RNA Populations Research Articles

Gaining insight into transcriptome-wide RNA population dynamics through the chemistry of 4-thiouridine.

Cellular RNA levels are the result of a juggling act between RNA transcription, processing, and degradation. By tuning one or more of these parameters, cells can rapidly alter the available pool of transcripts in response to stimuli. While RNA sequencing (RNA-seq) is a vital method to quantify RNA levels genome-wide, it is unable to capture the dynamics of different RNA populations at steady-state or distinguish between different mechanisms that induce changes to the steady-state (i.e., altered rate of transcription vs. degradation). The dynamics of different RNA populations can be studied by targeted incorporation of noncanonical nucleosides. 4-Thiouridine (s4 U) is a commonly used and versatile RNA metabolic label that allows the study of many properties of RNA metabolism from synthesis to degradation. Numerous experimental strategies have been developed that leverage the power of s4 U to label newly transcribed RNA in whole cells, followed by enrichment with activated disulfides or chemistry to induce C mutations at sites of s4 U during sequencing. This review presents existing methods to study RNA population dynamics genome-wide using s4 U metabolic labeling, as well as a discussion of considerations and challenges when designing s4 U metabolic labeling experiments. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Turnover and Surveillance > Regulation of RNA Stability.

Functional characterization of RNA fragments using high-throughput interactome screening

Populations of small eukaryotic RNAs, in addition to relatively well recognized molecules such as miRNAs or siRNAs, also contain fragments derived from all classes of constitutively expressed non-coding RNAs. It has been recently demonstrated that the formation and accumulation of RNA fragments (RFs) is cell-/tissue-specific and depends on internal and external stimuli. Unfortunately, the mechanisms underlying RF biogenesis and function remain unclear. To better understand them, we employed RNA pull-down and mass spectrometry methods to characterize the interactions of seven RFs originating from tRNA, snoRNA and snRNA. By integrating our results with publicly available data on physical protein-protein interactions, we constructed an RF interactome network. We determined that the RF interactome comprises proteins generally different from those that interact with their parental full length RNAs. Proteins captured by the RFs were involved in mRNA splicing, tRNA processing, DNA recombination/replication, protein biosynthesis and carboxylic acid metabolism. Our data suggest that RFs can be endogenous aptamer-like molecules and potential players in recently revealed RNA-protein regulatory networks. SignificanceIn the recent decade it has become evident that RNAs with well-known functions (for example tRNA, snoRNA or rRNA) can be cleaved to yield short fragments, whose role in cells remains only partially characterized. At the same time, unconventional interactions between mRNA and proteins without RNA-binding domains have been demonstrated, revealing novel layers of possible RNA-mediated regulation. Considering the above, we hypothesized that RNA fragments (RFs) can be endogenous aptamer-like molecules that unconventionally interact with proteins. In this study we identified protein partners of seven selected RFs. We found that RFs bind different set of proteins than their parental full length RNAs and identified proteins differentially bound by the particular RFs. These observations suggest biological relevance of the discovered interactions. Our data provide a novel perspective on the significance of RFs and point to this pool of molecules as to a rich collection of potential components of the recently discovered RNA-protein regulatory networks.

Expanding the Nucleoside Recoding Toolkit: Revealing RNA Population Dynamics with 6-Thioguanosine.

RNA-sequencing (RNA-seq) measures RNA abundance in a biological sample but does not provide temporal information about the sequenced RNAs. Metabolic labeling can be used to distinguish newly made RNAs from pre-existing RNAs. Mutations induced from chemical recoding of the hydrogen bonding pattern of the metabolic label can reveal which RNAs are new in the context of a sequencing experiment. These nucleotide recoding strategies have been developed for a single uridine analogue, 4-thiouridine (s4U), limiting the scope of these experiments. Here we report the first use of nucleoside recoding with a guanosine analogue, 6-thioguanosine (s6G). Using TimeLapse sequencing (TimeLapse-seq), s6G can be recoded under RNA-friendly oxidative nucleophilic-aromatic substitution conditions to produce adenine analogues (substituted 2-aminoadenosines). We demonstrate the first use of s6G recoding experiments to reveal transcriptome-wide RNA population dynamics.

