Total RNA Pool Research Articles

Reprogramming the translatome during daily light transitions as affected by cytosolic glyceraldehyde-3-phosphate dehydrogenases GAPC1/C2.

Dark-light and light-dark transitions during the day are switching points of leaf metabolism that strongly affect the regulatory state of the cells, and this change is hypothesized to affect the translatome. The cytosolic glyceraldehyde-3-phosphate dehydrogenases GAPC1 and GAPC2 function in glycolysis, and carbohydrate and energy metabolism, but GAPC1/C2 also shows moonlighting functions in gene expression and post-transcriptional regulation. In this study we examined the rapid reprogramming of the translatome that occurs within 10 min at the end of the night and the end of the day in wild-type (WT) Arabidopsis and a gapc1/c2 double-knockdown mutant. Metabolite profiling compared to the WT showed that gapc1/c2 knockdown led to increases in a set of metabolites at the start of day, particularly intermediates of the citric acid cycle and linked pathways. Differences in metabolite changes were also detected at the end of the day. Only small sets of transcripts changed in the total RNA pool; however, RNA-sequencing revealed major alterations in polysome-associated transcripts at the light-transition points. The most pronounced difference between the WT and gapc1/c2 was seen in the reorganization of the translatome at the start of the night. Our results are in line with the proposed hypothesis that GAPC1/C2 play a role in the control of the translatome during light/dark transitions.

Ethanol-induced transcriptional and translational changes in Aldh1l1-Egfp/Rpl10a cortical astrocyte cultures.

The role astrocytes play in brain development and function has garnered greater attention as the diversity of roles they are involved in has become apparent. We have previously shown that ethanol-exposed astrocytes alter neuronal neurite outgrowth in an in vitro co-culture system and that ethanol alters the astrocyte-produced extracellular matrix (ECM) in vitro, with similar alterations in vivo. In this study, we utilized the translating ribosome affinity purification (TRAP) procedure in Aldh1l1-EGFP/Rpl10a transgenic mouse primary cortical astrocyte cultures to transcriptionally and translationally profile the astrocyte response to ethanol. We found a large number of differences between the total RNA pool and the translating RNA pool, indicating that the transcriptional state of astrocytes may not always reflect the translational state of astrocytes. In addition, there was a considerable overlap between ethanol-dysregulated genes in the total RNA pool and the translating RNA pool. Comparisons to published datasets indicate the in vitro model used here is most similar to PD1 or PD7 in vivo cortical astrocytes, and the ethanol-regulated genes showed a significant overlap with models of chronic ethanol exposure in astrocytes, a model of third-trimester ethanol exposure in the hippocampus and cerebellum, and an acute model of ethanol exposure in the hippocampus. These findings will further our understanding of the effects of ethanol on astrocyte gene expression and protein translation and how these changes may alter brain development and support the use of in vitro astrocyte cultures as models of neonatal astrocytes.

Breast cancer cell secretome analysis to decipher miRNA regulating the tumor microenvironment and discover potential biomarkers

