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PubMed1989

The Leishmania kinetoplast-mitochondrion contains terminal uridylyltransferase and RNA ligase activities.

Purified mitochondria of Leishmania tarentolae contain 3'-terminal uridylyltransferase and RNA ligase activities which can be solubilized by detergent lysis of the organelle. Run-off transcription of maxicircle and minicircle DNA also occurs in intact and Triton-lysed mitochondria, using [32P]GTP as the labeled precursor. Heparin inhibits the solubilized terminal uridylyltransferase activity but does not affect the labeling of endogenous RNAs in intact mitochondria with [32P]UTP. Clarification of the mitochondrial Triton lysate causes an increase in terminal uridylyltransferase activity with exogenous substrates. These two activities are candidates for involvement in a post-transcriptional RNA editing process of mitochondrial transcripts.

Animals↗

PubMed1997

Multiple forms of AMPA-type glutamate receptor mRNA phenotypes in goldfish retina and tectum.

1. A goldfish AMPA-type glutamate receptor cDNA (GFGR49) was cloned from a goldfish retinal cDNA library. 2. The GFGR49 clone is an immature product of pre-mRNA, possessing the C-flip exon and flanking introns. 3. RNase protection assays revealed multiple mRNAs that contain the C-flip exon. In addition, these assays indicated differential processing of these RNAs between retina and brain. 4. Finally, RNase protection assays employing probes spanning the 3'-coding region of GFGR49 revealed at least two different mRNA phenotypes. 5. Comparison with the sequence of genomic DNA, which was obtained by polymerase chain reaction amplification, suggests that such multiple mRNA phenotypes are attributed to the use of alternative intron-exon splicing junctions and to RNA editing.

Amino Acid Sequence↗

PubMed2000

Structural genomics of RNA.

A detailed understanding of the functions and interactions of biological macromolecules requires knowledge of their molecular structures. Structural genomics, the systematic determination of all macromolecular structures represented in a genome, is focused at present exclusively on proteins. It is clear, however, that RNA molecules play a variety of significant roles in cells, including protein synthesis and targeting, many forms of RNA processing and splicing, RNA editing and modification, and chromosome end maintenance. To comprehensively understand the biology of a cell, it will ultimately be necessary to know the identity of all encoded RNAs, the molecules with which they interact and the molecular structures of these complexes. This report focuses on the feasibility of structural genomics of RNA, approaches to determining RNA structures and the potential usefulness of an RNA structural database for both predicting folds and deciphering biological functions of RNA molecules.

Computational Biology↗

PubMed1986

Major transcript of the frameshifted coxII gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA.

The mitochondrial cytochrome oxidase (cox) subunit II gene from trypanosomes contains a frameshift at amino acid 170. This gene is highly conserved in different trypanosome species, suggesting that it is functional. Sequence determination of coxII transcripts of T. brucei and C. fasciculata reveals four extra, reading frame-restoring nucleotides at the frameshift position that are not encoded in the DNA. Southern blot analysis of DNA of both trypanosome species failed to show the existence of a second version of the coxII gene. We conclude, therefore, that the extra nucleotides are added during or after transcription of the frameshift gene by an RNA-editing process.

Animals↗

PubMed2004

RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2.

Here we report that bacteriophage T4 RNA ligase 2 (Rnl2) is an efficient catalyst of RNA ligation at a 3'-OH/5'-PO(4) nick in a double-stranded RNA or an RNA.DNA hybrid. The critical role of the template strand in approximating the reactive 3'-OH and 5'-PO(4) termini is underscored by the drastic reductions in the RNA-sealing activity of Rnl2 when the duplex substrates contain gaps or flaps instead of nicks. RNA nick joining requires ATP and a divalent cation cofactor (either Mg or Mn). Neither dATP, GTP, CTP, nor UTP can substitute for ATP. We identify by alanine scanning seven functionally important amino acids (Tyr-5, Arg-33, Lys-54, Gln-106, Asp-135, Arg-155, and Ser-170) within the N-terminal nucleotidyl-transferase domain of Rnl2 and impute specific roles for these residues based on the crystal structure of the AMP-bound enzyme. Mutational analysis of 14 conserved residues in the C-terminal domain of Rnl2 identifies 3 amino acids (Arg-266, Asp-292, and Glu-296) as essential for ligase activity. Our findings consolidate the evolutionary connections between bacteriophage Rnl2 and the RNA-editing ligases of kinetoplastid protozoa.