Analysis By Deep Sequencing of Discontinued Neurotropic Yellow Fever Vaccine Strains

Deep sequencing of live-attenuated viral vaccines has focused on vaccines in current use. Here we report characterization of a discontinued live yellow fever (YF) vaccine associated with severe adverse events. The French neurotropic vaccine (FNV) strain of YF virus was derived empirically in 1930 by 260 passages of wild-type French viscerotropic virus (FVV) in mouse brain. The vaccine was administered extensively in French-speaking Africa until discontinuation in 1982, due to high rates of post-vaccination encephalitis in children. Using rare archive strains of FNV, viral RNAs were sequenced and analyzed by massively parallel, in silico methods. Diversity and specific population structures were compared in reference to the wild-type parental strain FVV, and between the vaccine strains themselves. Lower abundance of polymorphism content was observed for FNV strains relative to FVV. Although the vaccines were of lower diversity than FVV, heterogeneity between the vaccines was observed. Reversion to wild-type identity was variably observed in the FNV strains. Specific population structures were recovered from vaccines with neurotropic properties; loss of neurotropism in mice was associated with abundance of wild-type RNA populations. The analysis provides novel sequence evidence that FNV is genetically unstable, and that adaptation of FNV contributed to the neurotropic adverse phenotype.

Bacteriophage-derived dsRNA of natural origin alters functional profile of rat microglial cells exposed to hypoxia in vitro: Evaluation of metabolic and phenotypic markers

BackgroundMicroglial cells (MC) are thought to be major innate immune cells in the central nervous system. Hypoxia can cause pro- and anti-inflammatory MC activation. Toll-like receptors (TLRs) play an important role in the regulation of microglia activity and can skew metabolic profile of resting and activated MC. However, the data concerning the effect of TLR agonists on metabolic profile of MC activated by hypoxia are controversial and sparse. Larifan is a pharmaceutical product containing a heterogeneous population of natural origin double-stranded RNA (dsRNA). This study was aimed to investigate the effect of the treatment with Larifan on functional profile of rat MC exposed to hypoxia in vitro. MethodsRat MC were cultured under normoxic (21% O2) or hypoxic (3% O2) conditions for 24 h. After this, cells were treated with Larifan (200 μg/mL) for 18 h. To characterize MC functional profile, reactive oxygen species (ROS) generation, phagocytic activity, arginine metabolism as well as CD206 and CD14 expression were examined. ResultsHypoxic MC were characterized by sharply increased ROS generation, moderately boosted phagocytic activity, arginine metabolism was skewed towards increased arginase activity, CD14+ and CD206+ cell fractions were higher than those in normoxic cells. Treatment MC with Larifan resulted in further increase ROS generation along with the decrease of phagocytic activity that is characteristic for the acquisition of antigen-presenting properties by these cells. The level of CD206 expression was lowered in treated hypoxic MC. ConclusionLarifan promotes transition of hypoxic MC to the state of mature pro-inflammatory APC in vitro.

Initial Phase of Replication of Plus-Stranded RNA Viruses

The author presents theoretical mean-field analysis of the initial phase of the kinetics of intracellular replication of plus-stranded RNA viruses, which are abundant and include, e.g., the hepatitis C virus (HCV). The treatment is based on the conventional concept that the replication process of such viruses takes place at the membrane complexes formed with participation of viral proteins, e.g., NS5A in the HCV case. The key novel prediction supported by Monte Carlo calculations is that this scheme may be insufficient in order to describe the very initial phase of the process, because the initial intracellular viral RNA and protein populations may in this case go extinct rather than overcome the kinetic barrier for transition to the full-scale infection of a host cell. Practically, this means that in such situations, the conventional replication scenario should be complemented by another pathway, e.g., by replication outside the membrane without viral proteins, which operates in the very beginning.

Silencing efficiency of dsRNA fragments targeting Fusarium graminearum TRI6 and patterns of small interfering RNA associated with reduced virulence and mycotoxin production.

