Showing papers on "RNA published in 2016"
TL;DR: The results provide evidence that circular RNA produced from precursor mRNA may have a regulatory role in human cells and characterize one abundant circRNA derived from Exon2 of the HIPK3 gene, termed circHIPK3.
Abstract: Circular RNAs (circRNAs) represent a class of widespread and diverse endogenous RNAs that may regulate gene expression in eukaryotes. However, the regulation and function of human circRNAs remain largely unknown. Here we generate ribosomal-depleted RNA sequencing data from six normal tissues and seven cancers, and detect at least 27,000 circRNA candidates. Many of these circRNAs are differently expressed between the normal and cancerous tissues. We further characterize one abundant circRNA derived from Exon2 of the HIPK3 gene, termed circHIPK3. The silencing of circHIPK3 but not HIPK3 mRNA significantly inhibits human cell growth. Via a luciferase screening assay, circHIPK3 is observed to sponge to 9 miRNAs with 18 potential binding sites. Specifically, we show that circHIPK3 directly binds to miR-124 and inhibits miR-124 activity. Our results provide evidence that circular RNA produced from precursor mRNA may have a regulatory role in human cells.
1,537 citations
TL;DR: LshC2c2 is a RNA-guided RNase which requires the activity of its two HEPN domains, suggesting previously unidentified mechanisms of RNA targeting and degradation by CRISPR systems.
Abstract: The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated genes (Cas) adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the class 2 type VI CRISPR-Cas effector C2c2 and demonstrate its RNA-guided ribonuclease function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis shows that C2c2 is guided by a single CRISPR RNA and can be programmed to cleave single-stranded RNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains, mutations of which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.
1,522 citations
TL;DR: This review guides the reader through important aspects of non-coding RNA biology, including their biogenesis, mode of actions, physiological function, as well as their role in the disease context (such as in cancer or the cardiovascular system).
Abstract: Advances in RNA-sequencing techniques have led to the discovery of thousands of non-coding transcripts with unknown function. There are several types of non-coding linear RNAs such as microRNAs (miRNA) and long non-coding RNAs (lncRNA), as well as circular RNAs (circRNA) consisting of a closed continuous loop. This review guides the reader through important aspects of non-coding RNA biology. This includes their biogenesis, mode of actions, physiological function, as well as their role in the disease context (such as in cancer or the cardiovascular system). We specifically focus on non-coding RNAs as potential therapeutic targets and diagnostic biomarkers.
1,238 citations
TL;DR: A view of the RNA virosphere is presented that is more phylogenetically and genomically diverse than that depicted in current classification schemes and provide a more solid foundation for studies in virus ecology and evolution.
Abstract: Current knowledge of RNA virus biodiversity is both biased and fragmentary, reflecting a focus on culturable or disease-causing agents. Here we profile the transcriptomes of over 220 invertebrate species sampled across nine animal phyla and report the discovery of 1,445 RNA viruses, including some that are sufficiently divergent to comprise new families. The identified viruses fill major gaps in the RNA virus phylogeny and reveal an evolutionary history that is characterized by both host switching and co-divergence. The invertebrate virome also reveals remarkable genomic flexibility that includes frequent recombination, lateral gene transfer among viruses and hosts, gene gain and loss, and complex genomic rearrangements. Together, these data present a view of the RNA virosphere that is more phylogenetically and genomically diverse than that depicted in current classification schemes and provide a more solid foundation for studies in virus ecology and evolution.
1,151 citations
TL;DR: The long non-coding RNA X-inactive specific transcript (XIST) mediates the transcriptional silencing of genes on the X chromosome and is highly methylated with at least 78 N6-methyladenosine (m6A) residues, revealing a pathway of m6A formation and recognition required for XIST-mediated transcriptional repression.