Amino Acid Sequence↗

PubMed1998

Characterization of a novel trypanosomatid small nucleolar RNA.

Trypanosomes possess unique RNA processing mechanisms including trans- splicing of pre-mRNA and RNA editing of mitochondrial transcripts. The previous finding of a trimethylguanosine (TMG) capped U3 homologue in trypanosomes suggests that rRNA processing may be related to the processing in other eukaryotes. In this study, we describe the first trypanosomatid snoRNA that belongs to the snoRNAs that were shown to guide ribose methylation of rRNA. The RNA, identified in the monogenetic trypanosomatid Leptomonas collosoma, was termed snoRNA-2 and is encoded by a multi-copy gene. SnoRNA-2 is 85 nt long, it lacks a 5' cap and possesses the C and D boxes characteristic to all snoRNAs that bind fibrillarin. Computer analysis indicates a potential for base-pairing between snoRNA-2 and 5.8S rRNA, and 18S rRNA. The putative interaction domains obey the rules suggested for the interaction of guide snoRNA with its rRNA target for directing ribose methylation on the rRNA. However, mapping the methylated sites on the 5.8S rRNA and 18S rRNA indicates that the expected site on the 5.8S is methylated, whereas the site on the 18S is not. The proposed interaction with 5.8S rRNA is further supported by the presence of psoralen cross-link sites on snoRNA-2. GenBank search suggests that snoRNA-2 is not related to any published snoRNAs. Because of the early divergence of the Trypanosomatidae from the eukaryotic lineage, the presence of a methylating snoRNA that is encoded by a multi-copy gene suggests that methylating snoRNAs may have evolved in evolution from self-transcribed genes.

Animals↗

PubMed1991

The rice mitochondrial nad3 gene has an extended reading frame at its 5' end: nucleotide sequence analysis of rice trnS, nad3, and rps12 genes.

The nucleotide sequences of the tRNASer (trnS), pseudo-tRNA, NADH dehydrogenase subunit 3 (nad3), and ribosomal protein S12 (rps12) genes from rice mitochondrial DNA (mtDNA) were determined. Both trnS and nad3 were confirmed to be single copy genes by Southern blot analysis. The nad3 and rps12 genes were arranged in tandem, and the two were co-transcribed. The order of the above four genes in rice mtDNA differed from the linear order observed for the wheat and maize genes. In rice mitochondria, the trnS and pseudo-tRNA genes were found upstream of the cytochrome c oxidase subunit I gene, instead of the nad3 and rps12 genes as observed in maize and wheat. Additionally, while the rice nad3 and rps12 genes remain paired, they too are in a different sequence environment from the wheat and maize genes. The apparent split of the two pairs of genes indicates the occurrence of a mitochondrial intramolecular recombinational event. Another peculiarity is that the sequence upstream of the translational initiation codon of the rice nad3 gene is different from that of the wheat and maize versions. The ATG initiation codon of wheat and maize nad3 is replaced by TTG in the rice nad3. A subsequent deduction of the amino acid sequence, accompanied by a primer extension analysis, indicates that the predicted rice NAD3 protein has an additional 37 amino acid residues at its N-terminus compared to the wheat and maize NAD3 proteins. cDNA sequence analysis showed no introns or the occurrence of RNA editing at the newly replaced TTG codon.

Amino Acid Sequence↗

PubMed1991

Modification of Trypanosoma brucei mitochondrial rRNA by posttranscriptional 3' polyuridine tail formation.

