transcriptome editing
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Trypanosoma cruzi is a unicellular protistan parasitic species that is comprised of strains and isolates exhibiting high levels of genetic and metabolic variability. In the insect vector, it is known to be highly responsive to starvation, a signal for progression to a life stage in which it can infect mammalian cells. Most mRNAs encoded in its mitochondrion require the targeted insertion and deletion of uridines to become translatable transcripts. This study defined differences in uridine-insertion/deletion RNA editing among three strains and established the mechanism whereby abundances of edited (and, thus, translatable) mitochondrial gene products increase during starvation. Our approach utilized our custom T-Aligner toolkit to describe transcriptome-wide editing events and reconstruct editing products from high-throughput sequencing data. We found that the relative abundance of mitochondrial transcripts and the proportion of mRNAs that are edited varies greatly between analyzed strains, a characteristic that could potentially impact metabolic capacity. Starvation typically led to an increase in overall editing activity rather than affecting a specific step in the process. We also determined that transcripts CR3, CR4, and ND3 produce multiple open reading frames that, if translated, would generate different proteins. Finally, we quantitated the inherent flexibility of editing in T. cruzi and found it to be higher relative to that in a related trypanosomatid lineage. Over time, new editing domains or patterns could prove advantageous to the organism and become more widespread within individual transcriptomes or among strains.
- MeSH
- editace RNA MeSH
- messenger RNA genetika metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- RNA mitochondriální genetika metabolismus MeSH
- RNA protozoální genetika metabolismus MeSH
- RNA metabolismus MeSH
- savci genetika MeSH
- transkriptom MeSH
- Trypanosoma brucei brucei * genetika MeSH
- Trypanosoma cruzi * genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Incipient sympatric speciation in blind mole rat, Spalax galili, in Israel, caused by sharp ecological divergence of abutting chalk-basalt ecologies, has been proposed previously based on mitochondrial and whole-genome nuclear DNA. Here, we present new evidence, including transcriptome, DNA editing, microRNA, and codon usage, substantiating earlier evidence for adaptive divergence in the abutting chalk and basalt populations. Genetic divergence, based on the previous and new evidence, is ongoing despite restricted gene flow between the two populations. The principal component analysis, neighbor-joining tree, and genetic structure analysis of the transcriptome clearly show the clustered divergent two mole rat populations. Gene-expression level analysis indicates that the population transcriptome divergence is displayed not only by soil divergence but also by sex. Gene ontology enrichment of the differentially expressed genes from the two abutting soil populations highlights reproductive isolation. Alternative splicing variation of the two abutting soil populations displays two distinct splicing patterns. L-shaped FST distribution indicates that the two populations have undergone divergence with gene flow. Transcriptome divergent genes highlight neurogenetics and nutrition characterizing the chalk population, and energetics, metabolism, musculature, and sensory perception characterizing the abutting basalt population. Remarkably, microRNAs also display divergence between the two populations. The GC content is significantly higher in chalk than in basalt, and stress-response genes mostly prefer nonoptimal codons. The multiple lines of evidence of ecological-genomic and genetic divergence highlight that natural selection overrules the gene flow between the two abutting populations, substantiating the sharp ecological chalk-basalt divergence driving sympatric speciation.
- MeSH
- ekosystém MeSH
- mikro RNA metabolismus MeSH
- půda MeSH
- silikáty MeSH
- Spalax genetika metabolismus MeSH
- sympatrie * MeSH
- tok genů MeSH
- transkriptom * MeSH
- uhličitan vápenatý MeSH
- vznik druhů (genetika) * MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Uridine insertion/deletion (U-indel) editing of mitochondrial mRNA, unique to the protistan class Kinetoplastea, generates canonical as well as potentially non-productive editing events. While the molecular machinery and the role of the guide (g) RNAs that provide required information for U-indel editing are well understood, little is known about the forces underlying its apparently error-prone nature. Analysis of a gRNA:mRNA pair allows the dissection of editing events in a given position of a given mitochondrial transcript. A complete gRNA dataset, paired with a fully characterized mRNA population that includes non-canonically edited transcripts, would allow such an analysis to be performed globally across the mitochondrial transcriptome. To achieve this, we have assembled 67 minicircles of the insect parasite Leptomonas pyrrhocoris, with each minicircle typically encoding one gRNA located in one of two similar-sized units of different origin. From this relatively narrow set of annotated gRNAs, we have dissected all identified mitochondrial editing events in L. pyrrhocoris, the strains of which dramatically differ in the abundance of individual minicircle classes. Our results support a model in which a multitude of editing events are driven by a limited set of gRNAs, with individual gRNAs possessing an inherent ability to guide canonical and non-canonical editing.
