Alternative RNA structures formed during transcription depend on elongation rate and modify RNA processing

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Partially edited RNAs are intermediates of RNA editing in plant mitochondria

RNA editing in flowering plant mitochondria addresses several hundred specific C nucleotides in individual sequence contexts in mRNAs and tRNAs. Many of the in vivo steady state RNAs are edited at some sites but not at others. It is still unclear whether such incompletely edited RNAs can either be completed or are aborted. To learn more about the dynamics of the substrate recognition process, we investigated in vitro RNA editing at a locus in the atp4 mRNA where three editing sites are clustered within four nucleotides. A single cis-element of about 20 nucleotides serves in the recognition of at least two sites. Competition with this sequence element suppresses in vitro editing. Surprisingly, unedited and edited competitors are equally effective. Experiments with partially pre-edited substrates indicate that indeed the editing status of a substrate RNA does not affect the binding affinity of the specificity factor(s). RNA molecules in which all editing sites are substituted by either A or G still compete, confirming that editing site recognition can occur independently of the actual editing site. These results show that incompletely edited mRNAs can be substrates for further rounds of RNA editing, resolving a long debated question.     

RNA editing of 10 Didymium iridis mitochondrial genes and comparison with the homologous genes in Physarum polycephalum

Regions of the Didymium iridis mitochondrial genome were identified with similarity to typical mitochondrial genes; however, these regions contained numerous stop codons. We used RT-PCR and DNA sequencing to determine whether, through RNA editing, these regions were transcribed into mRNAs that could encode functional proteins. Ten putative gene regions were examined: atp1, atp6, atp8, atp9, cox1, cox2, cytb, nad4L, nad6, and nad7. The cDNA sequences of each gene could encode a functional mitochondrial protein that was highly conserved compared with homologous genes. The type of editing events and editing sequence features were very similar to those observed in the homologous genes of Physarum polycephalum, though the actual editing locations showed a variable degree of conservation. Edited sites were compared with encoded sites in D. iridis and P. polycephalum for all 10 genes. Edited sequence for a portion of the cox1 gene was available for six myxomycetes, which, when compared, showed a high degree of conservation at the protein level. Different types of editing events showed varying degrees of site conservation with C-to-U base changes being the least conserved. Several aspects of single C insertion editing events led to the preferential creation of hydrophobic amino acid codons that may help to minimize adverse effects on the resulting protein structure.     

Uridine insertion/deletion RNA editing in trypanosomatid mitochondria: In search of the editosome

The RNA ligase-containing or L-complex is the core complex involved in uridine insertion/deletion RNA editing in trypanosome mitochondria. Blue native gels of glycerol gradient-separated fractions of mitochondrial lysate from cells transfected with the TAP-tagged editing protein, LC-8 (TbMP44/KREPB5), show a ∼1 MDa L-complex band and, in addition, two minor higher molecular weight REL1-containing complexes: one (L*a) co-sedimenting with the L-complex and running in the gel at around 1.2 MDa; the other (L*b) showing a continuous increase in molecular weight from 1 MDa to particles sedimenting over 70S. The L*b-complexes appear to be mainly composed of L-complex components, since polypeptide profiles of L- and L*b-complex gradient fractions were similar in composition and L*b-complex bands often degraded to L-complex bands after manipulation or freeze–thaw cycles. The L*a-complex may be artifactual since this gel shift can be produced by various experimental manipulations. However, the nature of the change and any cellular role remain to be determined. The L*b-complexes from both lysate and TAP pull-down were sensitive to RNase A digestion, suggesting that RNA is involved with the stability of the L*b-complexes. The MRP1/2 RNA binding complex is localized mainly in the L*b-complexes in substoichiometric amounts and this association is RNase sensitive. We suggest that the L*b-complexes may provide a scaffold for dynamic interaction with other editing factors during the editing process to form the active holoenzyme or “editosome.”     

银杏叶绿体ndhF RNA编辑现象分析及C290位编辑对胁迫处理的响应

RNA编辑是一种转录后修饰加工过程, 通过碱基的插入、缺失或替换可改变氨基酸的种类, 增加蛋白质的疏水性和同源蛋白在不同物种间的保守性。该文通过DNA与cDNA序列的比对, 分析了裸子植物银杏(Ginkgo biloba)叶绿体功能基因ndhF的编辑现象, 该基因共含有21个编辑位点, 且这21个位点均为部分编辑。生物信息学分析及与其它物种比对结果表明, ndhF C290位编辑可能会影响该蛋白的正确折叠。进一步使用单克隆酶切方法测定了不同胁迫处理对ndhF C290位编辑效率的影响, 结果表明该位点的编辑效率对温度和黑暗敏感。     

