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PubMed1994

Complete RNA editing of unspliced and dicistronic transcripts of the intron-containing reading frame IRF170 from maize chloroplasts.

The maize plastome harbors within the rps4-rps14 gene cluster the reading frame IRF170, which is interrupted by two introns. Although the function of the encoded peptide of 170 amino acids is not known, the conservation of IRF170 homologs in other plastomes is a strong indication that IRF170 is a functional gene. Amplification and sequence analyses of IRF170 specific cDNAs reveals two C-to-U editing events occurring within each of the first two exons. This situation allows an analysis of the temporal order between editing and splicing of a chloroplast transcript. By using intron-specific primer combinations, cDNAs derived from partially or even unspliced IRF170 transcripts could be amplified which in all cases showed complete editing. Complete editing was also observed with a cDNA derived from a transcript in which the proximal rps4 and the 5' half of IRF170-encoded sequences were still linked. This demonstrates that editing of the IRF170 transcript is an early processing step preceding both splicing and cleavage to monocistronic mRNA.

Amino Acid Sequence↗

PubMed1997

Cloning of an Apobec-1-binding protein that also interacts with apolipoprotein B mRNA and evidence for its involvement in RNA editing.

Apolipoprotein (apo)B mRNA editing is mediated by a multiprotein editosome complex. Apobec-1 is the catalytic component of this complex, but other proteins involved in editing have not been identified. We used the yeast two-hybrid system to identify an apobec-1-interacting protein, ABBP-1. ABBP-1 contains 331 amino acid residues and is identical to a previously reported human type A/B hnRNP except for a 47-residue insertion at its C-terminal region. It contains typical RNP motifs at its N-terminal half and glycine-rich motifs in the C-terminal region. Northern blot analysis indicates that ABBP-1 mRNA is distributed in multiple human tissues. By deletion analysis, we mapped the apobec-1-binding region to the glycine-rich domain. ABBP-1 also binds to apoB mRNA transcripts around the editing site and can be UV-cross-linked to them in vitro. Immnodepletion of ABBP-1 from an active apoB mRNA editing tissue extract inhibits its editing activity. Down-regulation of ABBP-1 in an apobec-1-expressing HepG2 cell line by transfection with an antisense ABBP-1 cDNA construct leads to inhibition of endogenous apoB mRNA editing. We conclude that ABBP-1 is an apobec-1-interacting protein that may play an important role in apoB mRNA editing.

APOBEC-1 Deaminase↗

PubMed1992

In vitro guide RNA/mRNA chimaera formation in Trypanosoma brucei RNA editing.

The post-transcriptional processing of various mitochondrial transcripts in kinetoplastids, kRNA editing, adds and removes uridines, producing mature messenger RNAs. This editing seems to be directed by 'guide' RNAs (gRNAs) which are complementary to portions of the mature message. The editing mechanism has been proposed to entail transesterification. Detection of chimaeric gRNA-mRNA molecules, intermediates predicted by transesterification, support this model. We report here the in vitro formation of such chimaeras where endogenous gRNAs are covalently linked to added synthetic mRNA. Addition of gel-purified gRNAs to the standard reaction mix increases chimaera formation. This increase is not observed when the gRNA 3'-hydroxyl group is chemically modified, identifying this terminal hydroxyl as the reactive group. These results provide the first experimental evidence for an in vitro RNA editing event and support the involvement of transesterification as a chemical mechanism.

Animals↗

PubMed1999

Reduction of GluR2 RNA editing, a molecular change that increases calcium influx through AMPA receptors, selective in the spinal ventral gray of patients with amyotrophic lateral sclerosis.

Enhancement of calcium influx through the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor is a plausible mechanism underlying selective neuronal death in amyotrophic lateral sclerosis (ALS). The calcium conductance of the AMPA receptor is regulated by the GluR2 subunit that is edited at the glutamine/arginine residue site in the subunit assembly. We investigated the molecular changes of GluR2 mRNA in the spinal cord of ALS cases, those of cases with other neurological diseases, and those of normal cases using reverse transcription-polymerase chain reaction combined with restriction enzyme cleavage. We found that the editing efficiency was significantly lower only in the ventral gray of ALS cases (virtually 0% in 2 cases) than in any spinal region of the disease controls and normal controls. In addition, expression of GluR2 mRNA is lower in the ventral gray of the ALS cases and disease controls than in that of the normal controls. The above molecular changes of GluR2 mRNA in the ventral gray of ALS cases may enhance calcium influx through AMPA receptors, thereby promoting neuronal vulnerability. The decrement of GluR2 mRNA editing efficiency is unique to the ventral gray of ALS cases and may be closely linked to the etiology of ALS.