Deoxynivalenol (DON) contamination of cereal grains caused by Fusarium head blight may be addressed by future RNA interference (RNAi)-based gene silencing approaches. However, utilizing these approaches will require a greater understanding of the principles that govern RNAi effectiveness in the pathogen Fusarium graminearum. RNAi in higher eukaryotes, including fungi, involves processing double stranded RNA (dsRNA) into small interfering RNA (siRNA) that silence gene expression based on base pair complementarity. This study examined virulence, DON production, and the small RNA (sRNA) populations in response to RNAi-based silencing of TRI6, a transcription factor that positively regulates DON synthesis via control of TRI5 expression. Silencing was accomplished via the expression of transgenes encoding inverted repeats targeting various regions of TRI6 (RNAi vectors). Transgene expression was associated with novel, TRI6-specific siRNAs. For RNAi vectors targeting the majority of TRI6 sequence (~600 bp), a discontinuous, repeatable pattern was observed in which most siRNAs mapped to specific regions of TRI6. Targeting shorter regions (250–350 bp) did not alter the siRNA populations corresponding to that region of TRI6. No phased processing was observed. The 5' base of ~83% of siRNAs was uracil, consistent with DICER processing and ARGONAUTE binding preferences for siRNA. Mutant lines showed TRI6 siRNA-associated reductions of TRI5 expression on toxin inducing media and DON in infected wheat and barley spikes. Shorter RNAi vectors resulted in variable levels of silencing that were less than for the ~600 bp RNAi vector, with a 343 bp RNAi vector targeting the 5’ end of TRI6 having the best silencing efficiency. This work identifies efficient shorter region for silencing of TRI6 and describes the patterns of siRNA corresponding to those regions.

Automated in vitro evolution of a translation-coupled RNA replication system in a droplet flow reactor

Automation is a useful strategy to make laborious evolutionary experiments faster and easier. To date, several types of continuous flow reactors have been developed for the automated evolutionary experiments of viruses and bacteria. However, the development of a flow reactor applicable to compartmentalized in vitro self-replication systems is still a challenge. In this study, we demonstrate automated in vitro evolution of a translation-coupled RNA system in a droplet flow reactor for the first time. This reactor contains approximately 1010 micro-scale droplets (average diameter is approximately 0.8 μm), which continuously fuse and divide among each other at a controllable rate. In the droplets, an RNA (artificial genomic RNA) replicate through the translation of self-encoded RNA replicase with spontaneously appearing parasitic RNA. We performed two automated replication experiments for more than 400 hours with different mixing intensities. We found that several mutations displayed increased frequencies in the genomic RNA populations and the dominant RNA mutants acquired the ability to replicate faster or acquired resistance to the parasitic RNA, demonstrating that Darwinian evolution occurred during the long-term replication. The droplet flow reactor we developed can be a useful tool to perform in vitro evolutionary experiments of translation-coupled systems.

Bacterial Y RNAs: Gates, Tethers, and tRNA Mimics.

Y RNAs are noncoding RNAs (ncRNAs) that are present in most animal cells and also in many bacteria. These RNAs were discovered because they are bound by the Ro60 protein, a major target of autoantibodies in patients with some systemic autoimmune rheumatic diseases. Studies of Ro60 and Y RNAs in Deinococcus radiodurans, the first sequenced bacterium with a Ro60 ortholog, revealed that they function with 3'-to-5' exoribonucleases to alter the composition of RNA populations during some forms of environmental stress. In the best-characterized example, Y RNA tethers the Ro60 protein to the exoribonuclease polynucleotide phosphorylase, allowing this exoribonuclease to degrade structured RNAs more effectively. Y RNAs can also function as gates to regulate access of other RNAs to the Ro60 central cavity. Recent studies in the enteric bacterium Salmonella enterica serovar Typhimurium resulted in the discovery that Y RNAs are widely present in bacteria. Remarkably, the most-conserved subclass of bacterial Y RNAs contains a domain that mimics tRNA. In this review, we discuss the structure, conservation, and known functions of bacterial Y RNAs as well as the certainty that more bacterial Y RNAs and additional roles for these ncRNAs remain to be uncovered.

