Showing papers on "RNA published in 2011"
TL;DR: Using a quantitative model, the first genome-scale prediction of synthesis rates of mRNAs and proteins is obtained and it is found that the cellular abundance of proteins is predominantly controlled at the level of translation.
Abstract: Gene expression is a multistep process that involves the transcription, translation and turnover of messenger RNAs and proteins. Although it is one of the most fundamental processes of life, the entire cascade has never been quantified on a genome-wide scale. Here we simultaneously measured absolute mRNA and protein abundance and turnover by parallel metabolic pulse labelling for more than 5,000 genes in mammalian cells. Whereas mRNA and protein levels correlated better than previously thought, corresponding half-lives showed no correlation. Using a quantitative model we have obtained the first genome-scale prediction of synthesis rates of mRNAs and proteins. We find that the cellular abundance of proteins is predominantly controlled at the level of translation. Genes with similar combinations of mRNA and protein stability shared functional properties, indicating that half-lives evolved under energetic and dynamic constraints. Quantitative information about all stages of gene expression provides a rich resource and helps to provide a greater understanding of the underlying design principles.
5,635 citations
TL;DR: It is proposed that this "competing endogenous RNA" (ceRNA) activity forms a large-scale regulatory network across the transcriptome, greatly expanding the functional genetic information in the human genome and playing important roles in pathological conditions, such as cancer.
5,334 citations
TL;DR: FTO exhibits efficient oxidative demethylation activity of abundant N6-methyladenosine (m6A) residues in RNA in vitro, and it is shown that FTO partially colocalizes with nuclear speckles, supporting m6A in nuclear RNA as a physiological substrate of FTO.
Abstract: We report here that fat mass and obesity-associated protein (FTO) has efficient oxidative demethylation activity targeting the abundant N6-methyladenosine (m(6)A) residues in RNA in vitro. FTO knockdown with siRNA led to increased amounts of m(6)A in mRNA, whereas overexpression of FTO resulted in decreased amounts of m(6)A in human cells. We further show the partial colocalization of FTO with nuclear speckles, which supports the notion that m(6)A in nuclear RNA is a major physiological substrate of FTO.
2,647 citations
TL;DR: It is demonstrated that linc-MD1 exerts the same control over differentiation timing in human myoblasts, and that its levels are strongly reduced in Duchenne muscle cells, indicating that the ceRNA network plays an important role in muscle differentiation.
2,231 citations
TL;DR: Recent developments in RNA-seq methods have provided an even more complete characterization of RNA transcripts, including improvements in transcription start site mapping, strand-specific measurements, gene fusion detection, small RNA characterization and detection of alternative splicing events.
Abstract: In the few years since its initial application, massively parallel cDNA sequencing, or RNA-seq, has allowed many advances in the characterization and quantification of transcriptomes. Recently, several developments in RNA-seq methods have provided an even more complete characterization of RNA transcripts. These developments include improvements in transcription start site mapping, strand-specific measurements, gene fusion detection, small RNA characterization and detection of alternative splicing events. Ongoing developments promise further advances in the application of RNA-seq, particularly direct RNA sequencing and approaches that allow RNA quantification from very small amounts of cellular materials.
1,928 citations
TL;DR: The psRNATarget as mentioned in this paper target analysis server is designed for high-throughput analysis of next-generation data with an efficient distributed computing back-end pipeline that runs on a Linux cluster.
