scholarly journals Genome-wide RNA structurome reprogramming by acute heat shock globally regulates mRNA abundance

2018 ◽  
Vol 115 (48) ◽  
pp. 12170-12175 ◽  
Author(s):  
Zhao Su ◽  
Yin Tang ◽  
Laura E. Ritchey ◽  
David C. Tack ◽  
Mengmeng Zhu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Rna Structure ◽  
Time Course ◽  
Structural Changes ◽  
Dimethyl Sulfate ◽  
Rna Degradation ◽  
Crop Species ◽  
Genome Wide

The heat shock response is crucial for organism survival in natural environments. RNA structure is known to influence numerous processes related to gene expression, but there have been few studies on the global RNA structurome as it prevails in vivo. Moreover, how heat shock rapidly affects RNA structure genome-wide in living systems remains unknown. We report here in vivo heat-regulated RNA structuromes. We applied Structure-seq chemical [dimethyl sulfate (DMS)] structure probing to rice (Oryza sativa L.) seedlings with and without 10 min of 42 °C heat shock and obtained structural data on >14,000 mRNAs. We show that RNA secondary structure broadly regulates gene expression in response to heat shock in this essential crop species. Our results indicate significant heat-induced elevation of DMS reactivity in the global transcriptome, revealing RNA unfolding over this biological temperature range. Our parallel Ribo-seq analysis provides no evidence for a correlation between RNA unfolding and heat-induced changes in translation, in contrast to the paradigm established in prokaryotes, wherein melting of RNA thermometers promotes translation. Instead, we find that heat-induced DMS reactivity increases correlate with significant decreases in transcript abundance, as quantified from an RNA-seq time course, indicating that mRNA unfolding promotes transcript degradation. The mechanistic basis for this outcome appears to be mRNA unfolding at both 5′ and 3′-UTRs that facilitates access to the RNA degradation machinery. Our results thus reveal unexpected paradigms governing RNA structural changes and the eukaryotic RNA life cycle.

scholarly journals Characterizing the structure-function relationship of a naturally-occurring RNA thermometer

2017 ◽  
Author(s):  
Sarai Meyer ◽  
Julius B. Lucks
Keyword(s):  
Heat Shock ◽  
Rna Structure ◽  
Structural Changes ◽  
Structural Motif ◽  
Ribosome Binding ◽  
Heat Shock Responses ◽  
Function Relationship ◽  
Relationship Of

AbstractA wide number of bacteria have been found to govern virulence and heat shock responses using temperature-sensing RNAs known as RNA thermometers. A prime example is theagsAthermometer known to regulate the production of the AgsA heat shock protein inSalmonella entericausing a “fourU” structural motif. Using the SHAPE-Seq RNA structure-probing methodin vivoandin vitro, we found that the regulator functions by a subtle shift in equilibrium RNA structure populations that lead to a partial melting of the helix containing the ribosome binding site. We also demonstrate that ribosome binding to theagsAmRNA causes changes to the thermometer structure that appear to facilitate thermometer helix unwinding. These results demonstrate how subtle RNA structural changes can govern gene expression and illuminate the function of an important bacterial regulatory motif.

scholarly journals Best practices for genome-wide RNA structure analysis: combination of mutational profiles and drop-off information

2017 ◽  
Author(s):  
Eva Maria Novoa ◽  
Jean-Denis Beaudoin ◽  
Antonio J Giraldez ◽  
John S Mattick ◽  
Manolis Kellis
Keyword(s):  
Reverse Transcription ◽  
Rna Structure ◽  
Structural Information ◽  
Dimethyl Sulfate ◽  
Mutational Signatures ◽  
Chemically Modified ◽  
Chemical Probing ◽  
Genome Wide ◽  
Generation Sequencing

ABSTRACTGenome-wide RNA structure maps have recently become available through the coupling of in vivo chemical probing reagents with next-generation sequencing. Initial analyses relied on the identification of truncated reverse transcription reads to identify the chemically modified nucleotides, but recent studies have shown that mutational signatures can also be used. While these two methods have been employed interchangeably, here we show that they actually provide complementary information. Consequently, analyses using exclusively one of the two methodologies may disregard a significant portion of the structural information. We also show that the identity and sequence environment of the modified nucleotide greatly affect the odds of introducing a mismatch or causing reverse transcriptase drop-off. Finally, we identify specific mismatch signatures generated by dimethyl sulfate probing that can be exploited to remove false positives typically produced in RNA structurome analyses, and how these signatures vary depending on the reverse transcription enzyme used.

scholarly journals SPLICE-q: a Python tool for genome-wide quantification of splicing efficiency