Global profiling of hnRNP A2/B1-RNA binding on chromatin highlights LncRNA interactions

ABSTRACTLong noncoding RNAs (lncRNAs) often carry out their functions through associations with adaptor proteins. We recently identified heterogeneous ribonucleoprotein (hnRNP) A2/B1 as an adaptor of the human HOTAIR lncRNA. hnRNP A2 and B1 are splice isoforms of the same gene. The spliced version of HOTAIR preferentially associates with the B1 isoform, which we hypothesize contributes to RNA-RNA matching between HOTAIR and transcripts of target genes in breast cancer. Here we used enhanced cross-linking immunoprecipitation (eCLIP) to map the direct interactions between A2/B1 and RNA in breast cancer cells. Despite differing by only twelve amino acids, the A2 and B1 splice isoforms associate preferentially with distinct populations of RNA in vivo. Through cellular fractionation experiments we characterize the pattern of RNA association in chromatin, nucleoplasm, and cytoplasm. We find that a majority of interactions occur on chromatin, even those that do not contribute to co-transcriptional splicing. A2/B1 binding site locations on multiple RNAs hint at a contribution to the regulation and function of lncRNAs. Surprisingly, the strongest A2/B1 binding site occurs in a retained intron of HOTAIR, which interrupts an RNA-RNA interaction hotspot. In vitro eCLIP experiments highlight additional exonic B1 binding sites in HOTAIR which also surround the RNA-RNA interaction hotspot. Interestingly, a version of HOTAIR with the intron retained is still capable of making RNA-RNA interactions in vitro through the hotspot region. Our data further characterize the multiple functions of a repurposed splicing factor with isoform-biased interactions, and highlight that the majority of these functions occur on chromatin-associated RNA.

An RNA-binding protein Blanks plays important roles in defining small RNA and mRNA profiles in Drosophila testes

Drosophila Blanks is a testes-specific RNA-binding protein required for post-meiotic spermiogenesis. However, Blanks's role in regulating RNA populations in the testes remains unknown. We performed small RNA and mRNA high-throughput sequencing in blanks mutant testes and controls. We identified two miRNAs, one siRNA, and hundreds of mRNAs that are significantly upregulated or downregulated in blanks mutant testes. Pathway analysis revealed that differentially expressed mRNAs are involved in catabolic and metabolic processes, anion and cation transport, mating, and reproductive behavior. Our results reveal that Blanks plays important roles in defining testicular small RNA and mRNA profiles.

Abstract 491: Understanding the roles of miRNAs in melanoma susceptibility through miQTL study using miRNA transcriptomes from 106 individuals

Abstract microRNAs (miRNA) are important mediators of post-transcriptional gene regulation and play critical roles in tumorigenesis. In melanoma, miRNAs are involved in proliferation, migration, invasion and drug resistance modulating RAS/MAPK and MITF pathways and others. Melanoma arises in melanocytes, and understanding heritable regulation of miRNAs in primary melanocytes could enhance our knowledge of genes and pathways contributing to melanoma risk. While a handful of studies in limited tissue types have identified miRNA transcripts with expression levels that are correlated with common genotypes using an expression quantitative loci approach (miQTL), expression levels of miRNAs are highly tissue-specific, further correlating with tissue-specific mRNA expression. Given the lack of cell-type specific miQTL data, we established a miQTL dataset using primary cultures of human melanocytes from 106 newborn males mainly of European decent. To assess comprehensive miRNA transcript levels from these samples, we performed RNA sequencing of the small RNA population (single-end, stranded, 51bp). An average of 63M reads per sample were obtained, and subsequently processed following miRMaster pipeline (ccb-compute.cs.uni-saarland.de/mirmaster) to detect known and novel miRNAs. A total of 961 known and 1262 novel miRNA transcripts were expressed above the background noise (> 0.05 Read Per Million and >5 reads per transcript in >=10 samples). Genotypes of 106 individuals were also assessed by direct genotyping using Illumina OmniExpress arrays followed by imputation using Michigan Imputation Server to produce a total of 10,687,550 genotypes. Subsequent QTL analyses using FastQTL (fastqtl.sourceforge.net) and QTLtools (qtltools.github.io/qtltools) identified 33 known and 34 novel miRNA transcripts as cis-miQTLs (q-value < 0.05) and 2 known and 6 novel miRNAs as trans-miQTLs (FDR < 0.1), respectively. We also observed a subset of significant cis-miQTLs overlapping with marginally significant mRNA trans-eQTLs from the same RNA samples, suggesting miRNA-mediated trans regulation. Further identification of melanocyte-specific miQTLs and their target genes will provide better understanding of melanocyte-specific miRNA regulation network. To identify candidate melanoma susceptibility genes functioning through heritable miRNA expression, melanocyte miQTLs will also be overlaid with melanoma genome-wide association data. In addition to the results from these analyses, further analyses of differentially expressed miRNAs and target genes between our primary melanocytes and melanoma tumors (available through The Cancer Genome Atlas) will shed light on the altered gene networks in melanomagenesis that might have therapeutic relevance through miRNA-mediated intervention. Citation Format: Jiyeon Choi, Tongwu Zhang, Michael Kovacs, Mai Xu, Andrew Vu, NISC Comparative Sequencing Program, Stacie Loftus, William Pavan, Kevin Brown. Understanding the roles of miRNAs in melanoma susceptibility through miQTL study using miRNA transcriptomes from 106 individuals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 491.

