Circular RNAs are abundant,conserved, and associated with ALU repeats

Circular RNAs composed of exonic sequence have been described in a small number of genes. Thought to result from splicing errors, circular RNA species possess no known function. To delineate the universe of endogenous circular RNAs, we performed high-throughput sequencing (RNA-seq) of libraries prepared from ribosome-depleted RNA with or without digestion with the RNA exonuclease, RNase R. We identified >25,000 distinct RNA species in human fibroblasts that contained non-colinear exons (a “backsplice”) and were reproducibly enriched by exonuclease degradation of linear RNA. These RNAs were validated as circular RNA (ecircRNA), rather than linear RNA, and were more stable than associated linear mRNAs in vivo. In some cases, the abundance of circular molecules exceeded that of associated linear mRNA by >10-fold. By conservative estimate, we identified ecircRNAs from 14.4% of actively transcribed genes in human fibroblasts. Application of this method to murine testis RNA identified 69 ecircRNAs in precisely orthologous locations to human circular RNAs. Of note, paralogous kinases HIPK2 and HIPK3 produce abundant ecircRNA from their second exon in both humans and mice. Though HIPK3 circular RNAs contain an AUG translation start, it and other ecircRNAs were not bound to ribosomes. Circular RNAs could be degraded by siRNAs and, therefore, may act as competing endogenous RNAs. Bioinformatic analysis revealed shared features of circularized exons, including long bordering introns that contained complementary ALU repeats. These data show that ecircRNAs are abundant, stable, conserved and nonrandom products of RNA splicing that could be involved in control of gene expression.     

Codon-Anticodon Recognition in the Bacillus subtilis glyQS T Box Riboswitch: RNA-DEPENDENT CODON SELECTION OUTSIDE THE RIBOSOME*

Many amino acid-related genes in Gram-positive bacteria are regulated by the T box riboswitch. The leader RNA of genes in the T box family controls the expression of downstream genes by monitoring the aminoacylation status of the cognate tRNA. Previous studies identified a three-nucleotide codon, termed the “Specifier Sequence,” in the riboswitch that corresponds to the amino acid identity of the downstream genes. Pairing of the Specifier Sequence with the anticodon of the cognate tRNA is the primary determinant of specific tRNA recognition. This interaction mimics codon-anticodon pairing in translation but occurs in the absence of the ribosome. The goal of the current study was to determine the effect of a full range of mismatches for comparison with codon recognition in translation. Mutations were individually introduced into the Specifier Sequence of the glyQS leader RNA and tRNA^Gly anticodon to test the effect of all possible pairing combinations on tRNA binding affinity and antitermination efficiency. The functional role of the conserved purine 3′ of the Specifier Sequence was also verifiedin this study. We found that substitutions at the Specifier Sequence resulted in reduced binding, the magnitude of which correlates well with the predicted stability of the RNA-RNA pairing. However, the tolerance for specific mismatches in antitermination was generally different from that during decoding, which reveals a unique tRNA recognition pattern in the T box antitermination system.     

The yjdF riboswitch candidate regulates gene expression by binding diverse azaaromatic compounds

The yjdF motif RNA is an orphan riboswitch candidate that almost exclusively associates with the yjdF protein-coding gene in many bacteria. The function of the YjdF protein is unknown, which has made speculation regarding the natural ligand for this putative riboswitch unusually challenging. By using a structure-probing assay for ligand binding, we found that a surprisingly broad diversity of nitrogen-containing aromatic heterocycles, or “azaaromatics,” trigger near-identical changes in the structures adopted by representative yjdF motif RNAs. Regions of the RNA that undergo ligand-induced structural modulation reside primarily in portions of the putative aptamer region that are highly conserved in nucleotide sequence, as is typical for riboswitches. Some azaaromatic molecules are bound by the RNA with nanomolar dissociation constants, and a subset of these ligands activate riboswitch-mediated gene expression in cells. Furthermore, genetic elements most commonly adjacent to the yjdF motif RNA or to the yjdF protein-coding region are homologous to protein regulators implicated in mitigating the toxic effects of diverse phenolic acids or polycyclic compounds. Although the precise type of natural ligand sensed by yjdF motif RNAs remains unknown, our findings suggest that this riboswitch class might serve as part of a genetic response system to toxic or signaling compounds with chemical structures similar to azaaromatics.     

