Sequences of three Trypanosoma congolense maxicircle genes allow prediction of regions encoding transcripts that undergo extensive RNA editing.
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On the possibility of constructive neutral evolution.
The neutral theory often is presented as a theory of "noise" or silent changes at an isolated "molecular level," relevant to marking the steady pace of divergence, but not to the origin of biological structure, function, or complexity. Nevertheless, precisely these issues can be addressed in neutral models, such as those elaborated here with regard to scrambled ciliate genes, gRNA-mediated RNA editing, the transition from self-splicing to spliceosomal splicing, and the retention of duplicate genes. All of these are instances of a more general scheme of "constructive neutral evolution" that invokes biased variation, epistatic interactions, and excess capacities to account for a complex series of steps giving rise to novel structures or operations. The directional and constructive outcomes of these models are due not to neutral allele fixations per se, but to these other factors. Neutral models of this type may help to clarify the poorly understood role of nonselective factors in evolutionary innovation and directionality.
Amino Acid Sequence↗
Construction of a 2.5-Mb integrated physical and gene map of distal 21q22.3.
The gene-rich telomeric region of 21q harbors several loci relevant to human diseases including autoimmune polyglandular disease type I, nonsyndromic deafness, Knobloch syndrome, holoprosencephaly, and bipolar affective disorder. A contig of genomic clones in this region would facilitate the isolation of these genes. However, distal 21q22.3 has yet been poorly mapped, presumably due to the presence of sequences that are underrepresented in yeast artificial chromosome (YAC) libraries. We generated a framework of YACs and used these clones as starting points for the isolation of a combination of bacterial artificial chromosome clones, P1-derived artificial chromosome clones, and cosmid clones by chromosome walking procedures. These studies resulted in the construction of a high-resolution contig map spanning the 2.5-Mb region from PFKL to the telomere, approximately 2 Mb of which are covered by ready-to-sequence contigs. Within this map we determined the location and relative distance of 21 markers. These include 9 established genetic markers, the order of which is cen-PFKL-D21S154-D21S170-D21S171-D21S1903- D21S1897- D21S112-D21S1446-D21S1575-tel. Moreover, we established the precise map position of 13 genes and 4 ESTs including the recently isolated genes C21ORF2, SMT3H1, RNA editing deaminase 1 (ADARB1), folate transporter (SLC19A1), COL18A1, lanosterol synthase (LSS-PEN), pericentrin (PCNT), and arginine methyltransferase (HRMT1L1). This integrated map provides a useful resource for the mapping and isolation of disease genes and for the construction of a complete transcription map of distal 21q as well as for large-scale sequencing efforts.
Chromosome Mapping↗
Ligand-Mediated Reprogramming Redirects Liver-Tropic Ionizable Lipid Nanoparticles for Lung-Selective mRNA Delivery.
Systemic delivery of messenger RNA (mRNA) to target tissues and cells using lipid nanoparticles (LNPs) holds transformative potential for gene therapy. However, most clinically validated LNP exhibit strong liver tropism, and redirecting their organ specificity without redesigning entirely new chemistries remains challenging. Here we present a ligand-mediated lipid reprogramming approach that repurposes chemically defined, liver-tropic, ionizable lipids (lipidoids) for mRNA delivery beyond the liver. From a library of 90 degradable lipidoids, we identified 2-t6b as a potent liver-targeting platform. By site-specific displaying of small molecule ligands onto 2-t6b headgroup, we engineered a series of reconfigured lipidoids that achieve lung-specific targeting while retaining the parent delivery scaffold. Ligand7-2-t6b-lipid-functionalized LNP achieved over 200-fold higher mRNA translation in the lungs compared to the parent liver-tropic LNP. Proteomics and molecular docking analysis revealed enhanced binding of the modified lipid to vitronectin, a serum glycoprotein that improves integrin binding and thus promotes cellular uptake and translation efficiency. Ligand-mediated 2-t6b/ligand7 LNPs achieved outperformed efficacy and therapeutic potential in lung-specific genome editing relative to SORT-constructed 2-t6b LNP system. Our modular reprogramming strategy provides a generalizable framework to upgrade existing liver-biased LNPs into lung-selective mRNA carriers, advancing next-generation tissue-specific mRNA therapies for gene editing, protein replacement therapy, and regenerative medicine.