Abstract 2456: The PIWI-interacting RNA pathway in prostate is regulated by vitamin D and altered in cancer

Abstract Low serum vitamin D is a risk factor for aggressive prostate cancer. piRNAs and their interacting partners, PIWI-like (PIWIL) proteins, are aberrantly expressed in many cancers, but few studies have focused on piRNAs in the prostate. We used laser-capture microdissected (LCM) human prostate epithelium from frozen samples collected for a clinical trial of high dose vitamin D to examine the small non-coding RNA population by next-generation sequencing. The 8 patients for this study were selected for having either high (avg=48.7pg/ml) or low (avg=21.4pg/ml) prostatic vitamin D (1,25(OH)2D) levels. VDR ChIP-seq was performed as well, using human primary epithelial cells treated for 3hr with EtOH or 50nM vitamin D. In LCM-collected prostate epithelium unexpectedly piRNAs represented ~20% of the mapped reads. Of all the small RNA species investigated, piRNAs were the most affected by vitamin D status, increasing in abundance in tissues wish high vitamin D (p=0.036, t-test). The LCM data were compared to small-RNA sequencing data from benign prostate available from The Cancer Genome Atlas (TCGA), which represent whole prostate tissue and may contain stromal bias. The LCM-collected samples had a distinct, more consistent profile of small RNAs than TCGA samples. Two unambiguous piRNAs that were highly expressed in LCM data, but not in TCGA data, were verified as epithelial-specific in prostate by in situ hybridization. Several piRNA-biogenesis genes lie within 100kb of VDR ChIP peaks, including TDRKH, which was found to be vitamin D-regulated experimentally. The expression of piRNA-interacting PIWIL proteins was assessed by RT-qPCR, immunoblot and immunofluorescence on a tissue microarray containing paired benign and cancer tissues (N=23). Of the 4 human PIWIL proteins, PIWIL1, 2 and 4 were present in normal prostate epithelial cells by PCR. PIWIL1 protein was also detected in PrE and RWPE-1 benign cells, but PIWIL1 was not detected in the PC3 prostate cancer cell line. By immunofluorescence, PIWIL1 protein in prostate epithelium significantly decreased in prostate cancer (p=0.016, paired t-test). In conclusion, vitamin D status associated with the expression of piRNA in prostate epithelium in clinical trial samples. PIWIL1 protein levels were lower in prostate cancer, suggesting a pro-differentiation role for the PIWI pathway that may be affected in vitamin D-deficient patients. Citation Format: Bethany Baumann, Shang Gao, Giovanni Lugli, Larisa Nonn. The PIWI-interacting RNA pathway in prostate is regulated by vitamin D and altered in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2456.