Bacterial noncoding Y RNAs are widespread and mimic tRNAs

Many bacteria encode an ortholog of the Ro60 autoantigen, a ring-shaped protein that is bound in animal cells to noncoding RNAs (ncRNAs) called Y RNAs. Studies in Deinococcus radiodurans revealed that Y RNA tethers Ro60 to polynucleotide phosphorylase, specializing this exoribonuclease for structured RNA degradation. Although Ro60 orthologs are present in a wide range of bacteria, Y RNAs have been detected in only two species, making it unclear whether these ncRNAs are common Ro60 partners in bacteria. In this study, we report that likely Y RNAs are encoded near Ro60 in >250 bacterial and phage species. By comparing conserved features, we discovered that at least one Y RNA in each species contains a domain resembling tRNA. We show that these RNAs contain nucleotide modifications characteristic of tRNA and are substrates for several enzymes that recognize tRNAs. Our studies confirm the importance of Y RNAs in bacterial physiology and identify a new class of ncRNAs that mimic tRNA.     

A conserved motif of vertebrate Y RNAs essential for chromosomal DNA replication

Noncoding Y RNAs are required for the reconstitution of chromosomal DNA replication in late G1 phase template nuclei in a human cell-free system. Y RNA genes are present in all vertebrates and in some isolated nonvertebrates, but the conservation of Y RNA function and key determinants for its function are unknown. Here, we identify a determinant of Y RNA function in DNA replication, which is conserved throughout vertebrate evolution. Vertebrate Y RNAs are able to reconstitute chromosomal DNA replication in the human cell-free DNA replication system, but nonvertebrate Y RNAs are not. A conserved nucleotide sequence motif in the double-stranded stem of vertebrate Y RNAs correlates with Y RNA function. A functional screen of human Y1 RNA mutants identified this conserved motif as an essential determinant for reconstituting DNA replication in vitro. Double-stranded RNA oligonucleotides comprising this RNA motif are sufficient to reconstitute DNA replication, but corresponding DNA or random sequence RNA oligonucleotides are not. In intact cells, wild-type hY1 or the conserved RNA duplex can rescue an inhibition of DNA replication after RNA interference against hY3 RNA. Therefore, we have identified a new RNA motif that is conserved in vertebrate Y RNA evolution, and essential and sufficient for Y RNA function in human chromosomal DNA replication.     

Serum,insulin and phorbol esters stimulate rRNA and tRNA gene expression in both dividing and nondividing Drosophila cells

The expression of genes that code for the large ribosomal RNAs (rRNAs) and tRNAs can be regulated by calcium, serum, insulin and a tumor-promoting phorbol ester, TPA. These effectors can rapidly alter rRNA and tRNA synthesis in dividing and nondividing Drosophila cells. In an in vitro assay system of the nondividing cells of the male accessory glands, calcium, insulin and TPA were shown to increase both rRNA and tRNA synthesis. Exposure of actively dividing Drosophila culture cells to differing serum concentrations or TPA also altered rRNA and tRNA synthesis. Nuclear run-on assays demonstrate that the exposure of these cells to increased serum concentrations coordinately alters RNA polymerase I loading on both 18S and 28S rDNA. These data indicate that calcium, growth factors and a tumor-promoter each can signal changes in ribosomal and tRNA gene expression.     

Correlation between seed longevity and RNA integrity in the embryos of rice seeds

The mature embryos of rice seeds contain translatable mRNAs required for the initial phase of germination. To clarify the relationship between seed longevity and RNA integrity in embryos, germinability and stability of embryonic RNAs were analyzed using the seeds of japonica rice cultivars subjected to controlled deterioration treatment (CDT) or long periods of storage. Degradation of RNA from embryos of a japonica rice cultivar “Nipponbare” was induced by CDT before the decline of the germination rate and we observed a positive relationship between seed germinability and integrity of embryonic RNAs. Moreover, this relationship was confirmed in the experiments using aged seeds from the “Nipponbare”, “Sasanishiki” and “Koshihikari” rice cultivars. In addition, the RNA integrity number (RIN) values, calculated using electrophoresis data and Agilent Bioanalyzer software, had a positive correlation with germinability (R²=0.75). Therefore, the stability of embryonic RNAs required for germination is involved in maintaining seed longevity over time and RIN values can serve as a quantitative indicator to evaluate germinability in rice.     