- MeSH
- editace RNA * MeSH
- fylogeneze MeSH
- genom protozoální * MeSH
- guide RNA, Kinetoplastida chemie metabolismus MeSH
- messenger RNA metabolismus MeSH
- RNA mitochondriální metabolismus MeSH
- transkriptom MeSH
- Trypanosomatina genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
RNA editing by targeted insertion and deletion of uridine is crucial to generate translatable mRNAs from the cryptogenes of the mitochondrial genome of kinetoplastids. This type of editing consists of a stepwise cascade of reactions generally proceeding from 3' to 5' on a transcript, resulting in a population of partially edited as well as pre-edited and completely edited molecules for each mitochondrial cryptogene of these protozoans. Often, the number of uridines inserted and deleted exceed the number of nucleotides that are genome-encoded. Thus, analysis of kinetoplastid mitochondrial transcriptomes has proven frustratingly complex. Here we present our analysis of Leptomonas pyrrhocoris mitochondrial cDNA deep sequencing reads using T-Aligner, our new tool which allows comprehensive characterization of RNA editing, not relying on targeted transcript amplification and on prior knowledge of final edited products. T-Aligner implements a pipeline of read mapping, visualization of all editing states and their coverage, and assembly of canonical and alternative translatable mRNAs. We also assess T-Aligner functionality on a more challenging deep sequencing read input from Trypanosoma cruzi. The analysis reveals that transcripts of cryptogenes of both species undergo very complex editing that includes the formation of alternative open reading frames and whole categories of truncated editing products.
- MeSH
- editace RNA * MeSH
- genom mitochondriální genetika MeSH
- genom protozoální genetika MeSH
- izoformy RNA genetika metabolismus MeSH
- mitochondrie genetika metabolismus MeSH
- RNA mitochondriální genetika metabolismus MeSH
- RNA protozoální genetika metabolismus MeSH
- sestřih RNA MeSH
- stanovení celkové genové exprese metody MeSH
- Trypanosoma brucei brucei genetika metabolismus MeSH
- Trypanosomatina genetika metabolismus MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In eukaryotes mRNA transcripts are extensively processed by different post-transcriptional events such as alternative splicing and RNA editing in order to generate many different mRNAs from the same gene, increasing the transcriptome and then the proteome diversity. The most frequent RNA editing mechanism in mammals involves the conversion of specific adenosines into inosines by the ADAR family of enzymes. This editing event can alter the sequence and the secondary structure of RNA molecules, with consequences for final proteins and regulatory RNAs. Alteration in RNA editing has been connected to tumor progression and many other important human diseases. Analysis of many editing sites in various cancer types is expected to provide new diagnostic and prognostic markers and might contribute to early detection of cancer, the monitoring of response to therapy, and to the detection of minimal residual disease.