Assessment of an Organ-Specific de Novo Transcriptome of the Nematode Trap-Crop, Solanum sisymbriifolium.

Solanum sisymbriifolium, also known as “Litchi Tomato” or “Sticky Nightshade,” is an undomesticated and poorly researched plant related to potato and tomato. Unlike the latter species, S. sisymbriifolium induces eggs of the cyst nematode, Globodera pallida, to hatch and migrate into its roots, but then arrests further nematode maturation. In order to provide researchers with a partial blueprint of its genetic make-up so that the mechanism of this response might be identified, we used single molecule real time (SMRT) sequencing to compile a high quality de novo transcriptome of 41,189 unigenes drawn from individually sequenced bud, root, stem, and leaf RNA populations. Functional annotation and BUSCO analysis showed that this transcriptome was surprisingly complete, even though it represented genes expressed at a single time point. By sequencing the 4 organ libraries separately, we found we could get a reliable snapshot of transcript distributions in each organ. A divergent site analysis of the merged transcriptome indicated that this species might have undergone a recent genome duplication and re-diploidization. Further analysis indicated that the plant then retained a disproportionate number of genes associated with photosynthesis and amino acid metabolism in comparison to genes with characteristics of R-proteins or involved in secondary metabolism. The former processes may have given S. sisymbriifolium a bigger competitive advantage than the latter did.

Small non-coding RNA landscape is modified by GPAT2 silencing in MDA-MB-231 cells.

Glycerol-3-phosphate acyltransferase-2 is a member of “cancer-testis gene” family. Initially linked to lipid metabolism, this gene has been recently found involved also in PIWI-interacting RNAs biogenesis in germline stem cells. To investigate its role in piRNA metabolism in cancer, the gene was silenced in MDA-MB-231 breast cancer cells and small RNA sequencing was applied. PIWI-interacting RNAs and tRNA-derived fragments expression profiles showed changes following GPAT2 silencing. Interestingly, a marked shift in length distribution for both small RNAs was detected in GPAT2-silenced cells. Most downregulated PIWI-interacting RNAs are single copy in the genome, intragenic, hosted in snoRNAs and previously found to be upregulated in cancer cells. Putative targets of these PIWI-interacting RNAs are linked to lipid metabolism. Downregulated tRNA derived fragments derived from, so-called ‘differentiation tRNAs’, whereas upregulated ones derived from proliferation-linked tRNAs. miRNA amounts decrease after Glycerol-3-phosphate acyltransferase-2 silencing and functional enrichment analysis of deregulated miRNA putative targets point to mitochondrial biogenesis, IGF1R signaling and oxidative metabolism of lipids and lipoproteins. In addition, miRNAs known to be overexpressed in breast cancer tumors with poor prognosis where found downregulated in GPAT2-silenced cells. In conclusion, GPAT2 silencing quantitatively and qualitatively affects the population of PIWI-interacting RNAs, tRNA derived fragments and miRNAs which, in combination, result in a more differentiated cancer cell phenotype.

Intracellular miRNA or siRNA delivery and function

Nanocarrier-mediated delivery and release of short non-coding RNA (e.g., miRNA or siRNA) into the cells with subsequent suppression of the populations of some of the mRNAs and proteins is of interest in the context of the development of a new generation of drugs. Bearing in mind such applications, the author shows the specifics of the corresponding transient kinetics by using three generic models without and with feedback resulting in bistability in the gene expression. In the absence of feedback, the suppression of the mRNA and protein population is transient. In the case of bistable kinetics, non-coding RNA can induce transition from the initial steady state to another steady state. The duration of this transition can be much longer than the time scale characterizing the drop of the non-coding RNA population. Quantitatively, the effect of the delivered non-coding RNA on gene expression can be appreciable if the maximum non-coding RNA population in the cytoplasm is comparable to or above 1000. All these conclusions have been drawn on the basis of calculations performed with the kinetic parameters typical for human cells.