MicroRNA (miRNA/miR) 526 b- and miR655-overexpressed tumor cell-free secretions regulate the breast cancer tumor microenvironment (TME) by promoting tumor-associated angiogenesis, oxidative stress, and hypoxic responses. Additionally, premature miRNA (pri-miR526b and pri-miR655) are established breast cancer blood biomarkers. However, the mechanisms of how these miRNAs regulate the TME has yet to be investigated. Mass spectrometry analysis of miRNA-overexpressed cell lines MCF7-miR526b, MCF7-miR655, and miRNA-low MCF7-Mock cell-free secretomes identified 34 differentially expressed proteins coded by eight genes. In both miRNA-high cell secretomes, four markers are upregulated: YWHAB, SFN, TXNDC12, and MYL6B, and four are downregulated: PEA15, PRDX4, PSMB6, and FN1. All upregulated marker transcripts are significantly high in both total cellular RNA pool and cell-free secretions of miRNA-high cell lines, validated with quantitative RT-PCR. Bioinformatics tools were used to investigate these markers' roles in breast cancer. These markers' top gene ontology functions are related to apoptosis, oxidative stress, membrane transport, and motility supporting oncogenic miR526b- and miR655-induced functions. Gene transcription factor analysis tools were used to show how these miRNAs regulate the expression of each secretory marker. Data extracted from the Human Protein Atlas showed that YWHAB, SFN, and TXNDC12 expression could distinguish early and late-stage breast cancer in various breast cancer subtypes and are associated with poor patient survival. Additionally, immunohistochemistry analysis showed the expression of each marker in breast tumors. A stronger correlation between miRNA clusters and upregulated secretory markers gene expression was found in the luminal A tumor subtype. YWHAB, SFN, and MYL6B are upregulated in breast cancer patient's blood, showing biomarker potential. Of these identified novel miRNA secretory markers, SFN and YWHAB successfully passed all validations and are the best candidates to further investigate their roles in miRNA associated TME regulation. Also, these markers show the potential to serve as blood-based breast cancer biomarkers, especially for luminal-A subtypes.

Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides.

The discovery of previously unknown functional roles of RNA in biological systems has led to increased interest in revealing novel RNA molecules as therapeutic targets and the development of tools to better understand the role of RNA in cells. RNA metabolic labeling broadens the scope of studying RNA by incorporating of unnatural nucleobases and nucleosides with bioorthogonal handles that can be utilized for chemical modification of newly synthesized cellular RNA. Such labeling of RNA provides access to applications including measurement of the rates of synthesis and decay of RNA, cellular imaging for RNA localization, and selective enrichment of nascent RNA from the total RNA pool. Several unnatural nucleosides and nucleobases have been shown to be incorporated into RNA by endogenous RNA synthesis machinery of the cells. RNA metabolic labeling can also be performed in a cell-specific manner, where only cells expressing an essential enzyme incorporate the unnatural nucleobase into their RNA. Although several discoveries have been enabled by the current RNA metabolic labeling methods, some key challenges still exist: (i) toxicity of unnatural analogues, (ii) lack of RNA-compatible conjugation chemistries, and (iii) background incorporation of modified analogues in cell-specific RNA metabolic labeling. In this Account, we showcase work done in our laboratory to overcome these challenges faced by RNA metabolic labeling.To begin, we discuss the cellular pathways that have been utilized to perform RNA metabolic labeling and study the interaction between nucleosides and nucleoside kinases. Then we discuss the use of vinyl nucleosides for metabolic labeling and demonstrate the low toxicity of 5-vinyluridine (5-VUrd) compared to other widely used nucleosides. Next, we discuss cell-specific RNA metabolic labeling with unnatural nucleobases, which requires the expression of a specific phosphoribosyl transferase (PRT) enzyme for incorporation of the nucleobase into RNA. In the course of this work, we discovered the enzyme uridine monophosphate synthase (UMPS), which is responsible for nonspecific labeling with modified uracil nucleobases. We were able to overcome this background labeling by discovering a mutant uracil PRT (UPRT) that demonstrates highly specific RNA metabolic labeling with 5-vinyluracil (5-VU). Furthermore, we discuss the optimization of inverse-electron-demand Diels-Alder (IEDDA) reactions for performing chemical modification of vinyl nucleosides to achieve covalent conjugation of RNA without transcript degradation. Finally, we highlight our latest endeavor: the development of mutually orthogonal chemical reactions for selective labeling of 5-VUrd and 2-vinyladenosine (2-VAdo), which allows for potential use of multiple vinyl nucleosides for simultaneous investigation of multiple cellular processes involving RNA. We hope that our methods and discoveries encourage scientists studying biological systems to include RNA metabolic labeling in their toolkit for studying RNA and its role in biological systems.