Adult↗

PubMed1994

RNA editing of transcripts of a chimeric mitochondrial gene associated with cytoplasmic male-sterility in Brassica.

The orf224 gene is a chimeric open reading frame associated with the Polima or pol cytoplasmic male sterility of Brassica napus. The first 58 codons and 5' upstream region of orf224 are derived from a conventional mitochondrial gene, orfB, while the origin of the remaining portion of the gene is unknown. Transcripts of the orf224 gene were found to be edited at a single site in the region of the gene that does not correspond to a known sequence. Oligonucleotides corresponding to the edited and unedited forms were shown to hybridize specifically to respective in vitro orf224 transcripts. Analysis of floral mtRNA by this method indicated that virtually all orf224 transcripts of both sterile and fertile, nuclear-restored pol cytoplasm plants are edited. Our results indicate that transcripts of novel, CMS-associated genes may be edited, but that, at least in this case, the degree of editing does not appear to be directly related to the male-sterile phenotype.

Amino Acid Sequence↗

PubMed2001

The genomics of land plant chloroplasts: Gene content and alteration of genomic information by RNA editing.

The entire nucleotide sequence of the chloroplast genome has been determined from 12 land plants. The gene content and arrangement are relatively uniform from species to species, and the genome contains an average of 111 identified gene species (except Epifagus). Chloroplast genes can be classified into three main categories: Genes for the photosynthetic apparatus, those for the transcription/translation system, and those related to biosyntheses. The genes encoding components of the photosynthesis apparatus have been identified by protein chemical analyses from higher plants, Chlamydomonas and cyanobacteria, and then by chloroplast transformation techniques using tobacco and Chlamydomonas. The genes for subunits of RNA polymerases and of ribosomes were initially deduced similarity to those in E. coli, and later confirmed by protein analyses. Coding information is often modified at the level of transcripts by RNA editing (mostly C-U changes), resulting in amino acid substitutions and creation of novel reading frames. Perspectives of chloroplast genomics are discussed.

Journal Article↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed2000

Identification and characterization of the trnS/pseudo-tRNA/nad3/rps12 gene cluster from Coix lacryma-jobi L: organization, transcription and RNA editing.

During a study of mitochondrial sequence conservation between the liverwort Marchantia polymorpha and several Angiosperm species, as revealed by heterologous hybridization experiments, the trnS/pseudo-tRNA/nad3/rps12 gene cluster in Coix lacryma-jobi L., an Asian grass species from the Andropogoneae, was identified using the mitochondrial probe orf167 from M. polymorpha. The Coix gene cluster was cloned and sequenced, and its expression analyzed. The gene sequence and gene locus organization were found to be similar to the corresponding cluster in wheat and maize. Northern hybridization and reverse transcription-polymerase chain reaction analyses indicated that nad3 and rps12 genes were co-transcribed as a 1.25 kb RNA molecule. The transcript displayed 20 and six RNA edition sites, in the nad3 and rps12 genes, respectively, that changed the codon identities to amino acids, which are better conserved in different organisms. Twenty-three cDNA clones were analysed for the edition process and revealed different partial editing patterns without apparent sequential processing.

Journal Article↗

PubMed1997

RNA editing in kinetoplastid protozoa.

Mitochondrial transcripts in kinetoplastids undergo remarkable posttranscriptional editing by uridylate insertion and deletion. The often dramatic remodeling of pre-mRNA sequences is directed by small guide RNAs (gRNAs) to produce mature mRNAs. In vitro analyses of editing have been used to determine the mechanism of editing and show that editing occurs by a series of enzyme-catalyzed steps. They also show that chimeric gRNA/mRNA molecules are not editing intermediates as proposed but are aberrant end products of editing. The complexes and molecules that catalyze editing are now being identified and characterized. The origin of editing, its developmental regulation which helps control the switching between terminal respiratory systems during the life cycle of trypanosomes, and other areas for future study are discussed.

Animals↗

PubMed2003

Messenger RNA editing in mammals: new members of the APOBEC family seeking roles in the family business.

Alteration of mRNA sequence through base modification mRNA editing frequently generates protein diversity. Several proteins have been identified as being similar to C-to-U mRNA editing enzymes based on their structural domains and the occurrence of a catalytic domain characteristic of cytidine deaminases. In light of the hypothesis that these proteins might represent novel mRNA editing systems that could affect proteome diversity, we consider their structure, expression and relevance to biomedically significant processes or pathologies.