Abstract: The long non-coding RNA X-inactive specific transcript (XIST) mediates the transcriptional silencing of genes on the X chromosome. Here we show that, in human cells, XIST is highly methylated with at least 78 N^6-methyladenosine (m^6A) residues—a reversible base modification of unknown function in long non-coding RNAs. We show that m^6A formation in XIST, as well as in cellular mRNAs, is mediated by RNA-binding motif protein 15 (RBM15) and its paralogue RBM15B, which bind the m^6A-methylation complex and recruit it to specific sites in RNA. This results in the methylation of adenosine nucleotides in adjacent m^6A consensus motifs. Furthermore, we show that knockdown of RBM15 and RBM15B, or knockdown of methyltransferase like 3 (METTL3), an m^6A methyltransferase, impairs XIST-mediated gene silencing. A systematic comparison of m^6A-binding proteins shows that YTH domain containing 1 (YTHDC1) preferentially recognizes m^6A residues on XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m^6A. These data reveal a pathway of m^6A formation and recognition required for XIST-mediated transcriptional repression.
1,108 citations
TL;DR: The results shed light on sRNA biogenesis and its dietary regulation during posttesticular sperm maturation, and they also link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo.
Abstract: Several recent studies link parental environments to phenotypes in subsequent generations. In this work, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA (sRNA) levels in mature sperm, with decreased let-7 levels and increased amounts of 5′ fragments of glycine transfer RNAs (tRNAs). In testicular sperm, tRNA fragments are scarce but increase in abundance as sperm mature in the epididymis. Epididymosomes (vesicles that fuse with sperm during epididymal transit) carry RNA payloads matching those of mature sperm and can deliver RNAs to immature sperm in vitro. Functionally, tRNA-glycine-GCC fragments repress genes associated with the endogenous retroelement MERVL, in both embryonic stem cells and embryos. Our results shed light on sRNA biogenesis and its dietary regulation during posttesticular sperm maturation, and they also link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo.
928 citations
TL;DR: The mechanism of YTHDF2-mediated degradation of m6A-containing RNAs in mammalian cells is uncovered and is shown to be mediated by the CCR4–NOT deadenylase complex.
Abstract: Methylation at the N6 position of adenosine (m(6)A) is the most abundant RNA modification within protein-coding and long noncoding RNAs in eukaryotes and is a reversible process with important biological functions. YT521-B homology domain family (YTHDF) proteins are the readers of m(6)A, the binding of which results in the alteration of the translation efficiency and stability of m(6)A-containing RNAs. However, the mechanism by which YTHDF proteins cause the degradation of m(6)A-containing RNAs is poorly understood. Here we report that m(6)A-containing RNAs exhibit accelerated deadenylation that is mediated by the CCR4-NOT deadenylase complex. We further show that YTHDF2 recruits the CCR4-NOT complex through a direct interaction between the YTHDF2 N-terminal region and the SH domain of the CNOT1 subunit, and that this recruitment is essential for the deadenylation of m(6)A-containing RNAs by CAF1 and CCR4. Therefore, we have uncovered the mechanism of YTHDF2-mediated degradation of m(6)A-containing RNAs in mammalian cells.
871 citations
TL;DR: These findings identify circANRIL as a prototype of a circRNA regulating ribosome biogenesis and conferring atheroprotection, thereby showing that circularization of long non-coding RNAs may alter RNA function and protect from human disease.
Abstract: Circular RNAs (circRNAs) are broadly expressed in eukaryotic cells, but their molecular mechanism in human disease remains obscure. Here we show that circular antisense non-coding RNA in the INK4 locus (circANRIL), which is transcribed at a locus of atherosclerotic cardiovascular disease on chromosome 9p21, confers atheroprotection by controlling ribosomal RNA (rRNA) maturation and modulating pathways of atherogenesis. CircANRIL binds to pescadillo homologue 1 (PES1), an essential 60S-preribosomal assembly factor, thereby impairing exonuclease-mediated pre-rRNA processing and ribosome biogenesis in vascular smooth muscle cells and macrophages. As a consequence, circANRIL induces nucleolar stress and p53 activation, resulting in the induction of apoptosis and inhibition of proliferation, which are key cell functions in atherosclerosis. Collectively, these findings identify circANRIL as a prototype of a circRNA regulating ribosome biogenesis and conferring atheroprotection, thereby showing that circularization of long non-coding RNAs may alter RNA function and protect from human disease.
808 citations
TL;DR: It is shown that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity.
Abstract: Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear. Here we show that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behaviour of the evolutionarily unrelated CRISPR enzyme Cpf1 (ref. 11). The two RNase activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow sensitive detection of cellular transcripts.
