Viroids: the minimal non-coding RNAs with autonomous replication

Viroids are small (246-401 nucleotides), non-coding, circular RNAs able to replicate autonomously in certain plants. Viroids are classified into the families Pospiviroidae and Avsunviroidae, whose members replicate in the nucleus and chloroplast, respectively. Replication occurs by an RNA-based rolling-circle mechanism in three steps: (1). synthesis of longer-than-unit strands catalyzed by host DNA-dependent RNA polymerases forced to transcribe RNA templates, (2). processing to unit-length, which in family Avsunviroidae is mediated by hammerhead ribozymes, and (3). circularization either through an RNA ligase or autocatalytically. Disease induction might result from the accumulation of viroid-specific small interfering RNAs that, via RNA silencing, could interfere with normal developmental pathways.     

RNA end-labeling and RNA ligase activities can produce a circular rRNA in whole cell extracts from trypanosomes.

We have found two enzymatic activities in whole cell extracts from Trypanosoma brucei; an end-labeling reaction involving a single uridine at the 3' end of ribosomal RNAs (rRNAs) and an RNA ligase activity joining 5' monophosphates to 3' hydroxyl groups. The RNA ligase acts upon one of the small rRNAs (180 nucleotides) from the trypanosome ribosomal repeat unit, forming a circular RNA. The specific circularization of this small rRNA is probably dependent on the secondary structure of the molecule and is not detectable in vivo.     

Two reactions of Haloferax volcanii RNA splicing enzymes: joining of exons and circularization of introns

Archaeal RNA splicing involves at least two protein enzymes, a specific endonuclease and a specific ligase. The endonuclease recognizes and cleaves within a characteristic bulge-helix-bulge (BHB) structure formed by pairing of the regions near the two exon-intron junctions, producing 2',3'-cyclic phosphate and 5'-hydroxyl termini. The ligase joins the exons and converts the cyclic phosphate into junction phosphate. The ligated product contains a seven-base hairpin loop, in which the splice junction is in between the two 3' terminal residues of the loop. Archaeal splicing endonucleases are also involved in rRNA processing, cutting within the BHB structures formed by pairing of the 5' and 3' flanking regions of the rRNAs. Large free introns derived from pre-rRNAs have been observed as stable and abundant circular RNAs in certain Crenarchaeota, a kingdom in the domain Archaea. In the present study, we show that the cells of Haloferax volcanii, a Euryarchaeote, contain circular RNAs formed by 3',5'-phosphodiester linkage between the two termini of the introns derived from their pre-tRNAs. H. volcanii ligase, in vitro, can also circularize both endonuclease-cleaved introns, and non-endonuclease-produced substrates. Exon joining and intron circularization are mechanistically similar ligation reactions that can occur independently. The size of the ligated hairpin loop and position of the splice junction within this loop can be changed in in vitro ligation reactions. Overall, archaeal RNA splicing seems to involve two sets of two symmetric transesterification reactions each.     

环形RNA分子揭秘

环形RNA分子是一类不具有5'末端帽子和3'末端poly(A)尾巴、并以共价键形成环形结构的非编码RNA分子。利用针对环形结构RNA分子的实验和计算方法,并结合新一代高通量测序,现已在多种细胞内发现了大量环形RNA分子的稳定存在。目前,尽管环形RNA分子的产生机制及其代谢仍然不清楚,但是已有的研究表明环形RNA分子具有调控基因表达的功能。对环形RNA分子的产生机制和功能等的进一步研究,将为人们深入理解生命活动在转录水平的复杂性调控提供重要的分子基础和研究依据。     

Oligoribonucleotide circularization by 'template-mediated' ligation with T4 RNA ligase: synthesis of circular hammerhead ribozymes.

Circular hammerhead ribozymes were synthesized from linear oligoribonucleotides using T4 RNA ligase. Some of the precursors could not be efficiently circularized under standard conditions. For these molecules, the use of a DNA template allowed their efficient circularization. The template was designed to prevent the precursor from folding into an unsuitable structure. The template allowed circular ribozymes as small as 15 nucleotides in length to be efficiently synthesized at concentrations as high as 50 microM in the ligation reaction. The circular products retained their biological activity.     

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.     