RNA, Messenger↗
Extensive editing of both processed and preprocessed maxicircle CR6 transcripts in Trypanosoma brucei.
Transcripts from several genes encoded in the Trypanosoma brucei maxicircle genome are altered by posttranscriptional uridine insertion and deletion through a process called RNA editing. We find that transcripts from the CR6 gene are extensively edited by addition of 132 uridines and deletion of 28 uridines to produce a fully edited mRNA 47% larger than unedited mRNA. Two open reading frames (ORFs) and their initiation and termination codons are created by editing of CR6 mRNA. Both ORFs specify small, hydrophobic proteins with no homology to proteins in three databases. Both unedited and edited CR6 transcripts are more abundant in bloodstream form than in procyclic form parasites. cDNA clones spanning both CR6 and the downstream NADH dehydrogenase subunit 5 (ND5) gene were isolated, indicating that mature CR6 and ND5 transcripts arise from a common precursor. Sequencing of these cDNAs revealed 37 nucleotides of overlap between the 3' end of CR6 and the 5' end of ND5. In addition, the CR6 portion of many of these molecules was extensively edited, indicating that RNA editing can precede precursor processing. These results provide the first clear demonstration of polycistronic transcription of maxicircle genes, and suggest new mechanisms by which both RNA editing and precursor processing may regulate maxicircle gene expression.
Amino Acid Sequence↗
Trypanosoma cruzi mitochondrial maxicircles display species- and strain-specific variation and a conserved element in the non-coding region.
BACKGROUND: The mitochondrial DNA of kinetoplastid flagellates is distinctive in the eukaryotic world due to its massive size, complex form and large sequence content. Comprised of catenated maxicircles that contain rRNA and protein-coding genes and thousands of heterogeneous minicircles encoding small guide RNAs, the kinetoplast network has evolved along with an extreme form of mRNA processing in the form of uridine insertion and deletion RNA editing. Many maxicircle-encoded mRNAs cannot be translated without this post-transcriptional sequence modification. RESULTS: We present the complete sequence and annotation of the Trypanosoma cruzi maxicircles for the CL Brener and Esmeraldo strains. Gene order is syntenic with Trypanosoma brucei and Leishmania tarentolae maxicircles. The non-coding components have strain-specific repetitive regions and a variable region that is unique for each strain with the exception of a conserved sequence element that may serve as an origin of replication, but shows no sequence identity with L. tarentolae or T. brucei. Alternative assemblies of the variable region demonstrate intra-strain heterogeneity of the maxicircle population. The extent of mRNA editing required for particular genes approximates that seen in T. brucei. Extensively edited genes were more divergent among the genera than non-edited and rRNA genes. Esmeraldo contains a unique 236-bp deletion that removes the 5'-ends of ND4 and CR4 and the intergenic region. Esmeraldo shows additional insertions and deletions outside of areas edited in other species in ND5, MURF1, and MURF2, while CL Brener has a distinct insertion in MURF2. CONCLUSION: The CL Brener and Esmeraldo maxicircles represent two of three previously defined maxicircle clades and promise utility as taxonomic markers. Restoration of the disrupted reading frames might be accomplished by strain-specific RNA editing. Elements in the non-coding region may be important for replication, transcription, and anchoring of the maxicircle within the kinetoplast network.
Amino Acid Sequence↗
Functions and mechanisms of RNA editing.
RNA editing can be broadly defined as any site-specific alteration in an RNA sequence that could have been copied from the template, excluding changes due to processes such as RNA splicing and polyadenylation. Changes in gene expression attributed to editing have been described in organisms from unicellular protozoa to man, and can affect the mRNAs, tRNAs, and rRNAs present in all cellular compartments. These sequence revisions, which include both the insertion and deletion of nucleotides, and the conversion of one base to another, involve a wide range of largely unrelated mechanisms. Recent advances in the development of in vitro editing and transgenic systems for these varied modifications have provided a better understanding of similarities and differences between the biochemical strategies, regulatory sequences, and cellular factors responsible for such RNA processing events.
Amino Acid Sequence↗
The complete mitochondrial genome of the common sea slater, Ligia oceanica (Crustacea, Isopoda) bears a novel gene order and unusual control region features.