A Survey for Viruses and Viroids of Peach in Tennessee Orchards by RNA Sequencing

The prevalent viruses and viroids in a population of 100 peach trees grown in Tennessee (TN) orchards were investigated by RNA sequencing and analysis of a pool of ribosomal-depleted total RNA derived from these plants. The sequences of nine viruses (cherry green ring mottle virus; cherry necrotic rusty mottle virus; grapevine-associated ilarvirus, GaIV; nectarine virus M, NeVM; Prunus necrotic ringspot virus, PNRSV; tomato mosaic virus, ToMV; tomato ringspot virus, ToRSV; turnip vein-clearing virus, TVCV; and white clover mosaic virus, WClMV) and one viroid (peach latent mosaic viroid, PLMVd) were detected. The presence of ToMV, PNRSV, NeVM, ToRSV and PLMVd sequences were confirmed in the original samples by RT-PCR followed by Sanger sequencing. This study represents the first survey of viruses and viroids of peach trees in TN orchards. To the best of our knowledge, ToMV, NeVM, GaIV, TVCV, and WClMV have not been reported previously associated with peach trees elsewhere in the world.

Identification of Intrinsic RNA Binding Specificity of Purified Proteins by in vitro RNA Immunoprecipitation (vitRIP).

RNA-protein interactions are often mediated by dedicated canonical RNA binding domains. However, interactions through non-canonical domains with unknown specificity are increasingly observed, raising the question how RNA targets are recognized. Knowledge of the intrinsic RNA binding specificity contributes to the understanding of target selectivity and function of an individual protein. The presented in vitro RNA immunoprecipitation assay (vitRIP) uncovers intrinsic RNA binding specificities of isolated proteins using the total cellular RNA pool as a library. Total RNA extracted from cells or tissues is incubated with purified recombinant proteins, RNA-protein complexes are immunoprecipitated and bound transcripts are identified by deep sequencing or quantitative RT-PCR. Enriched RNA classes and the nucleotide frequency in these RNAs inform on the intrinsic specificity of the recombinant protein. The simple and versatile protocol can be adapted to other RNA binding proteins and total RNA libraries from any cell type or tissue. Graphic abstract: Figure 1. Schematic of the in vitro RNA immunoprecipitation (vitRIP) protocol.

Post-transcriptional regulation of several biological processes involved in latex production in Hevea brasiliensis.

BackgroundSmall RNAs modulate plant gene expression at both the transcriptional and post-transcriptional level, mostly through the induction of either targeted DNA methylation or transcript cleavage, respectively. Small RNA networks are involved in specific plant developmental processes, in signaling pathways triggered by various abiotic stresses and in interactions between the plant and viral and non-viral pathogens. They are also involved in silencing maintenance of transposable elements and endogenous viral elements. Alteration in small RNA production in response to various environmental stresses can affect all the above-mentioned processes. In rubber trees, changes observed in small RNA populations in response to trees affected by tapping panel dryness, in comparison to healthy ones, suggest a shift from a transcriptional to a post-transcriptional regulatory pathway. This is the first attempt to characterise small RNAs involved in post-transcriptional silencing and their target transcripts in Hevea.MethodsGenes producing microRNAs (MIR genes) and loci producing trans-activated small interfering RNA (ta-siRNA) were identified in the clone PB 260 re-sequenced genome. Degradome libraries were constructed with a pool of total RNA from six different Hevea tissues in stressed and non-stressed plants. The analysis of cleaved RNA data, associated with genomics and transcriptomics data, led to the identification of transcripts that are affected by 20–22 nt small RNA-mediated post-transcriptional regulation. A detailed analysis was carried out on gene families related to latex production and in response to growth regulators.ResultsCompared to other tissues, latex cells had a higher proportion of transcript cleavage activity mediated by miRNAs and ta-siRNAs. Post-transcriptional regulation was also observed at each step of the natural rubber biosynthesis pathway. Among the genes involved in the miRNA biogenesis pathway, our analyses showed that all of them are expressed in latex. Using phylogenetic analyses, we show that both the Argonaute and Dicer-like gene families recently underwent expansion. Overall, our study underlines the fact that important biological pathways, including hormonal signalling and rubber biosynthesis, are subject to post-transcriptional silencing in laticifers.