Crystal structure of an RNA polymerase ribozyme in complex with an antibody fragment

All models of the RNA world era invoke the presence of ribozymes that can catalyse RNA polymerization. The class I ligase ribozyme selected in vitro 15 years ago from a pool of random RNA sequences catalyses formation of a 3',5'-phosphodiester linkage analogous to a single step of RNA polymerization. Recently, the three-dimensional structure of the ligase was solved in complex with U1A RNA-binding protein and independently in complex with an antibody fragment. The RNA adopts a tripod arrangement and appears to use a two-metal ion mechanism similar to protein polymerases. Here, we discuss structural implications for engineering a true polymerase ribozyme and describe the use of the antibody framework both as a portable chaperone for crystallization of other RNAs and as a platform for exploring steps in evolution from the RNA world to the RNA-protein world.     

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.     

竞争性内源RNA的生物学功能及其调控

最新研究表明,RNA之间可以通过竞争结合共同的microRNA反应元件(microRNA response element, MRE)实现相互调节,这种调控模式构成竞争性内源RNA(Competing endogenous RNA, ceRNA)。已发现的ceRNA包括蛋白编码mRNA和非编码RNA,其中后者包括假基因转录物、长链非编码RNA(Long non-coding RNA, lncRNA)、环状RNA(Circular RNA, circRNA)等。文章主要从ceRNA分类的角度,阐述各类ceRNA构成的调控网络发挥的生物学功能在病理和生理相关过程中的作用,以及可能影响ceRNA调控有效性的因素。     

Use of domain enzymes from wheat RNA ligase for in vitro preparation of RNA molecules

Wheat RNA ligase can be dissected into three isolated domain enzymes that are responsible for its core ligase, 5′-kinase, and 2′,3′-cyclic phosphate 3′-phosphodiesterase activities, respectively. In the present study, we pursued a practical strategy using the domain enzymes for in vitro step-by-step ligation of RNA molecules. As a part of it, we demonstrated that a novel side reaction on 5′-tri/diphosphate RNAs is dependent on ATP, a 2′-phosphate-3′-hydroxyl end, and the ligase domain. Mass spectroscopy and RNA cleavage analyses strongly suggested that it is an adenylylation on the 5′ terminus. The ligase domain enzyme showed a high productivity for any of the possible 16 combinations of terminal bases and a high selectivity for the 5′-phosphate and 2′-phosphate-3′-hydroxyl ends. Two RNA molecules having 5′-hydroxyl and 2′,3′-cyclic monophosphate groups were ligated almost stoichiometrically after separate conversion of respective terminal phosphate states into reactive ones. As the product has the same terminal state as the starting material, the next rounds of ligation are also possible in principle. Thus, we propose a flexible method for in vitro RNA ligation.     

真菌环状RNA鉴定及研究展望

环状RNA是一类具有丰富调节潜力的闭合环状单链RNA,其可以通过竞争性结合内源RNA、充当蛋白质支架以及翻译模板等方式,从而参与基因表达调控。本文综述了真菌环状RNA的鉴定流程和难点,为后续解析环状RNA在真菌发育与代谢等过程中的调节作用提供理论支持。     

Efficient RNA 5'-adenylation by T4 DNA ligase to facilitate practical applications

We describe a simple procedure for RNA 5'-adenylation using T4 DNA ligase. The 5'-monophosphorylated terminus of an RNA substrate is annealed to a complementary DNA strand that has a 3'-overhang of 10 nucleotides. Then, T4 DNA ligase and ATP are used to synthesize 5'-adenylated RNA (5'-AppRNA), which should find use in a variety of practical applications. In the absence of an acceptor nucleic acid strand, the two-step T4 DNA ligase mechanism is successfully interrupted after the adenylation step, providing 40%-80% yield of 5'-AppRNA after PAGE purification with few side products (the yield varies with RNA sequence). Optimized reaction conditions are described for 5'-adenylating RNA substrates of essentially any length including long and structured RNAs, without need for sequestration of the RNA 3'-terminus to avoid circularization. The new procedure is applicable on the preparative nanomole scale. This 5'-adenylation strategy using T4 DNA ligase is a substantial improvement over our recently reported adenylation method that uses T4 RNA ligase, which often leads to substantial amounts of side products and requires careful optimization for each RNA substrate. Efficient synthetic access to 5'-adenylated RNA will facilitate a range of applications by providing substrates for in vitro selection; by establishing a new protocol for RNA 5'-capping; and by providing an alternative approach for labeling RNA with (32)P or biophysical probes at the 5'-terminus.     