Abstract: Plant endogenous non-coding short small RNAs (20–24 nt), including microRNAs (miRNAs) and a subset of small interfering RNAs (ta-siRNAs), play important role in gene expression regulatory networks (GRNs). For example, many transcription factors and development-related genes have been reported as targets of these regulatory small RNAs. Although a number of miRNA target prediction algorithms and programs have been developed, most of them were designed for animal miRNAs which are significantly different from plant miRNAs in the target recognition process. These differences demand the development of separate plant miRNA (and ta-siRNA) target analysis tool(s). We present psRNATarget, a plant small RNA target analysis server, which features two important analysis functions: (i) reverse complementary matching between small RNA and target transcript using a proven scoring schema, and (ii) target-site accessibility evaluation by calculating unpaired energy (UPE) required to ‘open’ secondary structure around small RNA’s target site on mRNA. The psRNATarget incorporates recent discoveries in plant miRNA target recognition, e.g. it distinguishes translational and post-transcriptional inhibition, and it reports the number of small RNA/target site pairs that may affect small RNA binding activity to target transcript. The psRNATarget server is designed for high-throughput analysis of next-generation data with an efficient distributed computing back-end pipeline that runs on a Linux cluster. The server front-end integrates three simplified user-friendly interfaces to accept user-submitted or preloaded small RNAs and transcript sequences; and outputs a comprehensive list of small RNA/target pairs along with the online tools for batch downloading, key word searching and results sorting. The psRNATarget server is freely available at http://plantgrn.noble.org/psRNATarget/.
1,599 citations
TL;DR: It is shown that the rate of gene expression evolution varies among organs, lineages and chromosomes, owing to differences in selective pressures: transcriptome change was slow in nervous tissues and rapid in testes, slower in rodents than in apes and monotremes, and rapid for the X chromosome right after its formation.
Abstract: Changes in gene expression are thought to underlie many of the phenotypic differences between species. However, large-scale analyses of gene expression evolution were until recently prevented by technological limitations. Here we report the sequencing of polyadenylated RNA from six organs across ten species that represent all major mammalian lineages (placentals, marsupials and monotremes) and birds (the evolutionary outgroup), with the goal of understanding the dynamics of mammalian transcriptome evolution. We show that the rate of gene expression evolution varies among organs, lineages and chromosomes, owing to differences in selective pressures: transcriptome change was slow in nervous tissues and rapid in testes, slower in rodents than in apes and monotremes, and rapid for the X chromosome right after its formation. Although gene expression evolution in mammals was strongly shaped by purifying selection, we identify numerous potentially selectively driven expression switches, which occurred at different rates across lineages and tissues and which probably contributed to the specific organ biology of various mammals.
1,130 citations
TL;DR: The generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP provide an approach for genetic encoding of fluorescent RNAs and create a palette that spans the visible spectrum.
Abstract: Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various applications, but a comparable approach for fluorescently tagging RNA is lacking. Here, we describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNA-fluorophore complexes create a palette that spans the visible spectrum. An RNA-fluorophore complex, termed Spinach, resembles enhanced GFP and emits a green fluorescence comparable in brightness with fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach for genetic encoding of fluorescent RNAs.
1,111 citations
TL;DR: The finding that the binding of STAU1 to m RNAs can be transactivated by lncRNAs uncovers an unexpected strategy that cells use to recruit proteins to mRNAs and mediate the decay of these mRNas.
Abstract: Staufen 1 (STAU1)-mediated messenger RNA decay (SMD) involves the degradation of translationally active mRNAs whose 3'-untranslated regions (3' UTRs) bind to STAU1, a protein that binds to double-stranded RNA. Earlier studies defined the STAU1-binding site within ADP-ribosylation factor 1 (ARF1) mRNA as a 19-base-pair stem with a 100-nucleotide apex. However, we were unable to identify comparable structures in the 3' UTRs of other targets of SMD. Here we show that STAU1-binding sites can be formed by imperfect base-pairing between an Alu element in the 3' UTR of an SMD target and another Alu element in a cytoplasmic, polyadenylated long non-coding RNA (lncRNA). An individual lncRNA can downregulate a subset of SMD targets, and distinct lncRNAs can downregulate the same SMD target. These are previously unappreciated functions of non-coding RNAs and Alu elements. Not all mRNAs that contain an Alu element in the 3' UTR are targeted for SMD even in the presence of a complementary lncRNA that targets other mRNAs for SMD. Most known trans-acting RNA effectors consist of fewer than 200 nucleotides, and these include small nucleolar RNAs and microRNAs. Our finding that the binding of STAU1 to mRNAs can be transactivated by lncRNAs uncovers an unexpected strategy that cells use to recruit proteins to mRNAs and mediate the decay of these mRNAs. We name these lncRNAs half-STAU1-binding site RNAs (1/2-sbsRNAs).