Valine-Induced Isoleucine Starvation in Escherichia coli K-12 Studied by Spike-In Normalized RNA Sequencing.

Escherichia coli cells respond to a period of famine by globally reorganizing their gene expression. The changes are known as the stringent response, which is orchestrated by the alarmone ppGpp that binds directly to RNA polymerase. The resulting changes in gene expression are particularly well studied in the case of amino acid starvation. We used deep RNA sequencing in combination with spike-in cells to measure global changes in the transcriptome after valine-induced isoleucine starvation of a standard E. coli K12 strain. Owing to the whole-cell spike-in method that eliminates variations in RNA extraction efficiency between samples, we show that ribosomal RNA levels are reduced during isoleucine starvation and we quantify how the change in cellular RNA content affects estimates of gene regulation. Specifically, we show that standard data normalization relying on sample sequencing depth underestimates the number of down-regulated genes in the stringent response and overestimates the number of up-regulated genes by approximately 40%. The whole-cell spike-in method also made it possible to quantify how rapidly the pool of total messenger RNA (mRNA) decreases upon amino acid starvation. A principal component analysis showed that the first two components together described 69% of the variability of the data, underlining that large and highly coordinated regulons are at play in the stringent response. The induction of starvation by sudden addition of high valine concentrations provoked prominent regulatory responses outside of the expected ppGpp, RpoS, and Lrp regulons. This underlines the notion that with the high resolution possible in deep RNA sequencing analysis, any different starvation method (e.g., nitrogen-deprivation, removal of an amino acid from an auxotroph strain, or valine addition to E. coli K12 strains) will produce measurable variations in the stress response produced by the cells to cope with the specific treatment.

Sequencing cell-type-specific transcriptomes with SLAM-ITseq.

Analysis of cell-type-specific transcriptomes is vital for understanding the biology of tissues and organs in the context of multicellular organisms. In this Protocol Extension, we combine a previously developed cell-type-specific metabolic RNA labeling method (thiouracil (TU) tagging) and a pipeline to detect the labeled transcripts by a novel RNA sequencing (RNA-seq) method, SLAMseq (thiol (SH)-linked alkylation for the metabolic sequencing of RNA). By injecting a uracil analog, 4-thiouracil, into transgenic mice that express cell-type-specific uracil phosphoribosyltransferase (UPRT), an enzyme required for 4-thiouracil incorporation into newly synthesized RNA, only cells expressing UPRT synthesize thiol-containing RNA. Total RNA isolated from a tissue of interest is then sequenced with SLAMseq, which introduces thymine to cytosine (T>C) conversions at the sites of the incorporated 4-thiouracil. The resulting sequencing reads are then mapped with the T>C-aware alignment software, SLAM-DUNK, which allows mapping of reads containing T>C mismatches. The number of T>C conversions per transcript is further analyzed to identify which transcripts are synthesized in the UPRT-expressing cells. Thus, our method, SLAM-ITseq (SLAMseq in tissue), enables cell-specific transcriptomics without laborious FACS-based cell sorting or biochemical isolation of the labeled transcripts used in TU tagging. In the murine tissues we assessed previously, this method identified ~5,000 genes that are expressed in a cell type of interest from the total RNA pool from the tissue. Any laboratory with access to a high-throughput sequencer and high-power computing can adapt this protocol with ease, and the entire pipeline can be completed in <5 d.

RPAD (RNase R treatment, polyadenylation, and poly(A)+ RNA depletion) method to isolate highly pure circular RNA.