Self-splicing of yeast mitochondrial ribosomal and messenger RNA precursors

We have previously shown linear and circular splicing intermediates resembling intermediates that result from self-splicing of ribosomal precursor RNA of Tetrahymena to be present in mitochondrial RNA. Here we show that splicing of yeast mitochondrial precursor RNA also occurs in vitro in the absence of mitochondrial proteins. The large ribosomal RNA gene, consisting of the intron and part of the flanking exon regions, was inserted behind the SP6 promoter in a recombinant plasmid and was transcribed in vitro. The resulting RNA shows self-catalyzed splicing via incorporation of GTP at the 5'-end of the excised intron, 5'- to 3'-exon ligation, and intron circularization. When purified mitochondrial RNA is incubated under similar conditions with alpha-32P-GTP, the excised ribosomal intron RNA is also labeled, as well as several other RNA species. Some of these RNAs are derived from excised introns from the multiply split gene coding for cytochrome oxidase subunit I.     

Stored polyadenylated RNA and loss of vigour in germinating wheat embryos

Loss of vigour in wheat seed is associated with lesions affecting the rate of disappearance of stored poly A⁺ RNA (presumptive mRNA) in the germinating embryo when germination takes place at a sub-optimal temperature. During germination in the presence of α-amanitin and consequent of de novo polyA⁺ RNA biosynthesis, the wheat embryo can degrade up to 70% of the stored poly A⁺ RNA of the quiescent embryo before any significant reduction in the rate of protein biosynthesis in the embryo becomes apparent. It is possible that two subpopulations of poly A⁺ RNA species exist in wheat embryos during early germination, one population being degraded rapidly upon rehydration of the embryo whilst the other population supports protein biosynthesis in the initial germination stages prior to degradation.     

A chloroplastic RNA ligase activity analogous to the bacterial and archaeal 2´-5' RNA ligase

Bacteria and archaea contain a 2'-5' RNA ligase that seals in vitro 2',3'-cyclic phosphodiester and 5'-hydroxyl RNA termini, generating a 2',5'-phosphodiester bond. In our search for an RNA ligase able to circularize the monomeric linear replication intermediates of viroids belonging to the family Avsunviroidae, which replicate in the chloroplast, we have identified in spinach (Spinacea oleracea L.) chloroplasts a new RNA ligase activity whose properties resemble those of the bacterial and archaeal 2'-5' RNA ligase. The spinach chloroplastic RNA ligase recognizes the 5'-hydroxyl and 2',3'-cyclic phosphodiester termini of Avocado sunblotch viroid and Eggplant latent viroid RNAs produced by hammerhead-mediated self-cleavage, yielding circular products linked through an atypical, most likely 2',5'-phosphodiester, bond. The enzyme neither requires divalent cations as cofactors, nor NTPs as substrate. The reaction apparently reaches equilibrium at a low ratio between the final circular product and the linear initial substrate. Even if its involvement in viroid replication seems unlikely, the identification of a 2'-5' RNA ligase activity in higher plant chloroplasts, with properties very similar to an analogous enzyme widely distributed in bacterial and archaeal proteomes, is intriguing and suggests an important biological role so far unknown.     

Circularization of linear viroid RNA via 2'-phosphomonoester, 3', 5'-phosphodiester bonds by a novel type of RNA ligase from wheat germ and Chlamydomonas.

A novel type of RNA ligase activity in extracts of wheat germ or Chlamydomonas requires 2', 3'-cyclic phosphate and 5'-phosphate ends for ligation to form a 2'-phosphomonoester, 3',5'-phosphodiester bond. Using 5'-3 2P-labeled linear PSTV, we demonstrate that RNase T1-nicked viroid predominantly forms (formula; see text) U-bonds. Natural linear PSTV, however, forms mainly (formula; see text) A-bonds upon enzymatic circularization. We show that natural linear PSTV RNA has nicks between C181 and A182, or between C348 and A349, and that consequently C181 and C348 carry 2',3'-cyclophosphate termini.