银杏叶绿体ndhF RNA编辑现象分析及C290位编辑对胁迫处理的响应

RNA编辑是一种转录后修饰加工过程,通过碱基的插入、缺失或替换可改变氨基酸的种类,增加蛋白质的疏水性和同源蛋白在不同物种间的保守性。该文通过DNA与cDNA序列的比对,分析了裸子植物银杏（Ginkgobiloba）叶绿体功能基因ndhF的编辑现象,该基因共含有21个编辑位点,且这21个位点均为部分编辑。生物信息学分析及与其它物种比对结果表明,ndhFC290位编辑可能会影响该蛋白的正确折叠。进一步使用单克隆酶切方法测定了不同胁迫处理对ndhFC290位编辑效率的影响,结果表明该位点的编辑效率对温度和黑暗敏感。     

Cis- and trans-splicing and RNA editing are required for the expression of nad2 in wheat mitochondria

In wheat mitochondria, the gene coding for subunit 2 of the NADH-ubiquinone oxidoreductase (nad2) is divided into five exons located in two distant genomic regions. The first two exons of the gene, a and b, lie 22 kb downstream of exons c, d, and e, on the same DNA strand. All introns of nad2 are group II introns. A trans-splicing event is required to join exons b and c. It involves base pairing of the two precursor RNAs in the stem of domain IV of the intron. A gene coding for tRNA^Tyr is located upstream of exon c. In addition to splicing processes, mRNA editing is also required for the correct expression of nad2. The mature mRNA is edited at 36 positions, distributed over its five exons, resulting in 28 codon modifications. Editing increases protein hydrophobicity and conservation. Received: 11 August 1997 / Accepted: 2 February 1998     

棉花叶绿体基因RNA编辑位点的测定及分析

陆生植物叶绿体RNA编辑是转录后基因表达调控的一种重要方式。该文在预测棉花（Gossypium hirsutum）叶绿体基因RNA编辑位点的基础上,选取中棉10（CRRI 10）为实验材料,采用PCR、RT-PCR及测序等方法,确定CRRI 10的27个叶绿体蛋白编码基因共有55个编辑位点,均是C→U的转换。与棉种柯字310（C310）的编辑位点比对后发现,CRRI 10多出accD-468和rpoC1-163两个编辑位点,同时缺失psbN-10。利用生物信息学分析这3个位点,rpoC1-163和psbN-10的编辑可能会改变各自蛋白的二级结构。对CRRI 10中55个编辑位点上游的顺式作用元件（？30–？1）分析显示,共有8组顺式作用元件的相似性达到60%或以上,推测各组中的编辑位点可能由相同的反式作用因子来识别。     

植物细胞器RNA编辑因子的功能及其作用机制 *

在植物线粒体和叶绿体转录本上,数百个胞嘧啶(C)位点经脱氨基反应变为尿嘧啶(U),这是一种在转录本水平上对遗传信息进行修饰或调控的机制.在植物细胞器中,RNA编辑过程需要不同家族的RNA编辑因子相互作用组装成复杂的编辑复合体,特异地识别编辑位点进行编辑.最初的研究发现,植物RNA编辑受到高特异性五环肽重复(pentatricopeptide repeat, PPR)蛋白的调控,目前在植物中发现400多种PPR家族蛋白,编辑作用复杂.之后对RNA编辑因子互作蛋白/多细胞器RNA编辑因子(RNA editing factor interacting proteins /multiple organellar RNA editing factors,RIP/MORF),细胞器RNA识别基序(organelle RNA recognition motif,ORRM),细胞器锌指蛋白(organelle zinc-finger,OZ)等的研究表明,这些非PPR蛋白组分可以与PPR蛋白形成编辑复合体,共同参与编辑,且RNA编辑复合体具有多样性.RNA编辑因子的缺失会引起植物的生长发育受阻,果实成熟延迟等,对RNA编辑因子的研究显得尤为重要.对植物中RNA编辑因子的功能及其作用机制研究进展进行综述,旨在为后续RNA编辑的研究提供一定的参考.     

Fold recognition,homology modeling,docking simulations,kinetics analysis and mutagenesis of ATP/CTP:tRNA nucleotidyltransferase from Methanococcus jannaschii

ATP/CTP:tRNA nucleotidyltransferases (NTases) and poly(A) polymerases (PAPs) belong to the same superfamily and their catalytic domains are remotely related. Based on the results of fold-recognition analysis and comparison of secondary structure patterns, we predicted that these two NTase families share three domains, corresponding to "palm," "fingers," and "fingernails" in the PAP crystal structure. A homology model of tRNA NTase from Methanococcus jannaschii was constructed. Energy minimization calculations of enzyme-nucleotide complexes and computer-aided docking of nucleotides onto the enzyme's surface were carried out to explore possible ATP and CTP binding sites. Theoretical models were used to guide experimental analysis. Recombinant His-tagged enzyme was expressed in Escherichia coli, and kinetic properties were characterized. The apparent K(M) for CTP was determined to be 38 microM, and the apparent K(M) for ATP was 21 microM. Three mutations of basic amino acids to alanine were created in a highly conserved region predicted to be in the vicinity of the nucleotide binding site. A deletion was also constructed to remove the C-terminal structural domain defined by the model; it retained about 1% of wild type enzymatic activity using CTP as co-substrate, confirming that detectable catalytic activity is exhibited by the N-terminal domain, as defined by the model. Our results suggest a mechanism of differential ATP and CTP binding, which explains how the tRNA NTase, having only one catalytic site, utilizes different nucleotide triphosphates depending on the nature of the tRNA substrate.     

