Biochemistry (Moscow) (v.76, #8)

RNA editing: breaking the dogma by A. A. Bogdanov; R. A. Zinovkin; A. A. Zamyatnin Jr. (867-868).

RNA Editing adds flavor to complexity by C. P. Godfried Sie; M. Kuchka (869-881).
A-to-I RNA editing results in the conversion of single adenosines into inosines, which alters coding and non-coding sequences in RNA molecules, increasing the complexity of the transcriptome. This modification is vital in a number of brain-specific coding transcripts, where the introduced alternative amino acids impact protein function substantially. Indeed, deviations from normal editing levels have been detected in tissues from individuals affected by neurological diseases and cancer, underscoring the importance of correct and regulated editing. Since the discovery of A-to-I RNA editing, considerable effort has been made to uncover additional editing targets and analyze the subsequent functional consequences for the recoded substrates. The effects of editing on non-coding RNAs (ncRNAs) such as microRNAs (miRNAs) or long ncRNAs are less well explored. ncRNAs act as regulators of gene expression through chromatin modification, imprinting, alternative splicing, and mRNA translation and stability. Editing has the potential to dynamically alter and diversify ncRNAs, thereby redirecting their functions. How editing intersects, interferes with, and modulates the roles of ncRNAs, possibly in response to external stimuli, therefore warrants a deeper look. This review discusses recent advances and new insights in the field.
Keywords: RNA editing; ADAR; inosine; deamination; ncRNAs; transcriptome complexity

Glutamate receptor RNA editing in health and disease by A. Barbon; S. Barlati (882-889).
RNA editing is a post-transcriptional process with an important role in gene modification. This editing process involves site-selective deamination of adenosine into inosine in the pre-mRNA, leading to the alteration of translation codons and splicing sites in nuclear transcripts, thereby enabling functionally distinct proteins to arise from a single gene. One important instance is the neuron editing of the ionotropic glutamate receptors (iGluRs). GluRs play a key role in excitatory synaptic transmission and plasticity in the central nervous system (CNS); their channel properties are largely dictated by the subunit composition of the tetrameric receptors. AMPA/kainate channels are assembled from GluA1-4 AMPA or GluK1-5 kainate receptor subunits. In particular, three of the four AMPA and two of the five kainate receptor subunits are subject to RNA editing. The editing positions have been named on the basis of the amino acid substitutions, such as the Q/R site in AMPA GluA2; the Q/R site in GluK1 and GluK2; the R/G site in GluA2, GluA3, and GluA4; and the I/V and Y/C sites in GluK2. These amino acid changes lead to profound alterations of the channel properties. This paper reviews the most relevant data showing the importance of glutamate receptor RNA editing in finely tuning glutamatergic neurotransmission in the normal CNS and following alterations of the editing process in association with disease phenotypes. Overall, these data indicate that a highly regulated process of glutamate receptor editing is of key importance in the proper function of neuronal cells and in their ability to adapt and modulate synaptic function.
Keywords: RNA editing; glutamate receptor; neurological disorders

The regulation of neuronal excitability is complex, as ion channels and neurotransmitter receptors are underlying a large variety of modulating effects. Alterations in the expression patterns of receptors or channel subunits as well as differential splicing contribute to the regulation of neuronal excitability. RNA editing is another and more recently explored mechanism to increase protein diversity, as the genomic recoding leads to new gene products with novel functional and pharmacological properties. In humans A-to-I RNA editing targets several neuronal receptors and channels, including GluR2/5/6 subunits, the Kv1.1 channel, and the 5-HT2C receptor. Our review summarizes that RNA editing of these proteins does not only change protein function, but also the pharmacology and presumably the drug therapy in human diseases.
Keywords: AMPA/kainate receptors; Q/R editing site; spider toxins; Kv1.1 channel; open channel block; serotonin receptor; GPCR coupling