Trypanosoma brucei mitochondrial transcripts can be posttranscriptionally processed by uridine addition or deletion. With editing of mRNAs, uridine addition and deletion create precisely altered reading frames. The addition of nonencoded uridines to mitochondrial guide RNAs results in a less precise modification. Although uridines are specifically added to the 3' termini, their number varies, which results in heterogeneous oligo(U) tails on guide RNAs. In this paper, we show that the mitochondrial 9S and 12S rRNAs are also modified by uridine addition. These modifications appear to have aspects in common with both RNA editing and oligo(U) tail formation. Metabolic labeling studies with intact mitochondria and [alpha-32P]UTP, in the absence of transcription, demonstrated the posttranscriptional timing of the event. T1 RNase comparison analyses of cytidine 3',5'-[5'-32P]biphosphate 3'-end-labeled and [alpha-32P]UTP metabolically labeled rRNAs, along with direct RNA sequencing of the 3' termini, identified the site of uridine addition and revealed the creation of an oligo(U) tail for both rRNAs. 12S and 9S rRNAs hybrid selected from total cell RNA exhibited the same modification, demonstrating the presence of this processing in vivo. Moreover, only 3'-poly(U)-tailed 9S and 12S rRNAs were detected in total cellular and mitochondrial RNAs, which suggests that they are the most abundant and probable mature forms. The 12S and 9S rRNA oligo(U) tails differed significantly from each other, with the 12S having a heterogeneous tail of 2 to 17 uridines and the 9S having a tail of precisely 11 uridines. The mechanism of formation and the function of the rRNA poly(U) tails remain to be determined.

Animals↗

PubMed1997

cDNA sequence analysis of cardiotoxin variants from Taiwan cobra.

Five cDNAs encoding cardiotoxin variants were constructed from the cellular RNA isolated from the venom glands of Naja naja atra by reverse transcription-polymerase chain reaction. A high degree of nucleotide sequence homology was observed between these variants and other determined ones. Among them, a novel cardiotoxin 6 had 61 amino acid residues rather than 60 ones that usually observed with Naja naja atra cardiotoxins. The other cardiotoxin variants were the homologues of cardiotoxins 1, V or N with one or two amino acid substitutions, respectively. These results probably reflect the involvement of RNA editing in the production of cardiotoxin variants in the venom of Taiwan cobra.

Amino Acid Sequence↗

PubMed1995

apobec-1, the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme, is a novel RNA-binding protein.

Apolipoprotein B (apoB) mRNA editing is mediated by an enzyme complex which includes the catalytic subunit, apobec-1. Recombinant GST/APOBEC-1 binds with high specificity to a rat apoB RNA template as demonstrated by UV cross-linking and electrophoretic mobility shift assay (EMSA). ApoB RNA binding was competed by poly(U), poly(A,U), and tRNA, but not by poly(A) or other homopolymeric ribonucleotides. UV cross-linking of GST/APOBEC-1 to an apoB RNA template was uninfluenced by the binding of proteins of approximately 60 and approximately 44 kDa, present in S100 extracts prepared from different sources. The binding of these proteins was similarly uninfluenced by the simultaneous binding of GST/APO-BEC-1. Moreover, the inclusion of heterologous S100 extracts in the RNA binding reactions completely abrogated the competitive displacement of GST/APOBEC-1 by tRNA. EMSA revealed the onset of RNA binding within 1-2 min, and its specificity was confirmed by a supershift with anti-GST/APOBEC-1 antisera. The structural specificity for apoB RNA binding, as inferred from EMSA, appears to be distinct from apoB RNA editing since wild-type chicken apoB RNA, which is not editable, and several mutant chicken apoB RNAs containing clustered mutations within the minimal apoB RNA editing cassette, bound with efficiency similar to the rat apoB RNA template. In conclusion, while the data suggest that apobec-1 binds AU-rich templates, the importance of this observation in the context of mammalian apoB mRNA editing remains unknown.

APOBEC-1 Deaminase↗

PubMed1995

Chicken double-stranded RNA adenosine deaminase has apparent specificity for Z-DNA.

A M(r) 140,000 protein has been purified from chicken lungs to apparent homogeneity. The protein binds with high affinity to a non-BNA conformation, which is most likely to the Z-DNA. The protein also has a binding site for double-stranded RNA (dsRNA). Peptide sequences from this protein show similarity to dsRNA adenosine deaminase, an enzyme that deaminates adenosine in dsRNA to form inosine. Assays for this enzyme confirm that dsRNA adenosine deaminase activity and Z-DNA binding are properties of the same molecule. The coupling of these two activities in a single molecule may indicate a distinctive mechanism of gene regulation that is, in part, dependent on DNA topology. As such, DNA topology, through its effects on the efficiency and extent of RNA editing may be important in the generation of new phenotypes during evolution.