Recent developments in high-throughput RNA sequencing methods coupled with innovative bioinformatic tools have uncovered thousands of circular (circ)RNAs. CircRNAs have emerged as a vast and novel class of regulatory RNAs with potential to modulate gene expression by acting as sponges for microRNAs (miRNAs) and RNA-binding proteins (RBPs). The biochemical enrichment of circRNAs by exoribonuclease treatment or by depletion of polyadenylated RNAs coupled with deep-sequencing is widely used for the systematic identification of circRNAs. Although these methods enrich circRNAs substantially, they do not eliminate efficiently non-polyadenylated and highly-structured RNAs. Here, we describe a method we termed RPAD, based on initial RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion. These joint interventions drastically depleted linear RNAs leading to isolation of highly pure circRNAs from total RNA pools. By facilitating the isolation of highly pure circRNAs, RPAD enables the elucidation of circRNA biogenesis, sequence, and function.

ТРАНСКРИПЦИОННЫЙ АНАЛИЗ ЭКСПРЕССИИ ГЕНОВ ОСТРОВКА VcB V. CHOLERAE МЕТОДОМ ПОЛНОГЕНОМНОГО СЕКВЕНИРОВАНИЯ

The aim of the study was to analyze the expression of V. cholerae genes that are part of the VcB island by means of full-genomic sequencing of the transcriptome. The VcB island is localized on the second chromosome in all toxigenic vibrios studied and is absent in the atoxigenic apiliated strains. Two strains of V. cholerae O1 ctxA+ tcpA+ and one strain V. cholerae O1 ctxA– tcpA– were studied. The pool of total RNA vibrios was isolated by a technique based on differential precipitation in the presence of lithium ions. In the total pool of sequenced RNA, RNA encoded in the order of 3 500 by known cholera vibrio genes was identified. In a pool of total RNA from two ctx + tcpA + strains RNA transcripts were found for the five genes included in the VcB island, excluding the VCA0282 gene, previously identified as the ISVch5-transposase. In the the pool of total RNA from the ctx– tcpA– strain no transcripts of the two genes previously described as VCA0282-transposase and VCA0283 were detected. A possible explanation may be the existence of copies of these genes in other parts of the genome of the ctxA– tcpA– V. cholerae.

Description and initial characterization of metatranscriptomic nidovirus-like genomes from the proposed new family Abyssoviridae, and from a sister group to the Coronavirinae, the proposed genus Alphaletovirus

Transcriptomics has the potential to discover new RNA virus genomes by sequencing total intracellular RNA pools. In this study, we have searched publicly available transcriptomes for sequences similar to viruses of the Nidovirales order. We report two potential nidovirus genomes, a highly divergent 35.9 kb likely complete genome from the California sea hare Aplysia californica, which we assign to a nidovirus named Aplysia abyssovirus 1 (AAbV), and a coronavirus-like 22.3 kb partial genome from the ornamented pygmy frog Microhyla fissipes, which we assign to a nidovirus named Microhyla alphaletovirus 1 (MLeV). AAbV was shown to encode a functional main proteinase, and a translational readthrough signal. Phylogenetic analysis suggested that AAbV represents a new family, proposed here as Abyssoviridae. MLeV represents a sister group to the other known coronaviruses. The importance of MLeV and AAbV for understanding nidovirus evolution, and the origin of terrestrial nidoviruses are discussed.

Characterizing the Role of Exoribonucleases in the Control of Microbial Gene Expression: Differential RNA-Seq.