In mammalian cells two active enzymes, ADAR1 and ADAR2, carry out A-to-I RNA editing. These two editases share many common features in their protein structures, catalytic activities, and substrate requirements. However, the phenotypes of the knockout animals are remarkably different, which indicate the distinct functions they play. The most striking effect of ADAR1 knockout is cell death and interruption of embryonic development that are not observed in ADAR2 knockout. Evidences have shown that ADAR1 plays critical roles in the differentiating cells in embryo and adult tissues to support the cell’s survival and permit their further differentiation and maturation. However, our knowledge in understanding of the mechanism by which ADAR1 exerts its unique effects is very limited. Many efforts had been made trying to understand why ADAR1 is so important that it is indispensible for animal survival, including studies that identify the RNA editing substrates and studies on non-editing mechanisms. The interest of this review is focused on the question why ADAR1 and not ADAR2 is required for cell survival. Therefore, only the data, published and unpublished, potentially connecting ADAR1 to its cell death effect is selectively cited and discussed here. The features of cell death caused by ADAR1 deletion are summarized. Potential involvement of interferon and protein kinase RNA-activated (PKR) pathways is proposed, but obviously more experimental evaluations are needed.
Keywords: ADAR1; apoptosis; cell differentiation; embryonic development; RNAi

Measuring RNA editing of serotonin 2C receptor by K. Iwamoto; M. Bundo; K. Kasai; T. Kato (912-914).
Pre-mRNA of serotonin 2C receptor (HTR2C, 5-hydroxytryptamine (serotonin) receptor 2C) undergoes A-to-I type RNA editing, which is a post-transcriptional event leading to the change of genomically encoded information. RNA editing generates various HTR2C isoforms, each of which has distinctive receptor activity. Postmortem, animal, and pharmacological studies have suggested that the altered RNA editing of HTR2C is involved in the pathophysiology of mental disorders, although results remain inconsistent. Here we review the techniques used for estimation of RNA editing of HTR2C. Among the techniques reported so far, a high-throughput sequencing-based method would be the most powerful method of choice for the large-scale experiments. Several different methods that were previously developed, such as pyrosequencing and capillary electrophoresis, should be suitable for validation as well as for rapid screening or exploratory purposes.
Keywords: ADAR; HTR2C; pyrosequencing; high-throughput sequencing

Identification of A-to-I RNA editing: Dotting the i’s in the human transcriptome by A. Kiran; G. Loughran; J. J. O’Mahony; P. V. Baranov (915-923).
The phenomenon of adenosine-to-inosine (A-to-I) RNA editing has attracted considerable attention from the scientific community due to its potential relationship to the evolution of cognition in animals. While A-to-I editing exists in all organisms with neurons, including those with primitive neuronal systems (hydra and nematodes), it is particularly frequent in organisms with a highly developed central nervous system (primates, especially humans). Diversification of RNA transcript sequences via A-to-I editing serves a number of different functional roles, such as altering the genome-templated identity of particular amino acids in proteins or altering splice site junctions and modulating regulation of alternatively spliced mRNA variants. Here we provide an overview of current computational and experimental methods for the high-throughput discovery of edited RNA nucleotides in the human transcriptome, as well as a survey of the existing RNA editing bioinformatics resources and an outlook of future perspectives.
Keywords: RNA editing; A-to-I; inosine; RNA processing; transcriptome diversity; RNA sequencing; human genome; editome; inosinome

RNA editing in plant organelles. Why make it easy? by B. Castandet; A. Araya (924-931).
Gene expression in plant organelles involves a number of distinct co- or posttranscriptional nucleic acid modifications: 5′ and 3′ RNA processing, cis- and trans-splicing, RNA stability, and RNA editing. All contribute to the steady-state RNA levels available for the translation of the reduced but essential organellar genetic information. Different from other maturation processes, RNA editing at the transcript level modifies the information encoded by organellar genes and is an essential step for the production of functional proteins. Editing changes are extensive in mitochondria from flowering plants with more than 400 cytidine-to-uridine changes that involve most transcripts, while in chloroplasts they are limited to some RNAs. An additional U-to-C RNA editing reaction is observed with the C-to-U transitions in fern and moss organelles. While RNA editing targets mostly concern coding regions, some events occur in untranslated regions. Whereas RNA editing is genetically and biochemically distinct from other RNA modification activities, evidence is growing for a tight connection between the different processing events. Although the understanding of this astonishing mechanism has increased since its discovery in 1989, some important questions remain unanswered. In this review we discuss the current knowledge on the different aspects of C-to-U, and to a lesser extent U-to-C, and look at RNA editing in plants with a particular emphasis on recent developments involving the role of pentatricopeptide repeat (PPR) proteins in this process.
Keywords: RNA editing; mRNA; mitochondrion; chloroplast; PPR genes