Limited RNA Editing in Exons of Mouse Liver and Adipose

Several studies have investigated RNA–DNA differences (RDD), presumably due to RNA editing, with conflicting results. We report a rigorous analysis of RDD in exonic regions in mice, taking into account critical biases in RNA-Seq analysis. Using deep-sequenced F1 reciprocal inbred mice, we mapped 40 million RNA-Seq reads per liver sample and 180 million reads per adipose sample. We found 7300 apparent hepatic RDDs using a multiple-site mapping procedure, compared with 293 RDD found using a unique-site mapping procedure. After filtering for repeat sequence, splice junction proximity, undirectional strand, and extremity read bias, 63 RDD remained. In adipose tissue unique-site mapping identified 1667 RDD, and after applying the same four filters, 188 RDDs remained. In both tissues, the filtering procedure increased the proportion of canonical (A-to-I and C-to-U) editing events. The genomic DNA of 12 RDD sites among the potential 63 hepatic RDD was tested by Sanger sequencing, three of which proved to be due to unreferenced SNPs. We validated seven liver RDD with Sequenom technology, including two noncanonical, Gm5424 C-to-I(G) and Pisd I(G)-to-A RDD. Differences in diet, sex, or genetic background had very modest effects on RDD occurrence. Only a small number of apparent RDD sites overlapped between liver and adipose, indicating a high degree of tissue specificity. Our findings underscore the importance of properly filtering for bias in RNA-Seq investigations, including the necessity of confirming the DNA sequence to eliminate unreferenced SNPs. Based on our results, we conclude that RNA editing is likely limited to hundreds of events in exonic RNA in liver and adipose.     

听原性惊厥易感大鼠下丘GluR2的表达及QR位点编辑水平

听原性惊厥易感大鼠是强直 -阵挛惊厥大发作的一种模型 .一般认为 ,下丘是听原性惊厥发作神经元网络的启动部位 .采用RT PCR、Western印迹、免疫组织化学等方法观察了听原性惊厥易感大鼠 (P77PMC)一次惊厥发作与惊厥点燃状态下AMPA受体亚基GluR2在下丘内表达的改变 ,并采用限制性酶切方法分析了GluR2Q R位点mRNA编辑水平的改变 .研究结果显示 ,一次惊厥发作后下丘内GluR2表达无明显改变 ,惊厥点燃后下丘内GluR2表达降低 ,一次惊厥发作及惊厥点燃状态下GluR2Q R位点处于编辑成熟状态 .提示 ,GluR2表达降低参与了点燃状态下的惊厥发作 ,在听原性惊厥易感大鼠惊厥发作机制中不涉及下丘内GluR2Q R位点编辑水平改变 .     

Computational analysis of RNA editing sites in plant mitochondrial genomes reveals similar information content and a sporadic distribution of editing sites

A computational analysis of RNA editing sites was performedon protein-coding sequences of plant mitochondrial genomes fromArabidopsis thaliana, Beta vulgaris, Brassica napus, and Oryzasativa. The distribution of nucleotides around edited and uneditedcytidines was compared in 41 nucleotide segments and included1481 edited cytidines and 21,390 unedited cytidines in the 4genomes. The distribution of nucleotides was examined in 1,2, and 3 nucleotide windows by comparison of nucleotide frequencyratios and relative entropy. The relative entropy analyses indicatethat information is encoded in the nucleotide sequences in the5 prime flank (–18 to –14, –13 to –10,–6 to –4, –2/–1) and the immediate 3prime flanking nucleotide (+1), and these regions may be importantin editing site recognition. The relative entropy was largewhen 2 or 3 nucleotide windows were analyzed, suggesting thatseveral contiguous nucleotides may be involved in editing siterecognition. RNA editing sites were frequently preceded by 2pyrimidines or AU and followed by a guanidine (HYCG) in themonocot and dicot mitochondrial genomes, and rarely precededby 2 purines. Analysis of chloroplast editing sites from a dicot,Nicotiana tabacum, and a monocot, Zea mays, revealed a similardistribution of nucleotides around editing sites (HYCA). Thesimilarity of this motif around editing sites in monocots anddicots in both mitochondria and chloroplasts suggests that amechanistic basis for this motif exists that is common in thesedifferent organelle and phylogenetic systems. The preferredsequence distribution around RNA editing sites may have an importantimpact on the acquisition of editing sites in evolution becausethe immediate sequence context of a cytidine residue may rendera cytidine editable or uneditable, and consequently determinewhether a T to C mutation at a specific position may be correctedby RNA editing. The distribution of editing sites in many protein-codingsequences is shown to be non-random with editing sites clusteredin groups separated by regions with no editing sites. The sporadicdistribution of editing sites could result from a mechanismof editing site loss by gene conversion utilizing edited sequenceinformation, possibly through an edited cDNA intermediate.