BACKGROUND: Sequence data and other characters from mitochondrial genomes (gene translocations, secondary structure of RNA molecules) are useful in phylogenetic studies among metazoan animals from population to phylum level. Moreover, the comparison of complete mitochondrial sequences gives valuable information about the evolution of small genomes, e.g. about different mechanisms of gene translocation, gene duplication and gene loss, or concerning nucleotide frequency biases. The Peracarida (gammarids, isopods, etc.) comprise about 21,000 species of crustaceans, living in many environments from deep sea floor to arid terrestrial habitats. Ligia oceanica is a terrestrial isopod living at rocky seashores of the european North Sea and Atlantic coastlines. RESULTS: The study reveals the first complete mitochondrial DNA sequence from a peracarid crustacean. The mitochondrial genome of Ligia oceanica is a circular double-stranded DNA molecule, with a size of 15,289 bp. It shows several changes in mitochondrial gene order compared to other crustacean species. An overview about mitochondrial gene order of all crustacean taxa yet sequenced is also presented. The largest non-coding part (the putative mitochondrial control region) of the mitochondrial genome of Ligia oceanica is unexpectedly not AT-rich compared to the remainder of the genome. It bears two repeat regions (4x 10 bp and 3x 64 bp), and a GC-rich hairpin-like secondary structure. Some of the transfer RNAs show secondary structures which derive from the usual cloverleaf pattern. While some tRNA genes are putative targets for RNA editing, trnR could not be localized at all. CONCLUSION: Gene order is not conserved among Peracarida, not even among isopods. The two isopod species Ligia oceanica and Idotea baltica show a similarly derived gene order, compared to the arthropod ground pattern and to the amphipod Parhyale hawaiiensis, suggesting that most of the translocation events were already present the last common ancestor of these isopods. Beyond that, the positions of three tRNA genes differ in the two isopod species. Strand bias in nucleotide frequency is reversed in both isopod species compared to other Malacostraca. This is probably due to a reversal of the replication origin, which is further supported by the fact that the hairpin structure typically found in the control region shows a reversed orientation in the isopod species, compared to other crustaceans.
Animals↗
Mitochondrial electroporation and in organello RNA editing of chimeric atp6 transcripts.
The Sorghum bicolor atp6-1 gene and chimeric atp6 genes with additional maize sequences were introduced into isolated maize mitochondria via electroporation. Transcripts isolated after in vitro incubation of the transformed organelles were then analysed for RNA editing. Transcripts of the S. bicolor atp6-1 gene, and the RNAs obtained from most of chimeric sorghum-maize atp6 gene constructs tested, were not edited. However, the transcript of one engineered chimeric gene comprising the 5'untranslated sequence and a segment of the N-terminal ORF of the maize atp6 combined with the sorghum atp6 core ORF and 3'untranslated sequence was found to be partially edited. We were able to exclude low RNA stability or insufficient editing capacity as the reason for failure to edit in the other instances. Instead, the data indicate that the maize sequence in the edited fusion transcript provides a structural motif or binding site for a transcript-specific editing factor.
Base Sequence↗
Editing of the GLuR-B ion channel RNA in vitro by recombinant double-stranded RNA adenosine deaminase.
Double-stranded RNA (dsRNA)-specific adenosine deaminase (DRADA) has been implicated as an enzyme responsible for the editing of RNA transcripts encoding glutamate-gated ion channel subunits (GLuR) in brain. In one case, the editing alters the gene-encoded glutamine (Q) to an arginine (R) located within the channel-forming domain of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor subunit GLuR-B. The result of editing at this site, called the 'Q/R' site, is a profound alteration of the Ca2+ permeability of the GLuR channel. Using recombinantly expressed DRADA proteins, we now demonstrate in vitro that DRADA is indeed involved in editing of the GLuR-B RNA. In addition to the formation of an RNA duplex structure involving exon and intron sequences, Q/R site-selective editing by DRADA also requires a cofactor protein(s) commonly present even in non-neuronal cells. The accuracy and efficiency of this RNA editing system appear to be determined by the quantitative balance between DRADA, cofactor and substrate GLuR-B RNA.