Differential RNA-Seq is a next-generation technology method to determine the significant transcriptomic differences between two and more samples. With this method it is possible to analyze the total RNA content of different samples making it the best global analysis method currently available to study the roles of exoribonucleases in the cell. These enzymes are responsible for the RNA processing and degradation in the cells and therefore affect the total RNA pool in ways not yet fully understood. In Escherichia coli there are three main degradative exoribonucleases RNase II, RNase R, and PNPase that degrade the RNA from the 3' to the 5'-end. These enzymes have several roles in the cell and even though they are degradative enzymes RNase II and PNPase can also protect some RNAs from degradation and PNPase can also act as an RNA polymerase under some conditions. The multiplicity of roles of these exoribonucleases leads to a very high number of transcripts that are affected by their absence in the cell. With the differential RNA-Seq it is possible to obtain a much deeper understanding of how these enzymes work and regulate the bacterial gene expression. In this chapter we have described a differential RNA-Seq data analysis protocol applied to the study of exoribonucleases. We also included the protocol for experimental validation of the RNA-Seq data using qPCR and motility assays. Although the methods described in this chapter were applied to the study of the exoribonucleases, they can also be used for other differential RNA-Seq studies.

Identifying intronic circRNAs: progress and challenges.

In an insightful editorial (1), Bogard and colleagues discussed a recent publication from our laboratory describing the method ‘RNase R treatment followed by Polyadenylation and poly(A) + RNA Depletion’ (RPAD) to isolate highly enriched circular RNAs (circRNAs) from heterogeneous total RNA pools (2).

Abstract P6-08-05: Genome-wide identification of transcripts regulated by estrogen in MCF-7 cells using BrU-seq

Abstract Estrogen promotes estrogen receptor (ER)-positive breast cancer cell growth by modulating transcription through ligand activated ER. The spectrum of early ER target genes has been identified through gene expression microarray and more recently with RNA-seq analyses. These methods, although comprehensive, fall short in identifying many ER-regulated transcripts which are either not present in microarray; too abundant to determine estrogen regulated changes among steady-state pools of RNAs; or have very short half-lives and low abundance such as some non-coding RNAs, including RNAs generated by enhancers bound by ER (eRNAs). Global run-on sequencing (GRO-seq) has been used to identify ER target genes and eRNAs in breast cancer cells. It is technically cumbersome and is performed under non-physiological condition. We have applied BrU-seq technique to identify global transcripts regulated by estrogen in MCF-7 breast cancer cells. The newly synthesized RNAs in MCF-7 cells treated with or without estrogen for various time points, were labeled with 5'-bromo-uridine (BrU). The labeled RNAs were purified by immunoprecipitation using an anti-BrdU antibody and then subjected to RNA sequencing analysis, which provides greater sensitivity than regular RNA-seq analysis from steady-state total RNA samples. Robust estrogen-regulated transcripts can be detected within 30 min of treatment. Significantly more transcripts (both coding and non-coding RNAs) were identified as estrogen targets using BrU-seq analysis than RNA-seq analysis from total RNA pools. Many of these estrogen-regulated transcripts are located close to ER binding sites identified from ER ChIP-seq analysis, indicating a functional role for these ER binding sites in regulating RNA transcription. For estrogen targets identified through both analyses, the magnitude of expression changes (both up- and down-regulation) is usually greater from BrU-seq than from the regular RNA-seq analysis. The dynamics of RNA synthesis and degradation for some unstable RNAs (such as eRNAs) can be interrogated through our data set. Also, RNA pol II elongation rate can be deduced from some long ER target genes (&amp;gt;100 kb) such as GREB1. In addition to identifying estrogen targets, BrU-seq should be a very useful technique in assessing transcriptional changes triggered by various signal transduction pathways. Citation Format: Sun J, Capobianco E, Tsinoremas N, Lippman M. Genome-wide identification of transcripts regulated by estrogen in MCF-7 cells using BrU-seq [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-05.