Towards annotating the plant epigenome: the Arabidopsis thaliana small RNA locus map

Based on 98 public and internal small RNA high throughput sequencing libraries, we mapped small RNAs to the genome of the model organism Arabidopsis thaliana and defined loci based on their expression using an empirical Bayesian approach. The resulting loci were subsequently classified based on their genetic and epigenetic context as well as their expression properties. We present the results of this classification, which broadly conforms to previously reported divisions between transcriptional and post-transcriptional gene silencing small RNAs, and to PolIV and PolV dependencies. However, we are able to demonstrate the existence of further subdivisions in the small RNA population of functional significance. Moreover, we present a framework for similar analyses of small RNA populations in all species.

Studying Stimulus‐Induced Changes in RNA Dynamics by Mutational Mapping through TimeLapse‐seq

The eukaryotic transcriptome is dynamically regulated through RNA transcription and decay allowing cells to respond to environmental stimuli in a rapid and specific manner. The bromodomain inhibitor JQ1, has been extensively studied in the context of cancer cells, as it blocks the activation of specific sets of oncogenes perturbing RNA population dynamics.1 Recently, JQ1 has additionally been found to repress pathogenic response genes in models of heart failure, however the mechanism of this action remains unclear.2 JQ1 is known to block the BET bromodomain reader proteins involved in chromatin remodeling, affecting the transcription of the target genes. We, therefore, aim to study the effect of JQ1 in heart failure on the transcriptional level. While RNA‐sequencing (RNA‐seq) provides a snapshot of the cellular transcriptome, temporal information is lost as rapid changes in the RNA population remain undetected among the excess of preexisting RNA. To gain insight into the role of JQ1‐induced pathways in cardiomyocytes, we are studying changes in RNA dynamics using TimeLapse‐seq3, recently developed nucleotide recoding chemistry that utilizes metabolic labeling in an enrichment‐free RNA‐seq approach. Through recoding of the hydrogen bonding pattern of the metabolic label, TimeLapse‐seq identifies new transcripts through apparent mutations in the RNA‐seq reads. We will describe progress using TimeLapse‐seq as a strategy to study differently regulated transcripts in disease and to deduce the mechanism of JQ1 in heart failure.Support or Funding InformationThis work was supported by the NIH NIGMS T32GM007223 (J.A.S. and E.E.D); NSF Graduate Research Fellowship (E.E.D); NIH New Innovator Award DP2 HD083992‐01 (M.D.S.), and a Searle scholarship (M.D.S.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The successes and future prospects of the linear antisense RNA amplification methodology.

It has been over a quarter of a century since the introduction of the linear RNA amplification methodology known as antisense RNA (aRNA) amplification. Whereas most molecular biology techniques are rapidly replaced owing to the fast-moving nature of development in the field, the aRNA procedure has become a base that can be built upon through varied uses of the technology. The technique was originally developed to assess RNA populations from small amounts of starting material, including single cells, but over time its use has evolved to include the detection of various cellular entities such as proteins, RNA-binding-protein-associated cargoes, and genomic DNA. In this Perspective we detail the linear aRNA amplification procedure and its use in assessing various components of a cell's chemical phenotype. This procedure is particularly useful in efforts to multiplex the simultaneous detection of various cellular processes. These efforts are necessary to identify the quantitative chemical phenotype of cells that underlies cellular function.

Localization of RNA and translation in the mammalian oocyte and embryo.

The tight correlation between mRNA distribution and subsequent protein localization and function indicate a major role for mRNA localization within the cell. RNA localization, followed by local translation, presents a mechanism for spatial and temporal gene expression regulation utilized by various cell types. However, little is known about mRNA localization and translation in the mammalian oocyte and early embryo. Importantly, fully-grown oocyte becomes transcriptionally inactive and only utilizes transcripts previously synthesized and stored during earlier development. We discovered an abundant RNA population in the oocyte and early embryo nucleus together with RNA binding proteins. We also characterized specific ribosomal proteins, which contribute to translation in the oocyte and embryo. By applying selected markers to mouse and human oocytes, we found that there might be a similar mechanism of RNA metabolism in both species. In conclusion, we visualized the localization of RNAs and translation machinery in the oocyte, that could shed light on this terra incognita of these unique cell types in mouse and human.