1,096 citations
TL;DR: ChIRP-seq of three lncRNAs reveal that RNA occupancy sites in the genome are focal, sequence-specific, and numerous, and generally applicable to illuminate the intersection of RNA and chromatin with newfound precision genome wide.
1,095 citations
TL;DR: It is found that TDP-43 autoregulates its synthesis, in part by directly binding and enhancing splicing of an intron in the 3′ untranslated region of its own transcript, thereby triggering nonsense-mediated RNA degradation.
Abstract: We used cross-linking and immunoprecipitation coupled with high-throughput sequencing to identify binding sites in 6,304 genes as the brain RNA targets for TDP-43, an RNA binding protein that, when mutated, causes amyotrophic lateral sclerosis. Massively parallel sequencing and splicing-sensitive junction arrays revealed that levels of 601 mRNAs were changed (including Fus (Tls), progranulin and other transcripts encoding neurodegenerative disease-associated proteins) and 965 altered splicing events were detected (including in sortilin, the receptor for progranulin) following depletion of TDP-43 from mouse adult brain with antisense oligonucleotides. RNAs whose levels were most depleted by reduction in TDP-43 were derived from genes with very long introns and that encode proteins involved in synaptic activity. Lastly, we found that TDP-43 autoregulates its synthesis, in part by directly binding and enhancing splicing of an intron in the 3' untranslated region of its own transcript, thereby triggering nonsense-mediated RNA degradation.
TL;DR: Tumour microvesicles contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer, including elevated levels of specific coding and non-coding RNA and DNA, mutated and amplified oncogene sequences and transposable elements.
Abstract: Tumour cells release an abundance of microvesicles containing a selected set of proteins and RNAs. Here, we show that tumour microvesicles also carry DNA, which reflects the genetic status of the tumour, including amplification of the oncogene c-Myc. We also find amplified c-Myc in serum microvesicles from tumour-bearing mice. Further, we find remarkably high levels of retrotransposon RNA transcripts, especially for some human endogenous retroviruses, such as LINE-1 and Alu retrotransposon elements, in tumour microvesicles and these transposable elements could be transferred to normal cells. These findings expand the nucleic acid content of tumour microvesicles to include: elevated levels of specific coding and non-coding RNA and DNA, mutated and amplified oncogene sequences and transposable elements. Thus, tumour microvesicles contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer.
TL;DR: Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation, it is found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo, highlighting the importance of T DP-43 for the regulation of splicing in the brain.
Abstract: TDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation (iCLIP), we found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo. Analysis of RNA binding by TDP-43 in brains from subjects with FTLD revealed that the greatest increases in binding were to the MALAT1 and NEAT1 noncoding RNAs. We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A substantial proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or have been implicated in neurological diseases, highlighting the importance of TDP-43 for the regulation of splicing in the brain.
TL;DR: This study identified and validated endogenous protein-coding transcripts that regulate PTEN, antagonize PI3K/AKT signaling, and possess growth- and tumor-suppressive properties and presents a road map for the prediction and validation of ceRNA activity and networks and thus imparts a trans-regulatory function to protein- coding mRNAs.
TL;DR: The salient structural and functional features of Hfq are described and possible mechanisms by which this protein can promote RNA interactions to catalyse specific and rapid regulatory responses in vivo are discussed.