Adenosine Deaminase↗

PubMed1994

Revision of the nucleotide sequence and RNA splicing pathway of the Neurospora mitochondrial gene encoding ATPase subunit 6.

Previous sequence analysis of the Neurospora oli2 (ATP6) mitochondrial gene suggested that, in addition to a typical Group-I intron, it contained an unusual, mostly-palindromic, 93-nucleotide intron. We report here revisions of the nucleotide sequence and analysis of the size and sequence of reverse-transcriptase PCR products that show: (1) the Group-I intron splice sites are located as predicted by previous DNA sequence analysis; (2) the putative 93-nt intron is not excised from the mature mRNA, and most of this sequence is actually in the 5' untranslated region. We conclude that the Neurospora ATP6 gene contains only one intron. Analysis of the cDNA sequence also confirms the non-universal nature of the Neurospora mitochondrial genetic code: a TGA codon inferred from the DNA sequence is present as UGA in the mRNA. This provides direct evidence that this codon is not altered, for example by RNA editing, to conform to the universal code.

Adenosine Triphosphatases↗

PubMed1990

The cytochrome oxidase subunit III gene in sunflower mitochondria is cotranscribed with an open reading frame conserved in higher plants.

The gene encoding subunit III of cytochrome oxidase (COXIII) has been identified in the sunflower mitochondrial genome. The COXIII coding region is located 570 bp downstream of a 477 bp open reading frame (ORFB). Sequence comparisons and hybridization experiments show that ORFB sequences are conserved in other plant mitochondrial genomes. Nucleotide and amino acid sequence comparisons suggest that RNA editing is required in sunflower mitochondria to synthesize a functional COXIII polypeptide.

Amino Acid Sequence↗

PubMed2003

Functional analysis of the KCS-like element of the interferon-inducible RNA-specific adenosine deaminase ADAR1 promoter.

The ADAR1 gene encodes an RNA-specific adenosine deaminase that alters the functional activity of both viral and cellular RNAs by posttranscriptional adenosine-to-inosine RNA editing. The interferon (IFN) responsive PI promoter of the ADAR1 gene possesses an IFN-stimulated response element (ISRE) responsible for IFN-inducibility, as well as an adjacent upstream sequence, designated kinase conserved sequence-like (KCS-l) element. The KCS-l element is similar to the 15-bp KCS element so far unique to the human and mouse RNA-dependent PKR kinase gene promoters. The KCS element of the PKR kinase (PKR) promoter is essential for both basal and IFN-inducible PKR promoter activity. We have now examined the functional properties of the KCS-l element of the ADAR1 PI promoter. Electrophoretic mobility shift assays (EMSAs) detected constitutively expressed nuclear proteins that bound selectively to the ADAR1 KCS-l element. Competition EMSA and antibody supershift assays indicated that ADAR1 KCS-l-binding proteins shared some properties with PKR KCS-binding proteins. However, transient transfection analyses performed with ADAR1 PI promoter constructs possessing deletion and substitution mutant forms of the KCS-l element revealed that the ADAR1 KCS-l element was not essential for either basal or IFN-inducible promoter activity. Substitution of the ADAR1 KCS-l element with the PKR KCS element increased both basal and inducible ADAR1 PI promoter activity. These results suggest that the KCS-l element of the ADAR1 PI promoter is not functionally equivalent to the KCS element of the PKR promoter.

Adenosine Deaminase↗

PubMed1999

T7 RNA polymerase-driven transcription in mitochondria of Leishmania tarentolae and Trypanosoma brucei.