A-to-I and C-to-U editing within transfer RNAs by A. A. H. Su; L. Randau (932-937).
A significant number of post-transcriptional changes occur during the generation of mature transfer RNAs (tRNAs). These changes within precursor-tRNA molecules include the processing of 5′ and 3′ termini, the introduction of modifications, and also RNA editing. In this review, we will detail the reported cases of A-to-I and C-to-U tRNA editing. The most widespread example is the A-to-I conversion of the tRNA anticodon wobble base mediated by TadA in prokaryotes and the heterodimeric ADAT2-ADAT3 complex in eukaryotes. Recently, the plant chloroplast adenosine-to-inosine tRNA editing enzyme has been discovered. The editing of C-to-U is much less prevalent within tRNA and is currently only known to occur in few organellar tRNA species and the cytoplasmic threonyl-tRNA in trypanosomatids. The responsible editing enzyme remains to be identified. Finally, an unusually widespread C-to-U editing scenario was discovered in the archaeon Methanopyrus kandleri. This editing is mediated by CDAT8, which is responsible for the restoration of the proper folding of thirty different tRNA species. The evolution of CDAT8 will be discussed.
Keywords: tRNA processing; CDAT8; tRNA editing; TadA; ADAT2-ADAT3

Changes in the levels of antiapoptotic protein B-cell lymphoma 2 (Bcl-2) protein has been reported in murine and human tuberculosis. We investigated the role of mitogen-activated protein kinase pathways in the production of Bcl-2 protein in THP-1 human monocytes infected with Mycobacterium tuberculosis H37Rv and H37Ra. Analysis of phosphorylation profiles of mitogen-activated protein kinase kinase-1, extracellular-signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, and p38 mitogen-activated protein kinase; B-cell lymphoma 2 kinetics; and tumor necrosis factor-α (TNF-α) secretion levels showed variation between the two strains. Mycobacterium tuberculosis H37Rv induced higher Bcl-2 and lower TNF-α levels, whereas H37Ra the reverse. The strains also differed in their usage of CD14 and human leukocyte antigen-DR receptors in mediating extracellular-signal regulated kinase 1/2 and p38 mitogen-activated protein kinase activation. Mycobacterium tuberculosis H37Rv- and H37Ra-induced Bcl-2 production was reduced by specific inhibitors of mitogen-activated protein kinase kinase-1 (PD98059) and p38 (SB203580), but increased by nuclear factor κB (NF-κB) inhibitor (BAY 11-7082). TNF-α production by both strains was reduced in the presence of specific inhibitors of mitogen-activated protein kinase kinase-1 (PD98059), p38 (SB203580), and NF-κB (BAY 11-7082). Furthermore, inhibition of NF-κB was accompanied by an increase in strain-induced extracellular-signal regulated kinase 1/2 phosphorylation. Collectively, these results indicate for the first time that the production of Bcl-2 and TNF-α by M. tuberculosis H37Rv/H37Ra-infected THP-1 human monocytes is mediated through mitogen-activated protein kinases and NF-κB.
Keywords: M. tuberculosis ; ERK; p38 MAPK; Bcl-2 production; NF-κB

Functional dissection of an enhancer-like element located within the second intron of the human U2AF1L4 gene by D. A. Didych; N. A. Smirnov; E. S. Kotova; S. B. Akopov; L. G. Nikolaev; E. D. Sverdlov (951-957).
A detailed functional and evolutionary analysis of an enhancer element of the human genome (enhancer 12) located in the second intron of the U2AF1L4 gene, which we identified earlier, is presented. Overlapping fragments of the studied genome region were analyzed for enhancer activity, and the site responsible for the activity of this element was identified using transient transfections of HeLa cells. Comparison of the enhancer 12 sequence with orthologous sequences from seven primate species revealed the existence of evolutionarily conserved sequences within this element. One of the identified conservative regions is likely responsible for the enhancer activity and is able to specifically interact in vitro with proteins of HeLa cell nuclear extract. The ability of orthologous primate sequences to compete with enhancer 12 for binding with HeLa cell nuclear extract proteins and to enhance the activity of the reporter gene in transient transfection of HeLa cells is demonstrated.
Keywords: human genome; regulation of gene expression; functional analysis; enhancer