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Verônica R. de Melo Costa ◽  
Julianus Pfeuffer ◽  
Annita Louloupi ◽  
Ulf A. V. Ørom ◽  
Rosario M. Piro
Keyword(s):  
Gene Expression ◽  
Cancer Progression ◽  
Time Course ◽  
Progressive Increase ◽  
Rna Seq ◽  
Transcript Processing ◽  
Aberrant Transcript ◽  
Genome Wide ◽  
Splicing Efficiency ◽  
Nascent Rna

Abstract Background Introns are generally removed from primary transcripts to form mature RNA molecules in a post-transcriptional process called splicing. An efficient splicing of primary transcripts is an essential step in gene expression and its misregulation is related to numerous human diseases. Thus, to better understand the dynamics of this process and the perturbations that might be caused by aberrant transcript processing it is important to quantify splicing efficiency. Results Here, we introduce SPLICE-q, a fast and user-friendly Python tool for genome-wide SPLICing Efficiency quantification. It supports studies focusing on the implications of splicing efficiency in transcript processing dynamics. SPLICE-q uses aligned reads from strand-specific RNA-seq to quantify splicing efficiency for each intron individually and allows the user to select different levels of restrictiveness concerning the introns’ overlap with other genomic elements such as exons of other genes. We applied SPLICE-q to globally assess the dynamics of intron excision in yeast and human nascent RNA-seq. We also show its application using total RNA-seq from a patient-matched prostate cancer sample. Conclusions Our analyses illustrate that SPLICE-q is suitable to detect a progressive increase of splicing efficiency throughout a time course of nascent RNA-seq and it might be useful when it comes to understanding cancer progression beyond mere gene expression levels. SPLICE-q is available at: https://github.com/vrmelo/SPLICE-q

Tissue specificity of type I collagen gene expression is determined at both transcriptional and post-transcriptional levels

1984 ◽  
Vol 4 (9) ◽  
pp. 1843-1852
Author(s):  
R J Focht ◽  
S L Adams
Keyword(s):  
Gene Expression ◽  
Rna Structure ◽  
Type I Collagen ◽  
Translational Efficiency ◽  
Type I ◽  
Collagen Gene ◽  
Differentiated Cells ◽  
Collagen Gene Expression

We analyzed the control of type I collagen synthesis in four kinds of differentiated cells from chicken embryos which synthesize very different amounts of the protein. Tendon, skin, and smooth muscle cells were found to have identical amounts of type I collagen RNAs; however, the RNAs had inherently different translatabilities, which were observed both in vivo and in vitro. Chondrocytes also had substantial amounts of type I collagen RNAs, even though they directed no detectable synthesis of the protein either in vivo or in vitro. Type I collagen RNAs in chondrocytes display altered electrophoretic mobilities, suggesting that in these cells the reduction in translational efficiency may be mediated in part by changes in the RNA structure. These data indicate that control of type I collagen gene expression is a complex process which is exerted at both transcriptional and post-transcriptional levels.

scholarly journals Identification and classification of cis-regulatory elements in the amphipod crustacean Parhyale hawaiensis

2021 ◽  
Author(s):  
Dennis A Sun ◽  
Nipam H Patel
Keyword(s):  
Gene Expression ◽  
Genome Annotation ◽  
Time Course ◽  
Regulatory Elements ◽  
Rna Seq ◽  
Genome Wide ◽  
Long Read ◽  
Amphipod Crustacean ◽  
Accessible Chromatin

AbstractEmerging research organisms enable the study of biology that cannot be addressed using classical “model” organisms. The development of novel data resources can accelerate research in such animals. Here, we present new functional genomic resources for the amphipod crustacean Parhyale hawaiensis, facilitating the exploration of gene regulatory evolution using this emerging research organism. We use Omni-ATAC-Seq, an improved form of the Assay for Transposase-Accessible Chromatin coupled with next-generation sequencing (ATAC-Seq), to identify accessible chromatin genome-wide across a broad time course of Parhyale embryonic development. This time course encompasses many major morphological events, including segmentation, body regionalization, gut morphogenesis, and limb development. In addition, we use short- and long-read RNA-Seq to generate an improved Parhyale genome annotation, enabling deeper classification of identified regulatory elements. We leverage a variety of bioinformatic tools to discover differential accessibility, predict nucleosome positioning, infer transcription factor binding, cluster peaks based on accessibility dynamics, classify biological functions, and correlate gene expression with accessibility. Using a Minos transposase reporter system, we demonstrate the potential to identify novel regulatory elements using this approach, including distal regulatory elements. This work provides a platform for the identification of novel developmental regulatory elements in Parhyale, and offers a framework for performing such experiments in other emerging research organisms.Primary Findings-Omni-ATAC-Seq identifies cis-regulatory elements genome-wide during crustacean embryogenesis-Combined short- and long-read RNA-Seq improves the Parhyale genome annotation-ImpulseDE2 analysis identifies dynamically regulated candidate regulatory elements-NucleoATAC and HINT-ATAC enable inference of nucleosome occupancy and transcription factor binding-Fuzzy clustering reveals peaks with distinct accessibility and chromatin dynamics-Integration of accessibility and gene expression reveals possible enhancers and repressors-Omni-ATAC can identify known and novel regulatory elements