The study of RNA editing and other molecular processes in the trypanosome mitochondrion would benefit greatly from the ability to insert and express exogenous DNA in the organelle. However, even with a method to introduce DNA, the current lack of knowledge about mitochondrial transcription would hinder efforts to obtain expression. To circumvent this problem, Leishmania tarentolae promastigotes and Trypanosoma brucei procyclic cells have been transfected with bacteriophage T7 RNA polymerase targeted to the mitochondrion. Mitochondria isolated from the transfectants contained active T7 RNA polymerase, as shown by a comigration in density gradients of mitochondrial marker enzymes and T7 polymerase activity. A DNA cassette under T7 control was introduced into isolated mitochondria from the transfectants by electroporation and the DNA was shown to be transcribed. This system should allow the transcription of foreign genes of choice within the mitochondrial matrix either in a transient assay using electroporation of DNA into isolated mitochondria, or in a stable assay using cells transfected with DNA by the biolistic gun method.

Animals↗

PubMed2003

Human fibrillarin forms a sub-complex with splicing factor 2-associated p32, protein arginine methyltransferases, and tubulins alpha 3 and beta 1 that is independent of its association with preribosomal ribonucleoprotein complexes.

Fibrillarin (FIB, Nop1p in yeast) is an RNA methyltransferase found not only in the fibrillar region of the nucleolus but also in Cajal bodies. FIB is essential for efficient processing of preribosomal RNA during ribosome biogenesis, although its precise function in this process and its role in Cajal bodies remain uncertain. Here, we demonstrate that the human FIB N-terminal glycine- and arginine-rich domain (residues 1-77) and its spacer region 1 (78-132) interact with splicing factor 2-associated p32 (SF2A-p32) and that the FIB methyltransferase-like domain (133-321) interacts with protein-arginine methyltransferase 5 (PRMT5, Janus kinase-binding protein 1). We also show that these proteins associate with several additional proteins, including PRMT1, tubulin alpha 3, and tubulin beta 1 to form a sub-complex that is principally independent of the association of FIB with preribosomal ribonucleoprotein complexes that co-immunoprecipitate with the sub-complex in human cells expressing FLAG-tagged FIB. Based on the physical association of FIB with SF2A-p32 and PRMTs, as well as the other reported results, we propose that FIB may coordinate both RNA and protein methylation during the processes of ribosome biogenesis in the nucleolus and RNA editing such as small nuclear (nucleolar) ribonucleoprotein biogenesis in Cajal bodies.

Chromosomal Proteins, Non-Histone↗

PubMed2004

Distant structural homology leads to the functional characterization of an archaeal PIN domain as an exonuclease.

Genome sequencing projects have focused attention on the problem of discovering the functions of protein domains that are widely distributed throughout living species but which are, as yet, largely uncharacterized. One such example is the PIN domain, found in eukaryotes, bacteria, and Archaea, and with suggested roles in signaling, RNase editing, and/or nucleotide binding. The first reported crystal structure of a PIN domain (open reading frame PAE2754, derived from the crenarchaeon, Pyrobaculum aerophilum) has been determined to 2.5 A resolution and is presented here. Mapping conserved residues from a multiple sequence alignment onto the structure identifies a putative active site. The discovery of distant structural homology with several exonucleases, including T4 phage RNase H and flap endonuclease (FEN1), further suggests a likely function for PIN domains as Mg2+-dependent exonucleases, a hypothesis that we have confirmed in vitro. The tetrameric structure of PAE2754, with the active sites inside a tunnel, suggests a mechanism for selective cleavage of single-stranded overhangs or flap structures. These results indicate likely DNA or RNA editing roles for prokaryotic PIN domains, which are strikingly numerous in thermophiles, and in organisms such as Mycobacterium tuberculosis. They also support previous hypotheses that eukaryotic PIN domains participate in RNAi and nonsense-mediated RNA degradation.

Amino Acid Sequence↗

PubMed1991

mRNA processing in the Trypanosomatidae.

Members of the Trypanosomatidae, which include the African trypanosomes, the American trypanosomes and the leishmanias, cause disease in vast proportions in man and his livestock and are a major detrimental factor to the social and economic well-being of the third world. Current research using the techniques of molecular biology has revealed two unusual types of mRNA processing in these protozoans; these are the addition of a shared leader sequence to the 5' ends of nuclear mRNAs by a mechanism of trans splicing, and the insertion and deletion of specific uridine residues in mitochondrial transcripts by RNA editing. The presence of these two mRNA processing pathways in the Trypanosomatidae has profound consequences for the organization and expression of their genetic information.

Amino Acid Sequence↗