LEA gene expression, RNA stability and pigment accumulation in three closely related Linderniaceae species differing in desiccation tolerance

Desiccation-tolerant plants (Craterostigma plantagineum and Lindernia brevidens) evolved a highly efficient strategies to prevent dehydration-induced irreversible damage. The protection system involves synthesis of LEA proteins, decrease of photosynthetic activity and activation of antioxidant systems. The regulation of these processes requires joint action of multiple proteins. Here, we present comparative analyses of accumulation of transcripts encoding components of the protection machinery, such as selected LEA proteins, enzymes of the chlorophyll degradation pathway and anthocyanin biosynthesis enzymes in total and polysomal RNA pools. The analyses revealed that desiccation-tolerant plants recruit mRNAs to ribosomes with higher efficiency than the desiccation-sensitive species L. subracemosa. Desiccation-tolerant species accumulated high amounts of LEA transcripts during dehydration and precisely controlled the amounts of chlorophyll keeping it at a level sufficient to activate photosynthesis after rehydration. In contrast, mRNA of L. subracemosa was prone to dehydration-induced degradation, decomposition of the photosynthetic apparatus and degradation of free chlorophyll. Thus, the results of the studies point to differences in the control of gene expression and degradation of chlorophyll in desiccation-tolerant versus desiccation-sensitive species when the plants were subjected to dehydration.

De novo Transcriptome Sequencing and Development of Abscission Zone-Specific Microarray as a New Molecular Tool for Analysis of Tomato Organ Abscission.

Abscission of flower pedicels and leaf petioles of tomato (Solanum lycopersicum) can be induced by flower removal or leaf deblading, respectively, which leads to auxin depletion, resulting in increased sensitivity of the abscission zone (AZ) to ethylene. However, the molecular mechanisms that drive the acquisition of abscission competence and its modulation by auxin gradients are not yet known. We used RNA-Sequencing (RNA-Seq) to obtain a comprehensive transcriptome of tomato flower AZ (FAZ) and leaf AZ (LAZ) during abscission. RNA-Seq was performed on a pool of total RNA extracted from tomato FAZ and LAZ, at different abscission stages, followed by de novo assembly. The assembled clusters contained transcripts that are already known in the Solanaceae (SOL) genomics and NCBI databases, and over 8823 identified novel tomato transcripts of varying sizes. An AZ-specific microarray, encompassing the novel transcripts identified in this study and all known transcripts from the SOL genomics and NCBI databases, was constructed to study the abscission process. Multiple probes for longer genes and key AZ-specific genes, including antisense probes for all transcripts, make this array a unique tool for studying abscission with a comprehensive set of transcripts, and for mining for naturally occurring antisense transcripts. We focused on comparing the global transcriptomes generated from the FAZ and the LAZ to establish the divergences and similarities in their transcriptional networks, and particularly to characterize the processes and transcriptional regulators enriched in gene clusters that are differentially regulated in these two AZs. This study is the first attempt to analyze the global gene expression in different AZs in tomato by combining the RNA-Seq technique with oligonucleotide microarrays. Our AZ-specific microarray chip provides a cost-effective approach for expression profiling and robust analysis of multiple samples in a rapid succession.

Probing N⁶-methyladenosine (m⁶A) RNA Modification in Total RNA with SCARLET.

Posttranscriptional N (6)-methyladenosine (m(6)A) RNA modification is indispensable for cell development and viability; however, functional investigation of m(6)A biological function has been hindered by the lack of methods for its precise identification and quantitation. Here, we describe a method that accurately identifies m(6)A position and modification fraction in human messenger RNA (mRNA) and long noncoding RNA (lncRNA) at single-nucleotide resolution, termed as "site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET)" (Fig. 1). This method combines two previously established techniques, site-specific cleavage and splint ligation, to probe the m(6)A RNA modification status at any mRNA/lncRNA site in the total RNA pool.