Cis- andtrans-acting factors regulating transcription of the BGT1 gene in response to hypertonicity

1998 ◽  
Vol 274 (4) ◽  
pp. F753-F761 ◽  
Author(s):  
Hiroshi Miyakawa ◽  
Seung Kyoon Woo ◽  
Ching-Pu Chen ◽  
Stephen C. Dahl ◽  
Joseph S. Handler ◽  
...  
Keyword(s):  
Dna Binding ◽  
Time Course ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Dimethyl Sulfate ◽  
Base Pairs ◽  
Biochemical Basis ◽  
Activation Of Transcription ◽  
Stimulation Of

We have previously identified a tonicity-responsive enhancer (TonE) in the promoter region of the canine BGT1 gene. TonE mediates hypertonicity-induced stimulation of transcription. Here, we characterize TonE and TonE binding proteins (TonEBPs) to provide a biochemical basis for cloning of the TonEBPs. Mutational analysis applied to both hypertonicity-induced stimulation of transcription and TonEBP binding reveals that TonE is 11 base pairs in length, with the consensus sequence of (C/T)GGAAnnn(C/T)n(C/T). Activity of the TonEBPs increases in response to hypertonicity with a time course similar to that of transcription of the BGT1 gene. Studies with inhibitors indicate that translation, but not transcription, is required for activation of the TonEBPs. Phosphorylation is required for the stimulation of transcription but not for activation of DNA binding by the TonEBPs. In vivo methylation by dimethyl sulfate reveals that the TonE site of the BGT1 gene is protected with a time course like that of activity of the TonEBPs and activation of transcription. Ultraviolet cross-linking indicates that the TonEBPs share a DNA binding subunit of 200 kDa.

scholarly journals Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays

Plant Methods ◽  
2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Snigdha Poddar ◽  
Jaclyn Tanaka ◽  
Jamie H. D. Cate ◽  
Brian Staskawicz ◽  
Myeong-Je Cho
Keyword(s):  
Gene Expression ◽  
Suspension Culture ◽  
Genome Editing ◽  
Time Course ◽  
Transient Gene Expression ◽  
Protoplast Isolation ◽  
Transient Transfection ◽  
Study Gene Expression ◽  
Rice Calli

Abstract Background An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. Results Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. Conclusion The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.

scholarly journals Microarray Based Functional Analysis of Myricetin and Proteomic Study on Its Anti-Inflammatory Property

2019 ◽  
Vol 2019 ◽  
pp. 1-13
Author(s):  
Tao Li ◽  
Jihe Zhu ◽  
Fangming Deng ◽  
Weiguo Wu ◽  
Zhibing Zheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Disease ◽  
Microarray Data ◽  
Inflammatory Cytokine ◽  
Gene Expressions ◽  
Genome Wide ◽  
Proteomic Study ◽  
Anti Inflammatory ◽  
First Time

Myricetin has been reported as a promising chemopreventive compound with multiple biofunctions. To evaluate its influence on gene expressions in genome-wide set and further investigate its anti-inflammatory property, the present study performed Gene Ontology and Ingenuity Pathway Analysis (IPA) to describe the basic gene expression characteristics by myricetin treatment in HepG2 cells, confirmed its multi-biofunction by real-time fluorescent quantitative PCR (RT-qPCR), and further verified its anti-inflammatory property by Western blotting and bio-plex-based cytokines assay. The IPA data showed that 337 gene expressions (48% of the top molecules) are disturbed over 2-fold, and the most possible biofunctions of myricetin are the effect on “cardiovascular disease, metabolic disease, and lipid metabolism,” via regulation of 28 molecules with statistic score of 46. RT-qPCR data confirmed the accuracy of microarray data, and cytokines assay results indicated that 6 of the total 27 inflammatory cytokine secretions were significantly inhibited by myricetin pretreatment, including TNF-α, IFN-γ, IL-1α, IL-1β, IL-2, and IL-6. The present study is the first time to elucidate the multi-function of myricetin in genome-wide set by IPA analysis and verify its anti-inflammatory property by proteomics of cytokines assay. Therefore, these results enrich the comprehensive bioactivities of myricetin and reveal that myricetin has powerful anti-inflammatory property, which provides encouragement for in vivo studies to verify its possible health benefits.

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