APOBEC-1 Deaminase↗

PubMed1991

RNA editing in brain controls a determinant of ion flow in glutamate-gated channels.

L-glutamate, the principal excitatory transmitter in the brain, gates ion channels mediating fast neurotransmission. Subunit components of two related classes of glutamate receptor channels have been characterized by cDNA cloning and shown to carry either an arginine or a glutamine residue in a defined position of their putative channel-forming segment. The arginine residue in this segment profoundly alters, and dominates, the properties of ion flow, as demonstrated for one channel class. We now show that the genomic DNA sequences encoding the particular channel segment of all subunits harbor a glutamine codon (CAG), even though an arginine codon (CGG) is found in mRNAs of three subunits. Multiple genes and alternative exons were excluded as sources for the arginine codon; hence, we propose that transcripts for three subunits are altered by RNA editing. This process apparently edits subunit transcripts of the two glutamate receptor classes with different efficiency and selectivity.

Amino Acid Sequence↗

PubMed2002

A putative RNA editing from U to C in a mouse mitochondrial transcript.

Recently, we isolated and characterized a new mouse mitochondrial RNA molecule containing the mitochondrial 16S RNA plus 121 nt joined to the 5' end of the RNA. This fragment arises from the L strand of the same gene and we have named this transcript chimeric RNA. At position 121 of the RNA there is a C, which, according to the sequence of the mitochondrial 16S RNA gene, should be a U. We hypothesized that this RNA is synthesized having a U at position 121, which is later substituted to a C by a putative editing reaction. Based on the presence of sites for the restriction endonucleases RsaI and Fnu4HI around position 121, both forms of the RNA were detected in mouse tissues. To confirm the presence of the non-edited and putative edited RNA, a fragment containing the first 154 nt of the RNA was amplified by RT-PCR and cloned. The substitution of U for C was demonstrated by sequencing these clones. In vitro transcription experiments demonstrated that the substitution of U for C is not due to artifact of amplification or cloning. Moreover, in mitochondria from testis only the non-edited form was found. This, together with other experimental evidence, demonstrated that the base substitution was not due to polymorphism of the mitochondrial 16S RNA gene. This is the first demonstration of a substitution reaction from U to C in a mammalian mitochondrial transcript.

Animals↗

PubMed2005

ADAR2-mediated editing of RNA substrates in the nucleolus is inhibited by C/D small nucleolar RNAs.

Posttranscriptional, site-specific adenosine to inosine (A-to-I) base conversions, designated as RNA editing, play significant roles in generating diversity of gene expression. However, little is known about how and in which cellular compartments RNA editing is controlled. Interestingly, the two enzymes that catalyze RNA editing, adenosine deaminases that act on RNA (ADAR) 1 and 2, have recently been demonstrated to dynamically associate with the nucleolus. Moreover, we have identified a brain-specific small RNA, termed MBII-52, which was predicted to function as a nucleolar C/D RNA, thereby targeting an A-to-I editing site (C-site) within the 5-HT2C serotonin receptor pre-mRNA for 2'-O-methylation. Through the subcellular targeting of minigenes that contain natural editing sites, we show that ADAR2- but not ADAR1-mediated RNA editing occurs in the nucleolus. We also demonstrate that MBII-52 forms a bona fide small nucleolar ribonucleoprotein particle that specifically decreases the efficiency of RNA editing by ADAR2 at the targeted C-site. Our data are consistent with a model in which C/D small nucleolar RNA might play a role in the regulation of RNA editing.

Adenosine Deaminase↗

PubMed1995

Kainate receptor subunits expressed in single cultured hippocampal neurons: molecular and functional variants by RNA editing.

To determine the kainate receptor subunits that are found in native kainate receptors, we have applied a multiplex PCR of cDNAs reverse transcribed from mRNA harvested from single cultured hippocampal neurons after electrophysiological recording. We found that all the cells showing rapidly desensitizing currents in response to kainate express the GluR6 subunit mRNA, and that some of them also express the GluR5 subunit mRNA. No GluR7, KA-1, or KA-2 subunit mRNAs were detected. Analysis of the editing sites of the GluR6 mRNA demonstrated that the three editing sites present in these subunits are edited to a different extent. Predominant expression of the unedited variant (Q) was observed, but edited and unedited variants may coexist in the same cell. In addition, we show that the Q/R site from the GluR6 subunit controls functional properties of native kainate receptors.

Animals↗

PubMed2005

Complex management: RNA editing in trypanosomes.

