Characterization of Five RNA Editing Sites in Shab Potassium Channels

RNA editing revises the genetic code at precise locations, creating single base changes in mRNA. These changes can result in altered coding potential and modifications to protein function. Sequence analysis of the Shab potassium channel of Drosophila melanogaster revealed five such RNA editing sites. Four are constitutively edited (I583V, T643A, Y660C and I681V) and one undergoes developmentally regulated editing (T671A). These sites are located in the S4, S5-S6 loop and the S6 segments of the channel. We examined the biophysical consequences of editing at these sites by creating point mutations, each containing the genomic (unedited) base at one of the five sites in the background of a channel in which all other sites are edited. We also created a completely unedited construct. The function of these constructs was characterized using two-microelectrode voltage clamp in Xenopus oocytes. Each individual 'unediting' mutation slowed the time course of deactivation and the rise time during channel activation. Two of the mutants exhibited significant hyperpolarized shifts in their midpoints of activation. Constructs that deactivated slowly also inactivated slowly, supporting a mechanism of closed-state inactivation. One of the editing sites, position 660, aligns with the Shaker 449 residue, which is known to be important in tetraethylammonium (TEA) block. The aromatic, genomically-encoded residue tyrosine at this position in Shab enhances TEA block 14 fold compared to the edited residue, cysteine. These results show that both the position of the RNA editing site and the identity of the substituted amino acid are important for channel function.     

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。     

Identification and Functional Characterization of Neo-Poly(A) Polymerase, an RNA Processing Enzyme Overexpressed in Human Tumors

Poly(A) polymerase (PAP) plays an essential role in polyadenylation of mRNA precursors, and it has long been thought that mammalian cells contain only a single PAP gene. We describe here the unexpected existence of a human PAP, which we call neo-PAP, encoded by a previously uncharacterized gene. cDNA was isolated from a tumor-derived cDNA library encoding an 82.8-kDa protein bearing 71% overall similarity to human PAP. Strikingly, the organization of the two PAP genes is nearly identical, indicating that they arose from a common ancestor. Neo-PAP and PAP were indistinguishable in in vitro assays of both specific and nonspecific polyadenylation and also endonucleolytic cleavage. Neo-PAP produced by transfection was exclusively nuclear, as demonstrated by immunofluorescence microscopy. However, notable sequence divergence between the C-terminal domains of neo-PAP and PAP suggested that the two enzymes might be differentially regulated. While PAP is phosphorylated throughout the cell cycle and hyperphosphorylated during M phase, neo-PAP did not show evidence of phosphorylation on Western blot analysis, which was unexpected in the context of a conserved cyclin recognition motif and multiple potential cyclin-dependent kinase (cdk) phosphorylation sites. Intriguingly, Northern blot analysis demonstrated that each PAP displayed distinct mRNA splice variants, and both PAP mRNAs were significantly overexpressed in human cancer cells compared to expression in normal or virally transformed cells. Neo-PAP may therefore be an important RNA processing enzyme that is regulated by a mechanism distinct from that utilized by PAP.     

RNA编辑功能和RNA干扰

RNA编辑被认为是生命体一种新的基因加工与修饰现象,是指DNA转录成RNA后除RNA剪切外的其他加工过程,以核苷酸的删除、插入或替换等方式改变遗传信息,揭示生物进化过程中基因修饰和调控的另一个重要途径,是对中心法则的重要补充.而RNAi是一种由dsRNA介导的,在转录水平、转录后水平和翻译水平上阻断基因表达的基因调节途径.着重介绍 RNA编辑功能、RNA编辑与RNA干扰关系.     

Characterization of the Inflammatory Microenvironment and Identification of Potential Therapeutic Targets in Wilms Tumors

The role of inflammation in cancer has been reported in various adult malignant neoplasms. By contrast, its role in pediatric tumors has not been as well studied. In this study, we have identified and characterized the infiltration of various inflammatory immune cells as well as inflammatory markers in Wilms tumor (WT), the most common renal malignancy in children. Formalin-fixed paraffin-embedded blocks from tumors and autologous normal kidneys were immunostained for inflammatory immune cells (T cells, B cells, macrophages, neutrophils, and mast cells) and inflammatory markers such as cyclooxygenase-2 (COX-2), hypoxia-inducible factor 1α, phosphorylated STAT3, phosphorylated extracellular signal–related kinases 1 and 2, inducible nitric oxide synthase, nitrotyrosine, and vascular endothelial growth factor expression. Overall, we found that there was predominant infiltration of tumor-associated macrophages in the tumor stroma where COX-2 was robustly expressed. The other tumor-associated inflammatory markers were also mostly localized to tumor stroma. Hence, we speculate that COX-2–mediated inflammatory microenvironment may be important in WT growth and potential therapies targeting this pathway may be beneficial and should be tested in clinical settings for the treatment of WTs in children.     