Abstract: Hfq is an RNA-binding protein that is common to diverse bacterial lineages and has key roles in the control of gene expression. By facilitating the pairing of small RNAs with their target mRNAs, Hfq affects the translation and turnover rates of specific transcripts and contributes to complex post-transcriptional networks. These functions of Hfq can be attributed to its ring-like oligomeric architecture, which presents two non-equivalent binding surfaces that are capable of multiple interactions with RNA molecules. Distant homologues of Hfq occur in archaea and eukaryotes, reflecting an ancient origin for the protein family and hinting at shared functions. In this Review, we describe the salient structural and functional features of Hfq and discuss possible mechanisms by which this protein can promote RNA interactions to catalyse specific and rapid regulatory responses in vivo.
TL;DR: It is demonstrated that the NS3 protease (NS3pro) domain alone can specifically bind to HCV-IRES RNA, predominantly in the SLIV region, and that this binding reduces translation in favor of RNA replication.
Abstract: HCV NS3 protein plays a central role in viral polyprotein processing and RNA replication. We demonstrate that the NS3 protease (NS3(pro)) domain alone can specifically bind to HCV-IRES RNA, predominantly in the SLIV region. The cleavage activity of the NS3 protease domain is reduced upon HCV-RNA binding. More importantly, NS3(pro) binding to the SLIV hinders the interaction of La protein, a cellular IRES-trans acting factor required for HCV IRES-mediated translation, resulting in inhibition of HCV-IRES activity. Although overexpression of both NS3(pro) as well as the full length NS3 protein decreased the level of HCV IRES mediated translation, replication of HCV replicon RNA was enhanced significantly. These observations suggest that the NS3(pro) binding to HCV IRES reduces translation in favor of RNA replication. The competition between the host factor (La) and the viral protein (NS3) for binding to HCV IRES might regulate the molecular switch from translation to replication of HCV.
TL;DR: Results suggest a model in which ANRIL binds to and recruits PRC2 to repress the expression of p15INK4B locus, which is bound by SUZ12, a component of polycomb repression complex 2 (PRC2), and is H3K27-trimethylated.
Abstract: A 42 kb region on human chromosome 9p21 encodes for three distinct tumor suppressors, p16INK4A, p14ARF and p15INK4B, and is altered in an estimated 30–40% of human tumors. The expression of the INK4A-ARF-INK4B gene cluster is silenced by polycomb during normal cell growth and is activated by oncogenic insults and during aging. How the polycomb is recruited to repress this gene cluster is unclear. Here, we show that expression of oncogenic Ras, which stimulates the expression of p15INK4B and p16INK4A, but not p14ARF, inhibits the expression of ANRIL (antisense non-coding RNA in the I NK4 locus), a 3.8 kb-long non-coding RNA expressed in the opposite direction from INK4A-ARF-INK4B. We show that the p15INK4B locus is bound by SUZ12, a component of polycomb repression complex 2 (PRC2), and is H3K27-trimethylated. Notably, depletion of ANRIL disrupts the SUZ12 binding to the p15INK4B locus, increases the expression of p15INK4B, but not p16INK4A or p14ARF, and inhibits cellular proliferation. Finally, RNA immunoprecipitation demonstrates that ANRIL binds to SUZ12 in vivo. Collectively, these results suggest a model in which ANRIL binds to and recruits PRC2 to repress the expression of p15INK4B locus.
TL;DR: Structural, mechanistic and molecular biological studies have started to reveal how these conserved proteins can perform such diverse functions and how accessory proteins have a central role in their regulation.
Abstract: RNA helicases of the DEAD box family are present in all eukaryotic cells and in many bacteria and Archaea. These highly conserved enzymes are required for RNA metabolism from transcription to degradation and are therefore important players in gene expression. DEAD box proteins use ATP to unwind short duplex RNA in an unusual fashion and remodel RNA-protein complexes, but they can also function as ATP-dependent RNA clamps to provide nucleation centres that establish larger RNA-protein complexes. Structural, mechanistic and molecular biological studies have started to reveal how these conserved proteins can perform such diverse functions and how accessory proteins have a central role in their regulation.