Most mitochondrial mRNAs in kinetoplastids require editing, that is, the posttranscriptional insertion and deletion of uridine nucleotides that are specified by guide RNAs and catalyzed by multiprotein complexes. Recent studies have identified many of the proteins in these complexes, in addition to some of their functions and interactions. Although much remains unknown, a picture of highly organized complexes is emerging that shows that the complex that catalyzes the central steps of editing is partitioned into distinct insertion and deletion editing subcomplexes. These subcomplexes coordinate hundreds of ordered catalytic steps that function to produce a single mature mRNA. The dynamic processes, which might entail interactions among multiprotein complexes and changes in their composition and conformation, remain to be elucidated.

Animals↗

PubMed2003

RNA editing and alternative splicing of human serotonin 2C receptor in schizophrenia.

Serotonin 2C receptor (5-HT2CR) heterogeneity in the brain occurs mostly from two different sources: (i) 5-HT2CR mRNA undergoes adenosine-to-inosine editing events at five positions, which leads to amino acid substitutions that produce receptor variants with different pharmacological properties; (ii) 5-HT2CR mRNA is alternatively spliced, resulting in a truncated mRNA isoform (5-HT2CR-tr) which encodes a non-functional serotonin receptor. 5-HT2CR mRNA editing efficiencies and the expression of the full-length and the truncated 5-HT2CR mRNA splice isoforms were analyzed in the prefrontal cortex of elderly subjects with schizophrenia vs. matched controls (ns = 15). No significant differences were found, indicating that there are no alterations in editing or alternative splicing of 5-HT2CRs that are associated with schizophrenia in persons treated with antipsychotic medications. Quantitation of 5-HT2CR and 5-HT2CR-tr mRNA variants revealed that the expression of 5-HT2CR-tr was approximately 50% of that observed for the full-length isoform.

Adenosine↗

PubMed1996

Guide RNA-directed uridine insertion RNA editing in vitro.

Guide RNAs (gRNAs) have been proposed to mediate uridine (U) addition/deletion editing of mitochondrial mRNAs in kinetoplastid protozoa. The Us are proposed to be derived either from UTP by two successive cleavage-ligations or transesterifications, or from the 3' end of the gRNA by the same mechanisms. We have demonstrated gRNA-dependent U insertions into a specific editing site of a pre-edited mRNA which was incubated in a mitochondrial extract from Leishmania tarentolae. The predominant number of U insertions was determined by the number of guiding nucleotides in the added gRNA, and the formation of a gRNA-mRNA anchor duplex was necessary for activity. UTP and alpha-beta bond hydrolysis of ATP were required, and the activity was inhibited above 50-100 mM KCl. A gRNA-independent insertion of up to approximately 13 Us occurred in the absence of the added cognate gRNA; the extent of this activity was affected by sequences upstream and downstream of the edited region. Heparin inhibited the gRNA-independent U insertion activity and had no effect on the gRNA-dependent activity. Blocking the 3' OH of the gRNA had little effect on the gRNA-dependent U insertion activity. The data are consistent with a cleavage-ligation model in which the Us are derived directly from UTP.

Animals↗

PubMed1996

RNA editing in the acceptor stem of squid mitochondrial tRNA(Tyr).

In squid (Loligo bleekeri) mitochondria, the two 3'-terminal nucleotides (G72-G73) of the tRNA(Tyr) gene overlap with the two 5'-terminal nucleotides (G1-G2) of the downstream tRNA(Cys) gene. To elucidate the processing mechanism(s) of the tRNA molecules derived from this region, tRNAs were analyzed by sequencing cDNAs synthesized from circularized tRNAs. Nucleotides G1-G2 in tRNA(Cys) appeared to be without post-transcriptional conversion, whereas CCA was post-transcriptionally added to the 3'-terminus. In contrast, in the majority of tRNAs(Tyr), G72-G73 were found to be converted to A72-A73, accompanied by the CCA addition. These results indicate that a precursor of tRNA(Tyr) is processed at U71 and two adenosines are attached prior to the CCA addition. Thus, we suggest that 5' processing of the precursor tRNA dominates 3' processing and maturation of the tRNA is mediated by a polyadenylylation enzyme in the mitochondria, a scenario which is consistent with the editing process proposed in land snail mitochondria. We also obtained intermediates, such as a premature tRNA lacking CCA that terminated at U71 and one with a single adenosine attached at position 72, which support the suggested maturation process. However, although we failed to detect a tRNA(Cys) lacking G1-G2 at the 5'-terminus, we obtained cDNAs for tRNA(Tyr) with G72-G73 and the CCA terminus. This inconsistent result suggests the co-existence of another process(es) in the maturation of these tRNA molecules in squid mitochondria.

Animals↗