Thermodynamics of RNA melting,one base pair at a time

The melting of base pairs is a ubiquitous feature of RNA structural transitions, which are widely used to sense and respond to cellular stimuli. A recent study employing solution nuclear magnetic resonance (NMR) imino proton exchange spectroscopy provides a rare base-pair-specific view of duplex melting in the Salmonella FourU RNA thermosensor, which regulates gene expression in response to changes in temperature at the translational level by undergoing a melting transition. The authors observe “microscopic” enthalpy–entropy compensation—often seen “macroscopically” across a series of related molecular species—across base pairs within the same RNA. This yields variations in base-pair stabilities that are an order of magnitude smaller than corresponding variations in enthalpy and entropy. A surprising yet convincing link is established between the slopes of enthalpy–entropy correlations and RNA melting points determined by circular dichroism (CD), which argues that unfolding occurs when base-pair stabilities are equalized. A single AG-to-CG mutation, which enhances the macroscopic hairpin thermostability and folding cooperativity and renders the RNA thermometer inactive in vivo, spreads its effect microscopically throughout all base pairs in the RNA, including ones far removed from the site of mutation. The authors suggest that an extended network of hydration underlies this long-range communication. This study suggests that the deconstruction of macroscopic RNA unfolding in terms of microscopic unfolding events will require careful consideration of water interactions.     

Phylogeny and evolution of chloroplast tRNAs in Adoxaceae

Chloroplasts are semiautonomous organelles found in photosynthetic plants. The major functions of chloroplasts include photosynthesis and carbon fixation, which are mainly regulated by its circular genomes. In the highly conserved chloroplast genome, the chloroplast transfer RNA genes (cp tRNA) play important roles in protein translation within chloroplasts. However, the evolution of cp tRNAs remains unclear. Thus, in the present study, we investigated the evolutionary characteristics of chloroplast tRNAs in five Adoxaceae species using 185 tRNA gene sequences. In total, 37 tRNAs encoding 28 anticodons are found in the chloroplast genome in Adoxaceae species. Some consensus sequences are found within the Ψ‐stem and anticodon loop of the tRNAs. Some putative novel structures were also identified, including a new stem located in the variable region of tRNA^Tyr in a similar manner to the anticodon stem. Furthermore, phylogenetic and evolutionary analyses indicated that synonymous tRNAs may have evolved from multiple ancestors and frequent tRNA duplications during the evolutionary process may have been primarily caused by positive selection and adaptive evolution. The transition and transversion rates are uneven among different tRNA isotypes. For all tRNAs, the transition rate is greater with a transition/transversion bias of 3.13. Phylogenetic analysis of cp tRNA suggested that the type I introns in different taxa (including eukaryote organisms and cyanobacteria) share the conserved sequences “U‐U‐x2‐C” and “U‐x‐G‐x2‐T,” thereby indicating the diverse cyanobacterial origins of organelles. This detailed study of cp tRNAs in Adoxaceae may facilitate further investigations of the evolution, phylogeny, structure, and related functions of chloroplast tRNAs.     

Intracellular mRNA cleavage by 3' tRNase under the direction of 2'-O-methyl RNA heptamers

Mammalian tRNA 3′ processing endoribonuclease (3′-tRNase) can cleave any RNA at any site under the direction of small guide RNA (sgRNA) in vitro. sgRNAs can be as short as heptamers, which are much smaller than small interfering RNAs of ~21 nt. Together with such flexibility in substrate recognition, the ubiquity and the constitutive expression of 3′-tRNase have suggested that this enzyme can be utilized for specific cleavage of cellular RNAs by introducing appropriate sgRNAs into living cells. Here we demonstrated that the expression of chloramphenicol acetyltransferase can be downregulated by an appropriate sgRNA which is introduced into Madin–Darby canine kidney epithelial cells as an expression plasmid or a synthetic 2′-O-methyl RNA. We also showed that 2′-O-methyl RNA heptamers can attack luciferase mRNAs with a high specificity and induce 3′-tRNase-mediated knock-down of the mRNAs in 293 cells. Furthermore, the MTT cell viability assay suggested that an RNA heptamer can downregulate the endogenous Bcl-2 mRNA in Sarcoma 180 cells. This novel sgRNA/3′-tRNase strategy for destroying specific cellular RNAs may be utilized for therapeutic applications.     

tRNA anticodon shifts in eukaryotic genomes

Embedded in the sequence of each transfer RNA are elements that promote specific interactions with its cognate aminoacyl tRNA-synthetase. Although many such “identity elements” are known, their detection is difficult since they rely on unique structural signatures and the combinatorial action of multiple elements spread throughout the tRNA molecule. Since the anticodon is often a major identity determinant itself, it is possible to switch between certain tRNA functional types by means of anticodon substitutions. This has been shown to have occurred during the evolution of some genomes; however, the scale and relevance of “anticodon shifts” to the evolution of the tRNA multigene family is unclear. Using a synteny-conservation–based method, we detected tRNA anticodon shifts in groups of closely related species: five primates, 12 Drosophila, six nematodes, 11 Saccharomycetes, and 61 Enterobacteriaceae. We found a total of 75 anticodon shifts: 31 involving switches of identity (alloacceptor shifts) and 44 between isoacceptors that code for the same amino acid (isoacceptor shifts). The relative numbers of shifts in each taxa suggest that tRNA gene redundancy is likely the driving factor, with greater constraint on changes of identity. Sites that frequently covary with alloacceptor shifts are located at the extreme ends of the molecule, in common with most known identity determinants. Isoacceptor shifts are associated with changes in the midsections of the tRNA sequence. However, the mutation patterns of anticodon shifts involving the same identities are often dissimilar, suggesting that alternate sets of mutation may achieve the same functional compensation.     

