  
Mirna Genes ◽  
Wide Range

AbstractBackgroundMature microRNAs (miRNAs) play an important role in repressing the expression of a wide range of protein coding transcripts by promoting their degradation or inhibiting their translation into functional proteins. The presence of segregating polymorphisms inside miRNA loci and their corresponding 3’UTR binding sites might disrupt canonical conserved miRNA-mRNA pairing, thus modifying gene expression patterns.ResultsWe aimed to investigate the variability of miRNA genes and their putative binding sites by analyzing whole-genome sequences from 120 pigs and wild boars from Europe and Asia. In total, 285 SNPs residing within miRNA loci were detected. From these, 221 were located in precursor regions, whereas 52 and 12 mapped to mature and seed regions, respectively. Moreover, a total of 109,724 polymorphisms were identified in predicted 7mer-m8 miRNA binding sites within porcine 3’UTRs. A principal components analysis revealed a clear genetic divergence between Asian and European samples, which was particularly strong for 3’UTR sequences. We also observed that miRNA genes show reduced polymorphism compared with other non-miRNA regions. To assess the potential consequences of miRNA polymorphisms, we sequenced the genomes of 5 Duroc pigs and, by doing so, we identified 15 miRNA SNPs that were genotyped in the offspring (N = 345) of the five boars. Association analyses between miRNA SNPs and hepatic and muscle microarray data allowed us to identify 4 polymorphisms displaying significant associations. Particularly interesting was the rs319154814 polymorphism (G/A), located in the apical loop of the ssc-miR-326 precursor sequence. This polymorphism is predicted to cause a subtle hairpin rearrangement that improves the accessibility to processing enzymatic factors.ConclusionsPorcine miRNA genes show a reduced variability, particularly in the seed region which plays a critical role in miRNA binding. Although it is generally assumed that SNPs mapping to the seed region are the ones with the strongest consequences on mRNA expression, we show that a SNP mapping to the apical region of ssc-miR-326 is associated with the mRNA expression of several of its predicted targets. This result suggests that porcine miRNA variability mapping within and outside the seed region could have important regulatory effects on gene expression.

scholarly journals hTFtarget: a comprehensive database for regulations of human transcription factors and their targets

2019 ◽  
Author(s):  
Qiong Zhang
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Dna Sequences ◽  
Chromatin Immunoprecipitation ◽  
Target Genes ◽  
Gene Expression Regulation ◽  
Epigenetic Modification ◽  
Wide Range ◽  
Chromatin Immunoprecipitation Sequencing ◽  
User Friendly

Transcription factors (TFs) as key regulators play crucial roles in biological processes. The identification of TF-target regulatory relationships is a key step for revealing functions of TFs and their regulations on gene expression. The accumulated data of Chromatin immunoprecipitation sequencing (ChIP-Seq) provides great opportunities to discover the TF-target regulations across different conditions. In this study, we constructed a database named hTFtarget, which integrated huge human TF target resources (7,190 ChIP-Seq samples of 659 TFs and high confident TF binding sites of 699 TFs) and epigenetic modification information to predict accurate TF-target regulations. hTFtarget offers the following functions for users to explore TF-target regulations: 1) Browse or search general targets of a query TF across datasets; 2) Browse TF-target regulations for a query TF in a specific dataset or tissue; 3) Search potential TFs for a given target gene or ncRNA; 4) Investigate co-association between TFs in cell lines; 5) Explore potential co-regulations for given target genes or TFs; 6) Predict candidate TFBSs on given DNA sequences; 7) View ChIP-Seq peaks for different TFs and conditions in genome browser. hTFtarget provides a comprehensive, reliable and user-friendly resource for exploring human TF-target regulations, which will be very useful for a wide range of users in the TF and gene expression regulation community. hTFtarget is available at http://bioinfo.life.hust.edu.cn/hTFtarget.

Activating FLT3 Mutations Display a Wide Range Of Transforming Potential and a Characteristic Pathway Profile Associated With Prognosis In Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1312-1312
Author(s):  
Hanna Janke ◽  
Friederike Schneider ◽  
Daniela Schumacher ◽  
Tobias Herold ◽  
Hopfner Karl-Peter ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Gene Expression Profile ◽  
Myeloid Leukemia ◽  
Normal Karyotype ◽  
Wide Range ◽  
Flt3 Mutations ◽  
Acute Myeloid