164 Translational regulation of gene expression in cardiomyocytes following exposure to oxidative stress

<h3></h3> Myocardial infarction leads to ischemia/reperfusion injury associated with an increase in oxidative stress. The cardiomyocyte response to oxidative stress is concentration-dependent: low concentrations may be protective, moderate levels promote apoptosis, and high levels lead to necrosis. These responses are associated with differential effects on cardiomyocyte mRNA expression. At the protein level, increasing levels of oxidative stress inhibit global protein synthesis, but some proteins continue to be synthesised. We investigated the effects of moderate and low levels of oxidative stress on translational regulation of mRNAs in cardiomyocytes. Total RNA and RNA associated with polysomes (i.e. mRNAs that are actively translated) were prepared from rat neonatal cardiomyocytes with or without treatment with H₂O₂ (0.2 mM, 1 h). RNA expression profiling was performed using Affymetrix microarrays and analysed using GeneSpring GX 13. 35 RNAs were significantly changed (&gt;1.5 fold; p &lt; 0.05, moderated T test with Benjamini and Hochberg false discovery rate correction) in the polysomes in response to H₂O₂, with a significant difference in the degree of change in polysomal vs total RNA. Of these, 9 were increased to a greater extent in the polysomes (indicative of selective translation) including the small G protein Rasd1 and the transcription factor Nfil3. Other mRNAs were not significantly different between polysomal and total RNA (e.g. Atf3, Hmox1), whereas genes such as Mdm2 were upregulated to a greater extent in the total RNA pool. Microarray data were validated by quantitative PCR. Data for all genes studied were consistent with the microarrays. In cardiomyocytes exposed to moderate oxidative stress (0.2 mM H₂O₂) or lower oxidative stress (0.1 mM H₂O₂), Rasd1 and Nfil3 were upregulated to a greater extent in the polysomes and Hmox1 showed a similar degree of upregulation in polysomal and total RNA pools. However, although Mdm2 mRNA was increased to a greater extent in the total pool with 0.2 mM H₂O₂, it was increased to a similar extent in polysomal and total RNA pools with 0.1 mM H₂O₂. Furthermore, although Atf3 mRNA was similarly upregulated in total and polysomal RNA pools with 0.2 mM H₂O₂, there was greater upregulation in the polysomes with 0.1 mM H₂O₂. Rasd1 protein was studied in cardiomyocytes exposed H₂O₂. Consistent with the mRNA expression data, Rasd1 was upregulated over ˜2 h. Thus, oxidative stress has concentration-dependent effects on protein translation of specific mRNAs. Rasd1 is of particular interest because it is associated with circadian rhythm regulation that may influence the response to myocardial infarction. Furthermore, a genetic polymorphism in the Rasd1 gene is associated with coronary artery disease and ischaemic stroke.

MicroRNA-27a Contributes to Rhabdomyosarcoma Cell Proliferation by Suppressing RARA and RXRA.

BackgroundRhabdomyosarcomas (RMS) are rare but very aggressive childhood tumors that arise as a consequence of a regulatory disruption in the growth and differentiation pathways of myogenic precursor cells. According to morphological criteria, there are two major RMS subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS) with the latter showing greater aggressiveness and metastatic potential with respect to the former. Efforts to unravel the complex molecular mechanisms underlying RMS pathogenesis and progression have revealed that microRNAs (miRNAs) play a key role in tumorigenesis.Methodology/Principal FindingsThe expression profiles of 8 different RMS cell lines were analyzed to investigate the involvement of miRNAs in RMS. The miRNA population from each cell line was compared to a reference sample consisting of a balanced pool of total RNA extracted from those 8 cell lines. Sixteen miRNAs whose expression discriminates between translocation-positive ARMS and negative RMS were identified. Attention was focused on the role of miR-27a that is up-regulated in the more aggressive RMS cell lines (translocation-positive ARMS) in which it probably acts as an oncogene. MiR-27a overexpressing cells showed a significant increase in their proliferation rate that was paralleled by a decrease in the number of cells in the G1 phase of the cell cycle. It was possible to demonstrate that miR-27a is implicated in cell cycle control by targeting the retinoic acid alpha receptor (RARA) and retinoic X receptor alpha (RXRA).ConclusionsStudy results have demonstrated that miRNA expression signature profiling can be used to classify different RMS subtypes and suggest that miR-27a may have a therapeutic potential in RMS by modulating the expression of retinoic acid receptors.