801 citations
TL;DR: Results suggest that in the m6A MTase complex, METTL3 primarily functions as the catalytic core, while METTL14 serves as an RNA-binding platform, reminiscent of the target recognition domain of DNA N6-adenine MTase.
Abstract: The structure of the METTL3–METTL14 complex, which mediates N6-adenosine methylation of RNA, suggests that the METTL3 subunit is the catalytic core while METTL14 serves to bind RNA. The various base modifications now known to occur in messenger RNA and long non-coding RNA are reversible, and are utilized to dynamically modify the function of the RNA. The N6-methyladenosine modification is removed by an enzyme complex comprising METTL3 and METTL14. Ping Yin and colleagues have solved structures of the methyltransferase domains of this heterodimeric complex with and without ligand. Surprisingly, the S-adenosyl methionine ligand was found only the METTL3 pocket, not in METTL14. This suggests a model in which there is a single catalytic subunit, with METTL3 functioning as an RNA binding platform. The reported structures provide unprecedented mechanistic insight into m6A RNA methylation and suggest new opportunities for the development of therapeutic agents. Chemical modifications of RNA have essential roles in a vast range of cellular processes1,2,3. N6-methyladenosine (m6A) is an abundant internal modification in messenger RNA and long non-coding RNA that can be dynamically added and removed by RNA methyltransferases (MTases) and demethylases, respectively2,3,4,5. An MTase complex comprising methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14) efficiently catalyses methyl group transfer6,7. In contrast to the well-studied DNA MTase8, the exact roles of these two RNA MTases in the complex remain to be elucidated. Here we report the crystal structures of the METTL3–METTL14 heterodimer with MTase domains in the ligand-free, S-adenosyl methionine (AdoMet)-bound and S-adenosyl homocysteine (AdoHcy)-bound states, with resolutions of 1.9, 1.71 and 1.61 A, respectively. Both METTL3 and METTL14 adopt a class I MTase fold and they interact with each other via an extensive hydrogen bonding network, generating a positively charged groove. Notably, AdoMet was observed in only the METTL3 pocket and not in METTL14. Combined with biochemical analysis, these results suggest that in the m6A MTase complex, METTL3 primarily functions as the catalytic core, while METTL14 serves as an RNA-binding platform, reminiscent of the target recognition domain of DNA N6-adenine MTase9,10. This structural information provides an important framework for the functional investigation of m6A.
719 citations
TL;DR: It is shown that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference and constitutes the most minimalistic of the CRISPR–Cas systems so far described.
Abstract: CRISPR-Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA(1). The well-studied types I, II ...
TL;DR: Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA, leading to reduced expression or altered function of mature miRNAs and certain microRNA precursors.
Abstract: Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. This A-to-I editing occurs not only in protein-coding regions of mRNAs, but also frequently in non-coding regions that contain inverted Alu repeats. Editing of coding sequences can result in the expression of functionally altered proteins that are not encoded in the genome, whereas the significance of Alu editing remains largely unknown. Certain microRNA (miRNA) precursors are also edited, leading to reduced expression or altered function of mature miRNAs. Conversely, recent studies indicate that ADAR1 forms a complex with Dicer to promote miRNA processing, revealing a new function of ADAR1 in the regulation of RNA interference.
TL;DR: This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation, and may be applicable to other plant species.
Abstract: Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research.
TL;DR: The initial functional analysis of a poorly characterized human lncRNA that is induced after DNA damage is described, introducing a mechanism that regulates the activity of a deeply conserved and highly dosage-sensitive family of RNA binding proteins and reveal unanticipated roles for a lnc RNA and PUMILIO proteins in the maintenance of genomic stability.
TL;DR: This Primer outlines the discovery, roles and regulation of circular RNAs, focussing on their potential functions during development and on the regulation of and functional roles played by these molecules.
Abstract: Just a few years ago, it had been assumed that the dominant RNA isoforms produced from eukaryotic genes were variants of messenger RNA, functioning as intermediates in gene expression. In early 2012, however, a surprising discovery was made: circular RNA (circRNA) was shown to be a transcriptional product in thousands of human and mouse genes and in hundreds of cases constituted the dominant RNA isoform. Subsequent studies revealed that the expression of circRNAs is developmentally regulated, tissue and cell-type specific, and shared across the eukaryotic tree of life. These features suggest important functions for these molecules. Here, we describe major advances in the field of circRNA biology, focusing on the regulation of and functional roles played by these molecules.