TL;DR: Exciting breakthroughs in understanding the mechanisms of the CRISPR-Cas system and its potential for biotechnological applications and understanding evolutionary dynamics are discussed.
Abstract: Bacteria and archaea have evolved defense and regulatory mechanisms to cope with various environmental stressors, including virus attack. This arsenal has been expanded by the recent discovery of the versatile CRISPR-Cas system, which has two novel features. First, the host can specifically incorporate short sequences from invading genetic elements (virus or plasmid) into a region of its genome that is distinguished by clustered regularly interspaced short palindromic repeats (CRISPRs). Second, when these sequences are transcribed and precisely processed into small RNAs, they guide a multifunctional protein complex (Cas proteins) to recognize and cleave incoming foreign genetic material. This adaptive immunity system, which uses a library of small noncoding RNAs as a potent weapon against fast-evolving viruses, is also used as a regulatory system by the host. Exciting breakthroughs in understanding the mechanisms of the CRISPR-Cas system and its potential for biotechnological applications and understanding evolutionary dynamics are discussed.
TL;DR: This study provides the first evidence for non‐coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels.
Abstract: MicroRNAs (miRNAs) are ∼22 nt non-coding RNAs that typically bind to the 3′ UTR of target mRNAs in the cytoplasm, resulting in mRNA destabilization and translational repression. Here, we report that miRNAs can also regulate gene expression by targeting non-coding antisense transcripts in human cells. Specifically, we show that miR-671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration-Related protein 1 (CDR1) locus in an Ago2-slicer-dependent manner. The resulting downregulation of circular antisense has a concomitant decrease in CDR1 mRNA levels, independently of heterochromatin formation. This study provides the first evidence for non-coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels.
TL;DR: The parameters remove destabilization of the anti region found in the ff99 force field and thus prevent formation of spurious ladder-like structural distortions in RNA simulations, and improve the description of the syn region and the syn–anti balance as well as enhance MD simulations of various RNA structures.
Abstract: We report a reparameterization of the glycosidic torsion χ of the Cornell et al. AMBER force field for RNA, χOL. The parameters remove destabilization of the anti region found in the ff99 force field and thus prevent formation of spurious ladder-like structural distortions in RNA simulations. They also improve the description of the syn region and the syn–anti balance as well as enhance MD simulations of various RNA structures. Although χOL can be combined with both ff99 and ff99bsc0, we recommend the latter. We do not recommend using χOL for B-DNA because it does not improve upon ff99bsc0 for canonical structures. However, it might be useful in simulations of DNA molecules containing syn nucleotides. Our parametrization is based on high-level QM calculations and differs from conventional parametrization approaches in that it incorporates some previously neglected solvation-related effects (which appear to be essential for obtaining correct anti/high-anti balance). Our χOL force field is compared with sev...
TL;DR: Exosomes in saliva, plasma and breast milk all contain RNA, confirming previous findings that exosomes from several sources contain RNA and supporting the notion thatExosomal RNA can be shuttled between cells.