Adenosine Deaminase↗
Editing of rps3/rpl16 transcripts creates a premature truncation of the rpl16 open reading frame.
Overlapping open reading frames corresponding to maize mitochondrial genes rps3 and rpl16 have been found in Petunia mitochondrial DNA. The DNA region associated with these two genes is part of the Petunia mitochondrial recombination repeat and is iterated three times. Analysis of transcripts from these genes shows that there is RNA editing of the coding regions and that one of the editing sites detected in the open reading frame overlap creates a premature stop codon in the rpl16 sequence. No transcripts were detected that were unedited at this site. Thus, in Petunia editing of rpl16 appears to render this gene nonfunctional.
Amino Acid Sequence↗
The dialectics of cancer: A theory of the initiation and development of cancer through errors in RNAi.
The recent discoveries of the RNA-mediated interference system in cells could explain all of the known features of human carcinogenesis. A key, novel idea, proposed here, is that the cell has the ability to recognise a mutated protein and/or mRNA. Secondly, the cell can generate its own short interfering RNA (siRNA) using an RNA polymerase to destroy mutated mRNA, even when only a single base pair in the gene has mutated. The anti-sense strand of the short RNA molecule (called sicRNA), targets the mutated mRNA of an oncogene or a tumour suppressor. The resulting double stranded RNA, using the RNA-induced silencing complex in the cytoplasm dices the mutated mRNA. In cancer-prone tissues, during cell mitosis, the sicRNA complex can move into the nucleus to target the mutated gene. The sicRNA, possibly edited by dsRNA-specific adenosine deaminase, converting adenosines to inosines, can be retained in the nucleus, with enhanced destructive capability. The sicRNA triggers the assembly of protein complexes leading to epigenetic modification of the promoter site of the mutant gene, specifically methylation of cytosines. In some instances, instead of methylation, the homologous DNA is degraded, leading to loss of heterozygosity. The factors controlling these two actions are unknown but the result is gene silencing or physical destruction of the mutant gene. The cell survives dependent on the functioning of the single, wild-type allele. An error in RNAi defence occurs when the sicRNA enters the nucleus and targets the sense strand of the wrong DNA. The sicRNA, because of the similarity of its short sequence and relaxed stringency, can target other RNAs, which are being transcribed. This can result in the methylation of the wrong promoter site of a gene or LOH of that region. In the vast majority of these cases, the aberrant hybridisations will have no effect on cell function or apoptosis eliminates non-viable cells. On a rare occasion, a preneoplastic cell is initiated when aberrant hybridisations switches on/off a gene involved in apoptosis, as well as a gene involved in cell proliferation and DNA damage surveillance. Genetic instability results when the sicRNA competes for a repeat sequence in the centromere or telomere, leading to gross chromosomal rearrangements. A malignancy develops when the sicRNAs fortuitously targets a microRNA (miRNA) or activates a transcription factor, resulting in the translation of a large number of new genes, alien to that tissue. This leads to dedifferentiation of the tissue, a resculpting of the histone code, chromosomal rearrangements, along a number of specific pathways, the gain of immortality and the dissemination of a metastatic cancer.
CpG Islands↗
Mitogenome assembly and phylogenetic relationships of Phalaris arundinacea.