RNA编辑的研究进展

RNA编辑,即通过碱基的插入、删除和替换对RNA进行的转录后加工过程,这一表观遗传现象也被认为是在RNA水平上对遗传信息进行修复的一种修正机制.本文主要综述了目前植物中基于PPR基因家族等编辑复合体以及动物中关于CRISPR/Cas系统的两种RNA编辑系统,并介绍了RNA编辑在植物生长发育过程中的重要作用,并展望了RN...     

Annexin Ⅰ蛋白在人前列腺癌中的表达及临床意义

目的:探讨Annexin Ⅰ蛋白在前列腺癌组织中的表达及其与前列腺癌发生、发展、转移及预后的关系.方法:回顾性分析110例前列腺癌及40例前列腺增生组织中Annexin Ⅰ蛋白的表达与前列腺癌Gleason分级、年龄、临床分期、转移及预后的相关性.结果:前列腺癌患者的Armexin Ⅰ表达水平显著地低于良性前列腺增生症患者(P＜0.05);Annexin Ⅰ在低分化癌中表达较高,与高、中分化癌相比有显著性差异(P＜0.05);而高、中分化癌之间Annexin Ⅰ表达均较低,两者之间无显著性差异(P＞0.05).前列腺癌Annexin Ⅰ蛋白表达与年龄无关,而与临床分期、淋巴结转移及预后有关(P＜0.05).结论:Annexin Ⅰ蛋白表达下调与前列腺癌发生发展和预后密切相关,可作为反映前列腺癌生物学行为和判断预后的生物标记物.     

Molecular and Functional Diversity of RNA Editing in Plant Mitochondria

RNA editing is a fundamental biochemical process relating to the modification of nucleotides in messenger RNAs of functional genes in cells. RNA editing leads to re-establishment of conserved amino acid residues for functional proteins in nuclei, chloroplasts, and mitochondria. Identification of RNA editing factors that contributes to target site recognition increases our understanding of RNA editing mechanisms. Significant progress has been made in recent years in RNA editing studies for both animal and plant cells. RNA editing in nuclei and mitochondria of animal cells and in chloroplast of plant cells has been extensively documented and reviewed. RNA editing has been also extensively documented on plant mitochondria. However, functional diversity of RNA editing factors in plant mitochondria is not overviewed. Here, we review the biological significance of RNA editing, recent progress on the molecular mechanisms of RNA editing process, and function diversity of editing factors in plant mitochondrial research. We will focus on: (1) pentatricopeptide repeat proteins in Arabidopsis and in crop plants; (2) the progress of RNA editing process in plant mitochondria; (3) RNA editing-related RNA splicing; (4) RNA editing associated flower development; (5) RNA editing modulated male sterile; (6) RNA editing-regulated cell signaling; and (7) RNA editing involving abiotic stress. Advances described in this review will be valuable in expanding our understanding in RNA editing. The diverse functions of RNA editing in plant mitochondria will shed light on the investigation of molecular mechanisms that underlies plant development and abiotic stress tolerance.     

编辑机器:锥虫RNA编辑复合体(译文)

包括底物前体mRNA和指导RNA（gRNA)在内的核糖核蛋白复合体的装配和解装配是RNA编辑的起始和增殖的关键。本文讨论了这些复合体的组成,以及它们的装配是如何调控RNA编辑的。     

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.     

Mechanism of U-Insertion RNA Editing in Trypanosome Mitochondria: Characterization of RET2 Functional Domains by Mutational Analysis

3′-Terminal uridylyl transferases (TUTases) selectively bind uridine 5′-triphosphate (UTP) and catalyze the addition of uridine 5′-monophosphate to the 3′-hydroxyl of RNA substrates in a template-independent manner. RNA editing TUTase 1 and RNA editing TUTase 2 (RET2) play central roles in uridine insertion/deletion RNA editing, which is an essential part of mitochondrial RNA processing in trypanosomes. Although the conserved N-terminal (catalytic) domain and C-terminal (nucleotide base recognition) domain are readily distinguished in all known TUTases, nucleotide specificity, RNA substrate preference, processivity, quaternary structures, and auxiliary domains vary significantly among enzymes of divergent biological functions. RET2 acts as a subunit of the RNA editing core complex to carry out guide-RNA-dependent U-insertion into mitochondrial mRNA. By correlating mutational effects on RET2 activity as recombinant protein and as RNA editing core complex subunit with RNAi-based knock-in phenotypes, we have assessed the UTP and RNA binding sites in RET2. Here we demonstrate functional conservation of key UTP-binding and metal-ion-coordinating residues and identify amino acids involved in RNA substrate recognition. Invariant arginine residues 144 and 435 positioned in the vicinity of the UTP binding site are critical for RET2 activity on single-stranded and double-stranded RNAs, as well as function in vivo. Recognition of a double-stranded RNA, which resembles a guide RNA/mRNA duplex, is further facilitated by multipoint contacts across the RET2-specific middle domain.