Galectins promote the interaction of influenza virus with its target cell by E. S. Chernyy; E. M. Rapoport; S. Andre; H. Kaltner; H. -J. Gabius; N. V. Bovin (958-967).
Influenza virus is known to bind sialoglycans located on the surface of the host cell. In addition, recent data suggest the involvement of other molecular targets in viral reception. Of note, a high density of terminal galactose residues is created on the surface of virions because of the influenza virus’ own neuraminidase activity. Thus, we suggested the possibility for an interaction of the influenza virus with galactose-binding proteins — galectins. In the present work we studied the influence of several galectins on the adhesion and further internalization of virus into the cell; six virus strains and three cell lines were studied. Chicken galectins CG-1A and -2 as well as human galectins HGal-1 and -8 promote virus binding in dose dependent manner, but they do not influence the internalization stage. Also, galectins are able to restore the ability of influenza virus to infect desialylated cells up to the level of native cells. When CG-1A in physiological concentrations was loaded onto viruses, the adhesion level was higher than in the case of on-cell loading. The effect of adhesion increase depends on the glycan structure of target-cell as well as of virus. The aggregated data suggest a promotional effect of galectins during the stage of influenza virus binding with the surface of target-cell.
Keywords: influenza virus; galectins; glycans

The NADH:ubiquinone oxidoreductase (respiratory complex I) activity of inside-out pig brain submitochondrial particles is inhibited by endogenous or externally added free fatty acids in time-dependent fashion. The rate and degree of the inhibition is dramatically increased by Ca2+. The Ca2+-promoted, fatty acid-induced inhibition is pH dependent, this being particularly evident at pH > 8.0. The inhibition is completely reversed by either EGTA or by bovine serum albumin (BSA). BSA prevents previously described (Kotlyar, A. B., Sled, V. D., and Vinogradov, A. D. (1992) Biochim. Biophys. Acta, 1098, 144–150) inhibitory effect of Ca2+ and alkaline pH on the de-active-to-active form transition of complex I. A possible mechanism of synergetic inhibition on complex I by Ca2+ and fatty acids is discussed.
Keywords: NADH:ubiquinone oxidoreductase; complex I; fatty acids; divalent cations; brain mitochondria

A minor isoform of the human RNA polymerase II subunit hRPB11 (POLR2J) interacts with several components of the translation initiation factor eIF3 by S. A. Proshkin; E. K. Shematorova; E. A. Souslova; G. M. Proshkina; G. V. Shpakovski (976-980).
Using the yeast two-hybrid (YTH) system we have uncovered interaction of the hRPB11cα minor isoform of Homo sapiens RNA polymerase II hRPB11 (POLR2J) subunit with three different subunits of the human translation initiation factor eIF3 (hEIF3): eIF3a, eIF3i, and eIF3m. One variant of eIF3m identified in the study is the product of translation of alternatively spliced mRNA. We have named a novel isoform of this subunit eIF3mβ. By means of the YTH system we also have shown that the new eIF3mβ isoform interacts with the eIF3a subunit. Whereas previously described subunit eIF3mα (GA17) has clear cytoplasmic localization, the novel eIF3mβ isoform is detected predominantly in the cell nucleus. The discovered interactions of the hRPB11cα isoform with several hEIF3 subunits demonstrate a new type coordination between transcription and the following (downstream) stages of gene expression (such as mRNA transport from nucleus to the active ribosomes in cytoplasm) in Homo sapiens and point out the possibility of existence of nuclear hEIF3 subcomplexes.
Keywords: Homo sapiens ; RNA polymerase II; hRPB11 (POLR2J); yeast two-hybrid system; hEIF3; transcription; translation