Abstract Background Internal tandem duplication (ITD) and pointmutations in the tyrosine kinase domain (TKD) of the receptor tyrosine kinase FLT3 occur in about 30% of patients with acute myeloid leukemia (AML). In contrast to the negative prognostic impact of FLT3-ITD in normal karyotype AML, FLT3 pointmutations occurring in the TKD and juxtamembrane (JM) region are less frequent and of unclear clinical impact. Although TKD mutations can induce resistance to tyrosine kinase inhibitors the individual transforming potential of FLT3 pointmutations has not been analysed in detail. In this study we have performed a comprehensive analysis of various FLT3 mutants in a comparative setting in vitro and analyzed gene expression profiles, and clinical outcome with respect to FLT3mutation status. Material and Methods We analyzed relapse and survival in 672 cytogenetically normal AML patients and the FLT3 status at diagnosis and relapse in 156 patients. In the murine Ba/F3 cell model we analyzed the transforming potential, subcellular localization, phosphorylation status and signaling properties of eight different FLT3 mutants. The investigated FLT3 mutations include three ITD of different length and insertion site, V592A in the JM region, common FLT3-TKD mutations D835V and D835Y as well as D839G and I867S in the second TKD. FLT3-D839G and -I867S were recently found in AML patients by our group during routine diagnostics but have not been functionally characterized before. The corresponding remission samples did not express these mutations. Further a gene expression profile analysis with respect to FLT3-ITD and -TKD mutation status and evaluation of differences in activation of predefined STAT5 target gene set was performed. Results In 672 normal karyotype AML patients FLT3-ITD, but not FLT3-TKD mutations were associated with an inferior relapse free and overall survival in multivariate analysis. In paired diagnosis-relapse samples FLT3-ITD showed higher stability (70%) compared to FLT3-TKD (30%). In vitro, FLT3-ITD induced a fully transformed phenotype in Ba/F3 cells, whereas FLT3 pointmutations showed a weaker but clearly transformed phenotype with gradual increase in proliferation and protection from apoptosis. The transforming capacity of the investigated mutants was associated with cell surface expression and tyrosine 591 phosphorylation of the FLT3 receptor. Western blot experiments revealed STAT5 activation only in FLT3-ITD transformed cells, further gene expression profile analyses displayed differences in predefined STAT5 target genes between FLT3-ITD and FLT3-TKD mutations. In contrast, FLT3-non-ITD mutants had an enhanced signal of AKT and MAPK activation. Further differences were found on mRNA level presenting deregulation of SOCS2, ENPP2, PRUNE2 and ART3 expression between FLT3-ITD, FLT3-TKD and FLT3-WT. Conclusion Although apparently divergent in response to treatment all functionally characterized mutants showed a clear gain-of-function phenotype with a wide range of transforming activity associated with clinical prognosis and signaling. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Survival of and In Situ Gene Expression by Vibrio vulnificus at Varying Salinities in Estuarine Environments

2007 ◽  
Vol 74 (1) ◽  
pp. 182-187 ◽  
Author(s):  
Melissa K. Jones ◽  
Elizabeth Warner ◽  
James D. Oliver
Keyword(s):  
Gene Expression ◽  
Vibrio Vulnificus ◽  
Natural Habitat ◽  
Term Survival ◽  
Long Term Survival ◽  
Wide Range ◽  
Opportunistic Human Pathogen

ABSTRACT The opportunistic human pathogen Vibrio vulnificus survives in a wide range of ecological environments, which demonstrates its ability to adapt to highly variable conditions. Survival and gene expression under various conditions have been extensively studied in vitro; however, little work has been done to evaluate this bacterium in its natural habitat. Therefore, this study monitored the long-term survival of V. vulnificus in situ and simultaneously evaluated the expression of stress (rpoS, relA, hfq, and groEL) and putative virulence (vvpE, smcR, viuB, and trkA) genes at estuarine sites of varying salinity. Additionally, the survival and gene expression of an rpoS and an oxyR mutant were examined under the same conditions. Differences between the sampling sites in the long-term survival of any strain were not seen. However, differences were seen in the expression of viuB, trkA, and relA but our findings differed from what has been previously shown in vitro. These results also routinely demonstrated that genes required for survival under in vitro stress or host conditions are not necessarily required for survival in the water column. Overall, this study highlights the need for further in situ evaluation of this bacterium in order to gain a true understanding of its ecology and how it relates to its natural habitat.

scholarly journals Regulation and Characterization of Two Nitroreductase Genes, nprA and nprB, of Rhodobacter capsulatus

2005 ◽  
Vol 71 (12) ◽  
pp. 7643-7649 ◽  
Author(s):  
Eva Pérez-Reinado ◽  
Rafael Blasco ◽  
Francisco Castillo ◽  
Conrado Moreno-Vivián ◽  
M. Dolores Roldán
Keyword(s):  
Gene Expression ◽  
Salicylic Acid ◽  
Rhodobacter Capsulatus ◽  
Sequence Data ◽  
Regulatory System ◽  
Wide Range ◽  
Nitrofuran Derivatives ◽  
Response To Stress

ABSTRACT Among photosynthetic bacteria, strains B10 and E1F1 of Rhodobacter capsulatus photoreduce 2,4-dinitrophenol (DNP), which is stoichiometrically converted into 2-amino-4-nitrophenol by a nitroreductase activity. The reduction of DNP is inhibited in vivo by ammonium, which probably acts at the level of the DNP transport system and/or physiological electron transport to the nitroreductase, since this enzyme is not inhibited by ammonium in vitro. Using the complete genome sequence data for strain SB1003 of R. capsulatus, two putative genes coding for possible nitroreductases were isolated from R. capsulatus B10 and disrupted. The phenotypes of these mutant strains revealed that both genes are involved in the reduction of DNP and code for two major nitroreductases, NprA and NprB. Both enzymes use NAD(P)H as the main physiological electron donor. The nitroreductase NprA is under ammonium control, whereas the nitroreductase NprB is not. In addition, the expression of the nprB gene seems to be constitutive, whereas nprA gene expression is inducible by a wide range of nitroaromatic and heterocyclic compounds, including several dinitroaromatics, nitrofuran derivatives, CB1954, 2-aminofluorene, benzo[a]pyrene, salicylic acid, and paraquat. The identification of two putative mar/sox boxes in the possible promoter region of the nprA gene and the induction of nprA gene expression by salicylic acid and 2,4-dinitrophenol suggest a role in the control of the nprA gene for the two-component MarRA regulatory system, which in Escherichia coli controls the response to some antibiotics and environmental contaminants. In addition, upregulation of the nprA gene by paraquat indicates that this gene is probably a member of the SoxRS regulon, which is involved in the response to stress conditions in other bacteria.

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