TL;DR: This chapter focuses on the study of miRNA-lncRNA interactions with either in silico or experimentally supported analyses, and proposes methodologies that can be appropriately adapted in order to become the backbone of advanced multistep functional miRNA analyses.
Abstract: Long noncoding RNAs (lncRNAs) are noncoding transcripts usually longer than 200 nts that have recently emerged as one of the largest and significantly diverse RNA families. The biological role and functions of lncRNAs are still mostly uncharacterized. Their target-mimetic, sponge/decoy function on microRNAs was recently uncovered. miRNAs are a class of noncoding RNA species (~22 nts) that play a central role in posttranscriptional regulation of protein coding genes by mRNA cleavage, direct translational repression and/or mRNA destabilization. LncRNAs can act as miRNA sponges, reducing their regulatory effect on mRNAs. This function introduces an extra layer of complexity in the miRNA-target interaction network. This chapter focuses on the study of miRNA-lncRNA interactions with either in silico or experimentally supported analyses. The proposed methodologies can be appropriately adapted in order to become the backbone of advanced multistep functional miRNA analyses.
TL;DR: Together, these features raise fundamental questions regarding the regulation of circRNA in cis and in trans, and its function, which are enriched in the brain and increase in abundance during fetal development.
TL;DR: Dwarf open reading frame (DWORF) is the only endogenous peptide known to activate the SERCA pump by physical interaction and provides a means for enhancing muscle contractility.
Abstract: Muscle contraction depends on release of Ca2+ from the sarcoplasmic reticulum (SR) and reuptake by the Ca2+adenosine triphosphatase SERCA. We discovered a putative muscle-specific long noncoding RNA that encodes a peptide of 34 amino acids and that we named dwarf open reading frame (DWORF). DWORF localizes to the SR membrane, where it enhances SERCA activity by displacing the SERCA inhibitors, phospholamban, sarcolipin, and myoregulin. In mice, overexpression of DWORF in cardiomyocytes increases peak Ca2+ transient amplitude and SR Ca2+ load while reducing the time constant of cytosolic Ca2+ decay during each cycle of contraction-relaxation. Conversely, slow skeletal muscle lacking DWORF exhibits delayed Ca2+ clearance and relaxation and reduced SERCA activity. DWORF is the only endogenous peptide known to activate the SERCA pump by physical interaction and provides a means for enhancing muscle contractility.
TL;DR: It is the diversity of RNA species detected through RNA-seq that holds new promise for the multi-faceted clinical applicability of RNA-based measures, including the potential of extracellular RNAs as non-invasive diagnostic indicators of disease.
Abstract: With the emergence of RNA sequencing (RNA-seq) technologies, RNA-based biomolecules hold expanded promise for their diagnostic, prognostic and therapeutic applicability in various diseases, including cancers and infectious diseases. Detection of gene fusions and differential expression of known disease-causing transcripts by RNA-seq represent some of the most immediate opportunities. However, it is the diversity of RNA species detected through RNA-seq that holds new promise for the multi-faceted clinical applicability of RNA-based measures, including the potential of extracellular RNAs as non-invasive diagnostic indicators of disease. Ongoing efforts towards the establishment of benchmark standards, assay optimization for clinical conditions and demonstration of assay reproducibility are required to expand the clinical utility of RNA-seq.
TL;DR: PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells, is developed and provides novel insights into the RNA structurome and interactome.
TL;DR: This review will summarize the current knowledge of the biological roles of mRNA caps in eukaryotic cells, different means that viruses and their host cells use to cap their RNA and the application of these capping machineries to synthesize functional mRNA.
