RNA-seq of S. meliloti strain ectopically overexpressing trpL and RIP-seq with 3xFLAG-tagged peptide peTrpL from S. meliloti
Ontology highlight
ABSTRACT:
ORGANISM(S): Sinorhizobium meliloti
PROVIDER: GSE118689 | GEO | 2020/05/18
REPOSITORIES: GEO
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Project description:RNA-seq of S. meliloti strain ectopically overexpressing trpL and RIP-seq with 3xFLAG-tagged peptide peTrpL from S. meliloti
| PRJNA486460 | ENA
Project description:We show that specific RNAs are coimmunoprecipitated in a tetracycline-dependent manner with the Flag-tagged peptide peTrpL of S. meliloti
2020-05-18 | GSE118688 | GEO
Project description:We show that the RNA leves of many genes in the S. meliloti overexpressing strain (OE) 2011 (pRK-RcsR1) are more similar to the levels of in wild type (WT) strain 2011 than in the empty vector control (EVC) strain 2011(pRK4352). Since the plasmid-containing strains were grown in the presence of 10 µg/ml tetracycline (Tc), while the WT was grown without Tc, the results suggest that the intrecellular Tc concentration in the OE strain are lower than in the EVC. This is explained by an increased efflux pump activity in the OE strain.
2020-05-18 | GSE118687 | GEO
Project description:Analysis of RNA coimmunoprecipitated with 3xFLAG-tagged peptide peTrpL from Sinorhizobium meliloti
| PRJNA486462 | ENA
Project description:Transcriptome analysis of Sinorhizobium meliloti strain ectopically overexpressing trpL
| PRJNA486461 | ENA
Project description:Associations between leguminous plants and symbiotic nitrogen-fixing rhizobia are a classic example of mutualism between a eukaryotic host and a specific group of prokaryotic microbes. Although this symbiosis is in part species specific, different rhizobial strains may colonize the same nodule. Some rhizobial strains are commonly known as better competitors than others, but detailed analyses that aim to predict rhizobial competitive abilities based on genomes are still scarce. Here, we performed a bacterial genome-wide association (GWAS) analysis to define the genomic determinants related to the competitive capabilities in the model rhizobial species Sinorhizobium meliloti. For this, 13 tester strains were green fluorescent protein (GFP) tagged and assayed versus 3 red fluorescent protein (RFP)-tagged reference competitor strains (Rm1021, AK83, and BL225C) in a Medicago sativa nodule occupancy test. Competition data and strain genomic sequences were employed to build a model for GWAS based on k-mers. Among the k-mers with the highest scores, 51 k-mers mapped on the genomes of four strains showing the highest competition phenotypes (>60% single strain nodule occupancy; GR4, KH35c, KH46, and SM11) versus BL225C. These k-mers were mainly located on the symbiosis-related megaplasmid pSymA, specifically on genes coding for transporters, proteins involved in the biosynthesis of cofactors, and proteins related to metabolism (e.g., fatty acids). The same analysis was performed considering the sum of single and mixed nodules obtained in the competition assays versus BL225C, retrieving k-mers mapped on the genes previously found and on vir genes. Therefore, the competition abilities seem to be linked to multiple genetic determinants and comprise several cellular components. IMPORTANCE Decoding the competitive pattern that occurs in the rhizosphere is challenging in the study of bacterial social interaction strategies. To date, the single-gene approach has mainly been used to uncover the bases of nodulation, but there is still a knowledge gap regarding the main features that a priori characterize rhizobial strains able to outcompete indigenous rhizobia. Therefore, tracking down which traits make different rhizobial strains able to win the competition for plant infection over other indigenous rhizobia will improve the strain selection process and, consequently, plant yield in sustainable agricultural production systems. We proved that a k-mer-based GWAS approach can efficiently identify the competition determinants of a panel of strains previously analyzed for their plant tissue occupancy using double fluorescent labeling. The reported strategy will be useful for detailed studies on the genomic aspects of the evolution of bacterial symbiosis and for an extensive evaluation of rhizobial inoculants.
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Project description:L7Ae is a universal archaeal protein that recognizes and stabilizes kink-turn (k-turn) motifs in RNA substrates. These structural motifs are widespread in nature and are found in many functional RNA species, including ribosomal RNAs. Synthetic biology approaches utilize L7Ae/k-turn interactions to control gene expression in eukaryotes. Here, we present results of comprehensive RNA immunoprecipitation sequencing (RIP-Seq) analysis of genomically tagged L7Ae from the hyperthermophilic archaeon Sulfolobus acidocaldarius A large set of interacting noncoding RNAs was identified. In addition, several mRNAs, including the l7ae transcript, were found to contain k-turn motifs that facilitate L7Ae binding. In vivo studies showed that L7Ae autoregulates the translation of its mRNA by binding to a k-turn motif present in the 5' untranslated region (UTR). A green fluorescent protein (GFP) reporter system was established in Escherichia coli and verified conservation of L7Ae-mediated feedback regulation in Archaea Mobility shift assays confirmed binding to a k-turn in the transcript of nop5-fibrillarin, suggesting that the expression of all C/D box sRNP core proteins is regulated by L7Ae. These studies revealed that L7Ae-mediated gene regulation evolved in archaeal organisms, generating new tools for the modulation of synthetic gene circuits in bacteria.IMPORTANCE L7Ae is an essential archaeal protein that is known to structure ribosomal RNAs and small RNAs (sRNAs) by binding to their kink-turn motifs. Here, we utilized RIP-Seq methodology to achieve a first global analysis of RNA substrates for L7Ae. Several novel interactions with noncoding RNA molecules (e.g., with the universal signal recognition particle RNA) were discovered. In addition, L7Ae was found to bind to mRNAs, including its own transcript's 5' untranslated region. This feedback-loop control is conserved in most archaea and was incorporated into a reporter system that was utilized to control gene expression in bacteria. These results demonstrate that L7Ae-mediated gene regulation evolved originally in archaeal organisms. The feedback-controlled reporter gene system can easily be adapted for synthetic biology approaches that require strict gene expression control.
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Project description:MicroRNAs (miRNAs) are key players in the regulation of neuronal processes by targeting a large network of target messenger RNAs (mRNAs). However, the identity and function of mRNAs targeted by miRNAs in specific cells of the brain are largely unknown. Here, we established an adeno-associated viral vector (AAV)-based neuron-specific Argonaute2:GFP-RNA immunoprecipitation followed by high-throughput sequencing to analyse the regulatory role of miRNAs in mouse hippocampal neurons. Using this approach, we identified more than two thousand miRNA targets in hippocampal neurons, regulating essential neuronal features such as cell signalling, transcription and axon guidance. Furthermore, we found that stable inhibition of the highly expressed miR-124 and miR-125 in hippocampal neurons led to significant but distinct changes in the AGO2 binding of target mRNAs, resulting in subsequent upregulation of numerous miRNA target genes. These findings greatly enhance our understanding of the miRNA targetome in hippocampal neurons.
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