Abstract: Exosomes are 30-100 nm membrane vesicles of endocytic origin produced by numerous cells. They can mediate diverse biological functions, including antigen presentation. Exosomes have recently been shown to contain functional RNA, which can be delivered to other cells. Exosomes may thus mediate biological functions either by surface-to-surface interactions with cells, or by the delivery of functional RNA to cells. Our aim was therefore to determine the presence of RNA in exosomes from human saliva, plasma and breast milk and whether these exosomes can be taken up by macrophages. Exosomes were purified from human saliva, plasma and breast milk using ultracentrifugation and filtration steps. Exosomes were detected by electron microscopy and examined by flow cytometry. Flow cytometry was performed by capturing the exosomes on anti-MHC class II coated beads, and further stain with anti-CD9, anti-CD63 or anti-CD81. Breast milk exosomes were further analysed for the presence of Hsc70, CD81 and calnexin by Western blot. Total RNA was detected with a Bioanalyzer and mRNA was identified by the synthesis of cDNA using an oligo (dT) primer and analysed with a Bioanalyzer. The uptake of PKH67-labelled saliva and breast milk exosomes by macrophages was examined by measuring fluorescence using flow cytometry and fluorescence microscopy. RNA was detected in exosomes from all three body fluids. A portion of the detected RNA in plasma exosomes was characterised as mRNA. Our result extends the characterisation of exosomes in healthy humans and confirms the presence of RNA in human saliva and plasma exosomes and reports for the first time the presence of RNA in breast milk exosomes. Our results also show that the saliva and breast milk exosomes can be taken up by human macrophages. Exosomes in saliva, plasma and breast milk all contain RNA, confirming previous findings that exosomes from several sources contain RNA. Furthermore, exosomes are readily taken up by macrophages, supporting the notion that exosomal RNA can be shuttled between cells.
TL;DR: The expanded future role of The RNA Modification Database will be to serve as a primary information portal for researchers across the entire spectrum of RNA-related research.
Abstract: Since its inception in 1994, The RNA Modification Database (RNAMDB, http://rna-mdb.cas.albany.edu/RNAmods/) has served as a focal point for information pertaining to naturally occurring RNA modifications. In its current state, the database employs an easy-to-use, searchable interface for obtaining detailed data on the 109 currently known RNA modifications. Each entry provides the chemical structure, common name and symbol, elemental composition and mass, CA registry numbers and index name, phylogenetic source, type of RNA species in which it is found, and references to the first reported structure determination and synthesis. Though newly transferred in its entirety to The RNA Institute, the RNAMDB continues to grow with two notable additions, agmatidine and 8-methyladenosine, appended in the last year. The RNA Modification Database is staying up-to-date with significant improvements being prepared for inclusion within the next year and the following year. The expanded future role of The RNA Modification Database will be to serve as a primary information portal for researchers across the entire spectrum of RNA-related research.
TL;DR: It is demonstrated that the induction of type I interferon by viruses deficient in 2′-O-methyltransferase was dependent on the cytoplasmic RNA sensor Mda5, which suggests that RNA modifications such as 2-methylation provide a molecular signature for the discrimination of self and non-self mRNA.
Abstract: The biological role of 2′-O-methylation of host and viral mRNA has remained elusive. Thiel and co-workers show that this modification modulates the induction of type I interferon and sensitivity to interferon.
TL;DR: Genome-wide analyses from several plant species have revealed that variation in miRNA foldback expression, structure, processing efficiency, and miRNA size have resulted in the unique functionality of MIRNA loci and resulting miRNAs.
Abstract: MicroRNAs (miRNAs) are small regulatory RNAs found in diverse eukaryotic lineages. In plants, a minority of annotated MIRNA gene families are conserved between plant families, while the majority are family- or species-specific, suggesting that most known MIRNA genes arose relatively recently in evolutionary time. Given the high proportion of young MIRNA genes in plant species, new MIRNA families are likely spawned and then lost frequently. Unlike highly conserved, ancient miRNAs, young miRNAs are often weakly expressed, processed imprecisely, lack targets, and display patterns of neutral variation, suggesting that young MIRNA loci tend to evolve neutrally. Genome-wide analyses from several plant species have revealed that variation in miRNA foldback expression, structure, processing efficiency, and miRNA size have resulted in the unique functionality of MIRNA loci and resulting miRNAs. Additionally, some miRNAs have evolved specific properties and functions that regulate other transcriptional or posttranscriptional silencing pathways. The evolution of miRNA processing and functional diversity underscores the dynamic nature of miRNA-based regulation in complex regulatory networks.
