Project description:ADARs are the primary factors underlying A-to-I editing in metazoans. We conducted the first global study of ADAR1-RNA interaction in human cells using CLIP-Seq. In contrast to the expected predominant binding of ADAR1 to Alu repeats, thousands of CLIP sites were located in non-Alu regions. This unexpectedly frequent non-Alu binding enabled discovery of transcriptome-wide functional and biophysical targets of ADAR1 in the regulation of mRNA processing including alternative 3' UTR usage and alternative splicing. In addition, a global analysis of ADAR1 binding to non-Alu regions also revealed its primary interaction with microRNA (miRNA) transcripts in the nucleus, which subsequently affected expression levels of mature miRNAs. A complex global picture was revealed regarding the dependence of this function on the double-stranded RNA binding domains or deaminase activity. Our study unfolded a broad landscape of the diverse functional roles of ADAR1. To identify ADAR binding dependent miRNA defferential expression profiles, U87MG cells were transfected with ADAR1 overexpression vector, RNA binding mutant (EAA and E912A), siRNA of ADAR1 or controls.
Project description:ADARs are the primary factors underlying A-to-I editing in metazoans. We conducted the first global study of ADAR1-RNA interaction in human cells using CLIP-Seq. In contrast to the expected predominant binding of ADAR1 to Alu repeats, thousands of CLIP sites were located in non-Alu regions. This unexpectedly frequent non-Alu binding enabled discovery of transcriptome-wide functional and biophysical targets of ADAR1 in the regulation of mRNA processing including alternative 3' UTR usage and alternative splicing. In addition, a global analysis of ADAR1 binding to non-Alu regions also revealed its primary interaction with microRNA (miRNA) transcripts in the nucleus, which subsequently affected expression levels of mature miRNAs. A complex global picture was revealed regarding the dependence of this function on the double-stranded RNA binding domains or deaminase activity. Our study unfolded a broad landscape of the diverse functional roles of ADAR1. To identify ADAR binding dependent miRNA defferential expression profiles, U87MG cells were transfected with ADAR1 overexpression vector, RNA binding mutant (EAA and E912A), siRNA of ADAR1 or controls.
Project description:ADARs are the primary factors underlying A-to-I editing in metazoans. We conducted the first global study of ADAR1-RNA interaction in human cells using CLIP-Seq. In contrast to the expected predominant binding of ADAR1 to Alu repeats, thousands of CLIP sites were located in non-Alu regions. This unexpectedly frequent non-Alu binding enabled discovery of transcriptome-wide functional and biophysical targets of ADAR1 in the regulation of mRNA processing including alternative 3' UTR usage and alternative splicing. In addition, a global analysis of ADAR1 binding to non-Alu regions also revealed its primary interaction with microRNA (miRNA) transcripts in the nucleus, which subsequently affected expression levels of mature miRNAs. A complex global picture was revealed regarding the dependence of this function on the double-stranded RNA binding domains or deaminase activity. Our study unfolded a broad landscape of the diverse functional roles of ADAR1.
Project description:ADARs are the primary factors underlying A-to-I editing in metazoans. We conducted the first global study of ADAR1-RNA interaction in human cells using CLIP-Seq. In contrast to the expected predominant binding of ADAR1 to Alu repeats, thousands of CLIP sites were located in non-Alu regions. This unexpectedly frequent non-Alu binding enabled discovery of transcriptome-wide functional and biophysical targets of ADAR1 in the regulation of mRNA processing including alternative 3' UTR usage and alternative splicing. In addition, a global analysis of ADAR1 binding to non-Alu regions also revealed its primary interaction with microRNA (miRNA) transcripts in the nucleus, which subsequently affected expression levels of mature miRNAs. A complex global picture was revealed regarding the dependence of this function on the double-stranded RNA binding domains or deaminase activity. Our study unfolded a broad landscape of the diverse functional roles of ADAR1.
Project description:Adenosine deaminases acting on RNA (ADARs) are the primary factors underlying adenosine to inosine (A-to-I) editing in metazoans. Here we report the first global study of ADAR1-RNA interaction in human cells using CLIP-seq. A large number of CLIP sites are observed in Alu repeats, consistent with ADAR1's function in RNA editing. Surprisingly, thousands of other CLIP sites are located in non-Alu regions, revealing functional and biophysical targets of ADAR1 in the regulation of alternative 3' UTR usage and miRNA biogenesis. We observe that binding of ADAR1 to 3' UTRs precludes binding by other factors, causing 3' UTR lengthening. Similarly, ADAR1 interacts with DROSHA and DGCR8 in the nucleus and possibly out-competes DGCR8 in primary miRNA binding, which enhances mature miRNA expression. These functions are dependent on ADAR1's editing activity, at least for a subset of targets. Our study unfolds a broad landscape of the functional roles of ADAR1.
Project description:Abscisic acid (ABA)-, stress-, and ripening-induced (ASR) proteins are involved in abiotic stress responses. However, the exact molecular mechanism underlying their function remains unclear. Notably, the direct targets of ASRs that confer drought stress tolerance have not yet been identified.In this study, we report that MaASR expression was induced by drought stress and MaASR overexpression in Arabidopsis strongly enhanced drought stress tolerance. Physiological analyses indicated that transgenic lines had higher survival rates, germination rates and proline content, and lower water loss rates (WLR) and malondialdehyde (MDA) content. MaASR-overexpressing lines also showed smaller leaves and reduced sensitivity to ABA. Further, microarray and chromatin immunoprecipitation-based sequencing (ChIP-seq) analysis revealed that MaASR participates in regulating photosynthesis, respiration, carbohydrate and phytohormone metabolism and signal transduction to confer plants with enhanced drought stress tolerance. Direct interactions of MaASR with promoters for the hexose transporter and Rho GTPase-activating protein (RhoGAP) genes were confirmed by electrophoresis mobility shift array (EMSA) analysis. Our results indicate that MaASR acts as a crucial regulator of photosynthesis, respiration, carbohydrate and phytohormone metabolism and signal transduction to mediate drought stress tolerance.