INTRODUCTION: As a perennial herb of Poaceae, Phalaris arundinacea plays key roles in grazing, production, and soil and water conservation because of its well-developed rhizomes and seed dispersal. We assembled and annotated the first mitogenome of P. arundinacea to support evolutionary and taxonomic research. METHODS: We assembled and annotated the first complete mitochondrial genome of P. arundinacea by integrating Illumina short reads with Nanopore long reads via a hybrid assembly strategy. The genome architecture was comprehensively characterized, encompassing codon usage bias, repetitive sequence organization, and inter-organellar genetic exchange with the chloroplast genome. RESULTS AND DISCUSSION: Assembly of the P. arundinacea mitogenome revealed two circular structures with a combined length of 526,717 bp. The genome comprised a set of 37 protein-coding genes (PCGs), 27 tRNAs, and 8 rRNAs, with the rRNA genes exhibiting full assembly (100% coverage). The mitochondrial genome contained 154 forward and 164 palindromic repeats, along with 25 tandem repeats and 124 simple sequence repeats (SSRs). Notably, 102 SSRs were distributed on contig1, predominantly in tetrameric form. Furthermore, 376 RNA editing sites were predicted. A total of 104 fragments were integrated into the mitochondrial genome from the chloroplast, amounting to 55,866 bp of transferred sequence. Finally, phylogenetic analysis of 28 plant mitogenomes placed P. arundinacea closest to species within the genus Poa (P. chaixii and P. pratensis). Comparative analysis of non-synonymous-to-synonymous substitution rate (Ka/Ks) ratios across divergent species revealed that the mitochondrial genome of P. arundinacea underwent stabilizing evolutionary dynamics, characterized by predominant purifying selection with several lineage-specific variations in selective pressure. Our findings support the close phylogenetic relationship between P. arundinacea and species of the genus Poa and provide a reference mitochondrial genome resource for future comparative studies within Phalaris that incorporate broader taxon sampling. These results support deeper phylogenetic investigations of P. arundinacea and facilitate future work on its germplasm characterization and applied use.
Phalaris arundinacea↗
Identification of candidate mitochondrial RNA editing ligases from Trypanosoma brucei.
Most mitochondrial genes of Trypanosoma brucei do not contain the necessary information to make translatable mRNAs. These transcripts must undergo RNA editing, a posttranscriptional process by which uridine residues are added and deleted from mitochondrial mRNAs. RNA editing is believed to be catalyzed by a ribonucleoprotein complex containing endonucleolytic, terminal uridylyl transferase (TUTase), 3' uridine-specific exonucleolytic (U-exo), and ligase activities. None of the catalytic enzymes for RNA editing have been identified. Here we describe the identification of two candidate RNA ligases (48 and 52 kDa) that are core catalytic components of the T. brucei ribonucleoprotein editing complex. Both enzymes share homology to the covalent nucleotidyl transferase superfamily and contain five key signature motifs, including the active site KXXG. In this report, we present data on the proposed 48 kDa RNA editing ligase. We have prepared polyclonal antibodies against recombinant 48 kDa ligase that specifically recognize the trypanosome enzyme. When expressed in trypanosomes as an epitope-tagged fusion protein, the recombinant ligase localizes to the mitochondrion, associates with RNA editing complexes, and adenylates with ATP. These findings provide strong support for the enzymatic cascade model for kinetoplastid RNA editing.
Adenosine Triphosphate↗
GP mRNA of Ebola virus is edited by the Ebola virus polymerase and by T7 and vaccinia virus polymerases.
The glycoprotein gene of Ebola virus contains a translational stop codon in the middle, thus preventing synthesis of full-length glycoprotein. Twenty percent of the mRNA isolated from Ebola virus-infected cells was shown to be edited, containing one additional nontemplate A in a stretch of seven consecutive A residues. Only the edited mRNA species encoded full-length glycoprotein, whereas the exact copies of the viral template coded for a smaller secreted glycoprotein. Expression of the glycoprotein by an in vitro transcription/translation system, by the vaccinia virus/T7 polymerase system, and by recombinant vaccinia virus revealed that full-length glycoprotein was synthesized not only when the edited glycoprotein gene (8A's) was used as a template for T7 and vaccinia virus polymerases, but also when the nonedited (genomic) glycoprotein gene was used. Analysis of mRNA produced by T7 and vaccinia virus polymerase from the 7A's construct revealed that 1-5% contained alterations at the same site that was also edited by the Ebola virus polymerase. Our data indicate that the editing site in the Ebola virus glycoprotein gene is recognized not only by Ebola virus polymerase but also by DNA-dependent RNA polymerases of different origin.
Amino Acid Sequence↗
Models for antigen receptor gene rearrangement. II. Multiple rearrangement in the TCR: allelic exclusion or inclusion?