TL;DR: It is identified that contaminants, including double-stranded RNA, in nucleoside-modified in vitro-transcribed RNA are responsible for innate immune activation and their removal by high performance liquid chromatography results in mRNA that does not induce IFNs and inflammatory cytokines and is translated at 10- to 1000-fold greater levels in primary cells.
Abstract: In vitro-transcribed mRNA has great therapeutic potential to transiently express the encoded protein without the adverse effects of viral and DNA-based constructs. Mammalian cells, however, contain RNA sensors of the innate immune system that must be considered in the generation of therapeutic RNA. Incorporation of modified nucleosides both reduces innate immune activation and increases translation of mRNA, but residual induction of type I interferons (IFNs) and proinflammatory cytokines remains. We identify that contaminants, including double-stranded RNA, in nucleoside-modified in vitro-transcribed RNA are responsible for innate immune activation and their removal by high performance liquid chromatography (HPLC) results in mRNA that does not induce IFNs and inflammatory cytokines and is translated at 10- to 1000-fold greater levels in primary cells. Although unmodified mRNAs were translated significantly better following purification, they still induced high levels of cytokine secretion. HPLC purified nucleoside-modified mRNA is a powerful vector for applications ranging from ex vivo stem cell generation to in vivo gene therapy.
TL;DR: It is demonstrated that external RNA controls are a useful resource for evaluating sensitivity and accuracy of RNA-seq experiments for transcriptome discovery and quantification and these quality metrics facilitate comparable analysis across different samples, protocols, and platforms.
Abstract: High-throughput sequencing of cDNA (RNA-seq) is a widely deployed transcriptome profiling and annotation technique, but questions about the performance of different protocols and platforms remain. We used a newly developed pool of 96 synthetic RNAs with various lengths, and GC content covering a 2(20) concentration range as spike-in controls to measure sensitivity, accuracy, and biases in RNA-seq experiments as well as to derive standard curves for quantifying the abundance of transcripts. We observed linearity between read density and RNA input over the entire detection range and excellent agreement between replicates, but we observed significantly larger imprecision than expected under pure Poisson sampling errors. We use the control RNAs to directly measure reproducible protocol-dependent biases due to GC content and transcript length as well as stereotypic heterogeneity in coverage across transcripts correlated with position relative to RNA termini and priming sequence bias. These effects lead to biased quantification for short transcripts and individual exons, which is a serious problem for measurements of isoform abundances, but that can partially be corrected using appropriate models of bias. By using the control RNAs, we derive limits for the discovery and detection of rare transcripts in RNA-seq experiments. By using data collected as part of the model organism and human Encyclopedia of DNA Elements projects (ENCODE and modENCODE), we demonstrate that external RNA controls are a useful resource for evaluating sensitivity and accuracy of RNA-seq experiments for transcriptome discovery and quantification. These quality metrics facilitate comparable analysis across different samples, protocols, and platforms.
TL;DR: R-loops formed over G-rich pause sites, followed by their resolution by senataxin, are key steps in the termination process of mammalian RNA polymerase II-transcribed genes.
TL;DR: Comparison of the spatial patterns surrounding HuR and miRNA binding sites provided functional evidence for HuR-dependent antagonism of proximal miRNA-mediated repression and it is concluded that HuR coordinates gene expression outcomes at multiple interconnected steps of RNA processing.
TL;DR: Rationally designed RNA assemblies can thus be used to construct functional architectures in vivo and increased hydrogen output as a function of scaffold architecture.
Abstract: The rules of nucleic acid base-pairing have been used to construct nanoscale architectures and organize biomolecules, but little has been done to apply this technology in vivo. We designed and assembled multidimensional RNA structures and used them as scaffolds for the spatial organization of bacterial metabolism. Engineered RNA modules were assembled into discrete, one-dimensional, and two-dimensional scaffolds with distinct protein-docking sites and used to control the spatial organization of a hydrogen-producing pathway. We increased hydrogen output as a function of scaffold architecture. Rationally designed RNA assemblies can thus be used to construct functional architectures in vivo.