RNA促进小鼠重组染色质白蛋白基因DNaseⅠ消化敏感性

同样的基因在不同的分化细胞中表达不同,基因的选择性表达问题涉及分化和衰老的本质。转录基因对DNaseⅠ(DNA酶Ⅰ)消化敏感,本文研究了RNA对小鼠重组染色质白蛋白基因DNaseⅠ消化敏感性的影响。分离BALB/c小鼠脑细胞核,加入终浓度为2mol/L的NaCl破坏核小体结构,加入不同量、不同来源的RNA,装透析袋,逐渐降低离子强度进行染色质重组。重组染色质中加入DNaseⅠ消化DNA,PCR扩增白蛋白基因的外显子1到外显子2约1200bp区段,PAGE电泳后,用银染色观察不同来源RNA促进DNaseⅠ对白蛋白基因的消化作用。不同组织来源（肝、肺、肾、脑）RNA对小鼠重组染色质中白蛋白基因DNaseⅠ消化敏感性均有促进作用,其中肝和肺RNA促进消化作用较强;酵母tRNA无显著促进消化作用;消化促进作用与RNA剂量有关。RNA能增加DNaseⅠ对白蛋白基因的消化敏感性且有组织（细胞）来源特异性。又委托丹麦Chemical R D 实验室合成2条与白蛋白基因互补的各23核苷酸的RNA,用其进行重组试验。结果表明,重组混合物中含有低至0.2μg/mL的RNA,即可以发挥显著的DNase I消化促进作用。     

CIRCexplorer3:A CLEAR Pipeline for Direct Comparison of Circular and Linear RNA Expression

Sequences of circular RNAs(circ RNAs) produced from back-splicing of exon(s) completely overlap with those from cognate linear RNAs transcribed from the same gene loci with the exception of their back-splicing junction(BSJ) sites.Therefore,examination of global circ RNA expression from RNA-seq datasets generally relies on the detection of RNA-seq fragments spanning BSJ sites,which is different from the quantification of linear RNA expression by normalized RNA-seq fragments mapped to whole gene bodies.Thus,direct comparison of circular and linear RNA expression from the same gene loci in a genome-wide manner has remained challenging.Here,we update the previously-reported CIRCexplorer pipeline to version 3 for circular and linear RNA expression analysis from ribosomal-RNA depleted RNA-seq(CIRCexplorer3-CLEAR).A new quantitation parameter,fragments per billion mapped bases(FPB),is applied to evaluate circular and linear RNA expression individually by fragments mapped to circ RNA-specific BSJ sites or to linear RNA-specific splicing junction(SJ) sites.Comparison of circular and linear RNA expression levels is directly achieved by dividing FPBcircby FPBlinearto generate a CIRCscore,which indicates the relative circ RNA expression level using linear RNA expression level as the background.Highlyexpressed circ RNAs with low cognate linear RNA expression background can be readily identified by CIRCexplorer3-CLEAR for further investigation.CIRCexplorer3-CLEAR is publically available at https://github.com/Yang Lab/CLEAR.     

Review of Non-coding RNAs and the epigenetic regulation of gene expression: A book edited by Kevin Morris

Advances in sequencing and detection technology over the past two decades, highlighted by the data explosion brought about by the human genome project, have transformed what was previously assumed to be a relatively simple genetic landscape into a new picture where the so-called “dark matter” of the genome has stolen the spotlight from the not so hip protein-coding genes. The simplified central dogma of molecular biology, in which a gene encodes for a protein via a messenger RNA (mRNA), is still at the core of genetics but is now caught in a much more complex web of regulation by the genomic region previously known as “junk” DNA. Books such as Non-coding RNAs and epigenetic regulation of gene expression, published by Caister Academic Press, become essential guidelines to help us understand the current status of the very fast paced field of RNA research, which has only just started to uncover the roles of non-coding RNAs (ncRNAs) in the regulation of gene expression.