Abstract: The 5' m7G cap is an evolutionarily conserved modification of eukaryotic mRNA. Decades of research have established that the m7G cap serves as a unique molecular module that recruits cellular proteins and mediates cap-related biological functions such as pre-mRNA processing, nuclear export and cap-dependent protein synthesis. Only recently has the role of the cap 2'O methylation as an identifier of self RNA in the innate immune system against foreign RNA has become clear. The discovery of the cytoplasmic capping machinery suggests a novel level of control network. These new findings underscore the importance of a proper cap structure in the synthesis of functional messenger RNA. In this review, we will summarize the current knowledge of the biological roles of mRNA caps in eukaryotic cells. We will also discuss different means that viruses and their host cells use to cap their RNA and the application of these capping machineries to synthesize functional mRNA. Novel applications of RNA capping enzymes in the discovery of new RNA species and sequencing the microbiome transcriptome will also be discussed. We will end with a summary of novel findings in RNA capping and the questions these findings pose.
Journal Article•
TL;DR: The discovery of extensive transcription of long noncoding RNAs (lncRNAs) provide an important new perspective on the centrality of RNA in gene regulation as mentioned in this paper, and they discuss genome-scale strategies to
Abstract: The discovery of extensive transcription of long noncoding RNAs (lncRNAs) provide an important new perspective on the centrality of RNA in gene regulation I will discuss genome-scale strategies to
TL;DR: This work identified 1,174 binding sites within 529 HeLa cell RBPs, discovering numerous RNA-binding domains (RBDs), which display a high degree of evolutionary conservation and incidence of Mendelian mutations, suggestive of important functional roles.
TL;DR: It is shown that m( 1)A is prevalent in Homo sapiens mRNA, which shows an m(1)A/A ratio of ∼0.02%, and it is shownthat m(2)A in mRNA is reversible by ALKBH3, a known DNA/RNA demethylase.
Abstract: N(1)-Methyladenosine (m(1)A) is a prevalent post-transcriptional RNA modification, yet little is known about its abundance, topology and dynamics in mRNA. Here, we show that m(1)A is prevalent in Homo sapiens mRNA, which shows an m(1)A/A ratio of ∼0.02%. We develop the m(1)A-ID-seq technique, based on m(1)A immunoprecipitation and the inherent ability of m(1)A to stall reverse transcription, as a means for transcriptome-wide m(1)A profiling. m(1)A-ID-seq identifies 901 m(1)A peaks (from 600 genes) in mRNA and noncoding RNA and reveals a prominent feature, enrichment in the 5' untranslated region of mRNA transcripts, that is distinct from the pattern for N(6)-methyladenosine, the most abundant internal mammalian mRNA modification. Moreover, m(1)A in mRNA is reversible by ALKBH3, a known DNA/RNA demethylase. Lastly, we show that m(1)A methylation responds dynamically to stimuli, and we identify hundreds of stress-induced m(1)A sites. Collectively, our approaches allow comprehensive analysis of m(1)A modification and provide tools for functional studies of potential epigenetic regulation via the reversible and dynamic m(1)A methylation.
TL;DR: It is demonstrated that nuclease-inactive S. pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells and establishes RCas9 as a means to track RNA inliving cells in a programmable manner without genetically encoded tags.
TL;DR: Understanding the structure, function, and interactions of the RNA-synthesizing machinery of coronaviruses will be key to rationalizing their evolutionary success and the development of improved control strategies.
Abstract: Coronaviruses are animal and human pathogens that can cause lethal zoonotic infections like SARS and MERS. They have polycistronic plus-stranded RNA genomes and belong to the order Nidovirales, a diverse group of viruses for which common ancestry was inferred from the common principles underlying their genome organization and expression, and from the conservation of an array of core replicase domains, including key RNA-synthesizing enzymes. Coronavirus genomes (~26-32 kilobases) are the largest RNA genomes known to date and their expansion was likely enabled by acquiring enzyme functions that counter the commonly high error frequency of viral RNA polymerases. The primary functions that direct coronavirus RNA synthesis and processing reside in nonstructural protein (nsp) 7 to nsp16, which are cleavage products of two large replicase polyproteins translated from the coronavirus genome. Significant progress has now been made regarding their structural and functional characterization, stimulated by technical advances like improved methods for bioinformatics and structural biology, in vitro enzyme characterization, and site-directed mutagenesis of coronavirus genomes. Coronavirus replicase functions include more or less universal activities of plus-stranded RNA viruses, like an RNA polymerase (nsp12) and helicase (nsp13), but also a number of rare or even unique domains involved in mRNA capping (nsp14, nsp16) and fidelity control (nsp14). Several smaller subunits (nsp7-nsp10) act as crucial cofactors of these enzymes and contribute to the emerging "nsp interactome." Understanding the structure, function, and interactions of the RNA-synthesizing machinery of coronaviruses will be key to rationalizing their evolutionary success and the development of improved control strategies.