Project description:ARHGAP36 is an atypical Rho GTPase-activating protein (GAP) family member that drives both spinal cord development and tumorigenesis, acting in part through an N-terminal motif that suppresses protein kinase A and activates Gli transcription factors. ARHGAP36 also contains isoform-specific N-terminal sequences, a central GAP-like module, and a unique C-terminal domain, and the functions of these regions remain unknown. Here we have mapped the ARHGAP36 structure-activity landscape using a deep sequencing-based mutagenesis screen and truncation mutant analyses. Using this approach, we have discovered several residues in the GAP homology domain that are essential for Gli activation and a role for the C-terminal domain in counteracting an N-terminal autoinhibitory motif that is present in certain ARHGAP36 isoforms. In addition, each of these sites modulates ARHGAP36 recruitment to the plasma membrane or primary cilium. Through comparative proteomics, we also have identified proteins that preferentially interact with active ARHGAP36, and we demonstrate that one binding partner, prolyl oligopeptidase-like protein, is a novel ARHGAP36 antagonist. Our work reveals multiple modes of ARHGAP36 regulation and establishes an experimental framework that can be applied towards other signaling proteins.
Project description:Introduction Multiple sclerosis (MS), a non-contagious and chronic disease of the central nervous system, is an unpredictable and indirectly inherited disease affecting different people in different ways. Using Omics platforms genomics, transcriptomics, proteomics, epigenomics, interactomics, and metabolomics database, it is now possible to construct sound systems biology models to extract full knowledge of the MS and recognize the pathway to uncover the personalized therapeutic tools. Methods In this study, we used several Bayesian Networks in order to find the transcriptional gene regulation networks that drive MS disease. We used a set of BN algorithms using the R add-on package bnlearn. The BN results underwent further downstream analysis and were validated using a wide range of Cytoscape algorithms, web based computational tools and qPCR amplification of blood samples from 56 MS patients and 44 healthy controls. The results were semantically integrated to improve understanding of the complex molecular architecture underlying MS, distinguishing distinct metabolic pathways and providing a valuable foundation for the discovery of involved genes and possibly new treatments. Results Results show that the LASP1, TUBA1C, and S100A6 genes were most likely playing a biological role in MS development. Results from qPCR showed a significant increase (P < 0.05) in LASP1 and S100A6 gene expression levels in MS patients compared to that in controls. However, a significant down regulation of TUBA1C gene was observed in the same comparison. Conclusion This study provides potential diagnostic and therapeutic biomarkers for enhanced understanding of gene regulation underlying MS.
Project description:Imbalance between the accumulation and removal of nitric oxide and its derivatives is a challenge faced by all plants at the cellular level, and is especially important under stress conditions. Exposure of plants to various biotic and abiotic stresses causes rapid changes in cellular redox tone potentiated by the rise in reactive nitrogen species that serve as signaling molecules in mediating defensive responses. To understand mechanisms mediated by these signaling molecules, we performed a large-scale analysis of the Arabidopsis transcriptome induced by nitrosative stress. We generated an average of 84 and 91 million reads from three replicates each of control and 1 mM S-nitrosocysteine (CysNO)-infiltrated Arabidopsis leaf samples, respectively. After alignment, more than 95% of all reads successfully mapped to the reference and 32,535 genes and 55,682 transcripts were obtained. CysNO infiltration caused differential expression of 6436 genes (3448 up-regulated and 2988 down-regulated) and 6214 transcripts (3335 up-regulated and 2879 down-regulated) 6 h post-infiltration. These differentially expressed genes were found to be involved in key physiological processes, including plant defense against various biotic and abiotic stresses, hormone signaling, and other developmental processes. After quantile normalization of the FPKM values followed by student's T-test (P < 0.05) we identified 1165 DEGs (463 up-regulated and 702 down-regulated) with at least 2-folds change in expression after CysNO treatment. Expression patterns of selected genes involved in various biological pathways were verified using quantitative real-time PCR. This study provides comprehensive information about plant responses to nitrosative stress at transcript level and would prove helpful in understanding and incorporating mechanisms associated with nitrosative stress responses in plants.
Project description:RNA-binding proteins coordinate the fates of multiple RNAs, but the principles underlying these global interactions remain poorly understood. We elucidated regulatory mechanisms of the RNA-binding protein HuR, by integrating data from diverse high-throughput targeting technologies, specifically PAR-CLIP, RIP-chip, and whole-transcript expression profiling. The number of binding sites per transcript, degree of HuR association, and degree of HuR-dependent RNA stabilization were positively correlated. Pre-mRNA and mature mRNA containing both intronic and 3' UTR binding sites were more highly stabilized than transcripts with only 3' UTR or only intronic binding sites, suggesting that HuR couples pre-mRNA processing with mature mRNA stability. We also observed HuR-dependent splicing changes and substantial binding of HuR in polypyrimidine tracts of pre-mRNAs. Comparison of the spatial patterns surrounding HuR and miRNA binding sites provided functional evidence for HuR-dependent antagonism of proximal miRNA-mediated repression. We conclude that HuR coordinates gene expression outcomes at multiple interconnected steps of RNA processing.