This series of papers addresses the effects of continuous Ag receptor gene rearrangement in lymphocytes on allelic exclusion. The previous paper discussed light chain gene rearrangement and receptor editing in B cells, and showed that these processes are ordered on three different levels. This order, combined with the constraints imposed by a strong negative selection, was shown to lead to effective allelic exclusion. In the present paper, we discuss rearrangement of TCR genes. In the TCR alpha-chain, allelic inclusion may be the rule rather than the exception. Several previous models, which attempted to explain experimental observations, such as the fractions of cells containing two productive TCRalpha rearrangements, did not sufficiently account for TCR gene organization, which limits secondary rearrangement, and for the effects of subsequent thymic selection. We present here a detailed, comprehensive computer simulation of TCR gene rearrangement, incorporating the interaction of this process with other aspects of lymphocyte development, including cell division, selection, cell death, and maturation. Our model shows how the observed fraction of T cells containing productive TCRalpha rearrangements on both alleles can be explained by the parameters of thymic selection imposed over a random rearrangement process.
Alleles↗
Multiple major increases and decreases in mitochondrial substitution rates in the plant family Geraniaceae.
BACKGROUND: Rates of synonymous nucleotide substitutions are, in general, exceptionally low in plant mitochondrial genomes, several times lower than in chloroplast genomes, 10-20 times lower than in plant nuclear genomes, and 50-100 times lower than in many animal mitochondrial genomes. Several cases of moderate variation in mitochondrial substitution rates have been reported in plants, but these mostly involve correlated changes in chloroplast and/or nuclear substitution rates and are therefore thought to reflect whole-organism forces rather than ones impinging directly on the mitochondrial mutation rate. Only a single case of extensive, mitochondrial-specific rate changes has been described, in the angiosperm genus Plantago. RESULTS: We explored a second potential case of highly accelerated mitochondrial sequence evolution in plants. This case was first suggested by relatively poor hybridization of mitochondrial gene probes to DNA of Pelargonium hortorum (the common geranium). We found that all eight mitochondrial genes sequenced from P. hortorum are exceptionally divergent, whereas chloroplast and nuclear divergence is unexceptional in P. hortorum. Two mitochondrial genes were sequenced from a broad range of taxa of variable relatedness to P. hortorum, and absolute rates of mitochondrial synonymous substitutions were calculated on each branch of a phylogenetic tree of these taxa. We infer one major, approximately 10-fold increase in the mitochondrial synonymous substitution rate at the base of the Pelargonium family Geraniaceae, and a subsequent approximately 10-fold rate increase early in the evolution of Pelargonium. We also infer several moderate to major rate decreases following these initial rate increases, such that the mitochondrial substitution rate has returned to normally low levels in many members of the Geraniaceae. Finally, we find unusually little RNA editing of Geraniaceae mitochondrial genes, suggesting high levels of retroprocessing in their history. CONCLUSION: The existence of major, mitochondrial-specific changes in rates of synonymous substitutions in the Geraniaceae implies major and reversible underlying changes in the mitochondrial mutation rate in this family. Together with the recent report of a similar pattern of rate heterogeneity in Plantago, these findings indicate that the mitochondrial mutation rate is a more plastic character in plants than previously realized. Many molecular factors could be responsible for these dramatic changes in the mitochondrial mutation rate, including nuclear gene mutations affecting the fidelity and efficacy of mitochondrial DNA replication and/or repair and--consistent with the lack of RNA editing--exceptionally high levels of "mutagenic" retroprocessing. That the mitochondrial mutation rate has returned to normally low levels in many Geraniaceae raises the possibility that, akin to the ephemerality of mutator strains in bacteria, selection favors a low mutation rate in plant mitochondria.
Base Sequence↗
A-to-I pre-mRNA editing in Drosophila is primarily involved in adult nervous system function and integrity.
Specific A-to-I RNA editing, like that seen in mammals, has been reported for several Drosophila ion channel genes. Drosophila possesses a candidate editing enzyme, dADAR. Here, we describe dADAR deletion mutants that lack ADAR activity in extracts. Correspondingly, all known Drosophila site-specific RNA editing (25 sites in three ion channel transcripts) is abolished. Adults lacking dADAR are morphologically wild-type but exhibit extreme behavioral deficits including temperature-sensitive paralysis, locomotor uncoordination, and tremors which increase in severity with age. Neurodegeneration accompanies the increase in phenotypic severity. Surprisingly, dADAR mutants are not short-lived. Thus, A-to-I editing of pre-mRNAs in Drosophila acts predominantly through nervous system targets to affect adult nervous system function, integrity, and behavior.
Adenosine Deaminase↗