01 Jan 2016
TL;DR: The EBV-encoded RNA (EBER 1 and EBER 2) were found to be associated with protein(s) associated with systemic lupus erythematosus-associated antibody anti-La.
Abstract: Primate cells harboring the Epstein-Barr virus (EBV) genome synthesize large amounts of two small RNAs: EBER 1 and EBER 2 (EBV-encoded RNA). These RNAs are approxi- mately 180 nucleotides long, possess 5' pppA termini, and lack poly(A). They have different TI and pancreatic RNase digestion fingerprints. They are not found in normal B lymphocytes, in transformed B lymphocytes that lack EBV DNA, in T lymphocytes transformed by Herpesvirus ateles, or in a variety of other nonlym- phoid mammalian cells. Hybridization analyses indicate that EBER l and EBER 2 are encoded by the EcoRI-J fragment of EBV (B95-8) DNA. In vivo both RNAs are associated with protein(s), al- lowing their specific precipitation by the systemic lupus erythe- matosus-associated antibody anti-La. The La antigen in uninfected mammalian cells consists of a heterogeneous class of small ribonu- cleoprotein particles, some of whose RNA components exhibit se- quence homology with a highly repetitive, interspersed class of hu- man DNA designated the Alu family. Possible functions for EBER 1 and EBER 2 in infection and cell transformation by EBV and their potential relationship to the pathogenesis of systemic lupus erythematosus are discussed.
TL;DR: It is found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction, and serves an important role in the secretion of miRNAs in exosomes by HEK293T cells.
Abstract: Exosomes are small vesicles that are secreted from metazoan cells and may convey selected membrane proteins and small RNAs to target cells for the control of cell migration, development and metastasis. To study the mechanisms of RNA packaging into exosomes, we devised a purification scheme based on the membrane marker CD63 to isolate a single exosome species secreted from HEK293T cells. Using immunoisolated CD63-containing exosomes we identified a set of miRNAs that are highly enriched with respect to their cellular levels. To explore the biochemical requirements for exosome biogenesis and RNA packaging, we devised a cell-free reaction that recapitulates the species-selective enclosure of miR-223 in isolated membranes supplemented with cytosol. We found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction. Furthermore, YBX1 serves an important role in the secretion of miRNAs in exosomes by HEK293T cells.
TL;DR: Single-cell RNA sequencing was used to determine the transcriptomes of 1,492 human pancreatic α, β, δ, and PP cells from non-diabetic and type 2 diabetes organ donors and identified cell-type-specific genes and pathways as well as 245 genes with disturbed expression intype 2 diabetes.
TL;DR: A computational predictor of mammalian m(6)A site named SRAMP, which combines three random forest classifiers that exploit the positional nucleotide sequence pattern, the K-nearest neighbor information and the position-independent nucleotide pair spectrum features, respectively.
Abstract: N(6)-methyladenosine (m(6)A) is a prevalent RNA methylation modification involved in the regulation of degradation, subcellular localization, splicing and local conformation changes of RNA transcripts. High-throughput experiments have demonstrated that only a small fraction of the m(6)A consensus motifs in mammalian transcriptomes are modified. Therefore, accurate identification of RNA m(6)A sites becomes emergently important. For the above purpose, here a computational predictor of mammalian m(6)A site named SRAMP is established. To depict the sequence context around m(6)A sites, SRAMP combines three random forest classifiers that exploit the positional nucleotide sequence pattern, the K-nearest neighbor information and the position-independent nucleotide pair spectrum features, respectively. SRAMP uses either genomic sequences or cDNA sequences as its input. With either kind of input sequence, SRAMP achieves competitive performance in both cross-validation tests and rigorous independent benchmarking tests. Analyses of the informative features and overrepresented rules extracted from the random forest classifiers demonstrate that nucleotide usage preferences at the distal positions, in addition to those at the proximal positions, contribute to the classification. As a public prediction server, SRAMP is freely available at http://www.cuilab.cn/sramp/.