- MeSH
- adenosindeaminasa genetika chemie metabolismus MeSH
- apolipoprotein B-100 chemie metabolismus MeSH
- apolipoprotein B-48 chemie metabolismus MeSH
- editace RNA fyziologie genetika MeSH
- glutamátové receptory chemie metabolismus MeSH
- lidé MeSH
- messenger RNA chemie metabolismus MeSH
- nádory metabolismus MeSH
- tyrosinfosfatasy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- odvolaná publikace MeSH
- přehledy MeSH
Diplonemids are highly abundant heterotrophic marine protists. Previous studies showed that their strikingly bloated mitochondrial genome is unique because of systematic gene fragmentation and manifold RNA editing. Here we report a comparative study of mitochondrial genome architecture, gene structure and RNA editing of six recently isolated, phylogenetically diverse diplonemid species. Mitochondrial gene fragmentation and modes of RNA editing, which include cytidine-to-uridine (C-to-U) and adenosine-to-inosine (A-to-I) substitutions and 3' uridine additions (U-appendage), are conserved across diplonemids. Yet as we show here, all these features have been pushed to their extremes in the Hemistasiidae lineage. For example, Namystynia karyoxenos has its genes fragmented into more than twice as many modules than other diplonemids, with modules as short as four nucleotides. Furthermore, we detected in this group multiple A-appendage and guanosine-to-adenosine (G-to-A) substitution editing events not observed before in diplonemids and found very rarely elsewhere. With >1,000 sites, C-to-U and A-to-I editing in Namystynia is nearly 10 times more frequent than in other diplonemids. The editing density of 12% in coding regions makes Namystynia's the most extensively edited transcriptome described so far. Diplonemid mitochondrial genome architecture, gene structure and post-transcriptional processes display such high complexity that they challenge all other currently known systems.
Kinetoplastids are flagellated protozoans, whose members include the pathogens Trypanosoma brucei, T. cruzi and Leishmania species, that are considered among the earliest diverging eukaryotes with a mitochondrion. This organelle has become famous because of its many unusual properties, which are unique to the order Kinetoplastida, including an extensive kinetoplast DNA network and U-insertion/deletion type RNA editing of its mitochondrial transcripts. In the last decade, considerable progress has been made in elucidating the complex machinery of RNA editing. Moreover, our understanding of the structure and replication of kinetoplast DNA has also dramatically improved. Much less however, is known, about the developmental regulation of RNA editing, its integration with other RNA maturation processes, stability of mitochondrial mRNAs, or evolution of the editing process itself. Yet the profusion of genomic data recently made available by sequencing consortia, in combination with methods of reverse genetics, hold promise in understanding the complexity of this exciting organelle, knowledge of which may enable us to fight these often medically important protozoans.
- MeSH
- editace RNA MeSH
- exprese genu MeSH
- financování organizované MeSH
- genetická transkripce MeSH
- genom protozoální MeSH
- Kinetoplastida genetika MeSH
- kinetoplastová DNA chemie MeSH
- messenger RNA metabolismus MeSH
- mitochondriální geny MeSH
- mitochondrie genetika MeSH
- RNA protozoální metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- přehledy MeSH
We review current studies of plant mitochondrial transcriptomes performed by RNA-seq, highlighting methodological challenges unique to plant mitochondria. We propose ways to improve read mapping accuracy and sensitivity such as modifying a reference genome at RNA editing sites, using splicing- and ambiguity-competent aligners, and masking chloroplast- or nucleus-derived sequences. We also outline modified RNA-seq methods permitting more accurate detection and quantification of partially edited sites and the identification of transcription start sites on a genome-wide scale. The application of RNA-seq goes beyond genome-wide determination of transcript levels and RNA maturation events, and emerges as an elegant resource for the comprehensive identification of editing, splicing, and transcription start sites. Thus, improved RNA-seq methods customized for plant mitochondria hold tremendous potential for advancing our understanding of plant mitochondrial evolution and cyto-nuclear interactions in a broad array of plant species.
BACKGROUND: Species within the angiosperm genus Silene contain the largest mitochondrial genomes ever identified. The enormity of these genomes (up to 11 Mb in size) appears to be the result of increased non-coding DNA, which represents >99 % of the genome content. These genomes are also fragmented into dozens of circular-mapping chromosomes, some of which contain no identifiable genes, raising questions about if and how these 'empty' chromosomes are maintained by selection. To assess the possibility that they contain novel and unannotated functional elements, we have performed RNA-seq to analyze the mitochondrial transcriptome of Silene noctiflora. RESULTS: We identified regions of high transcript abundance in almost every chromosome in the mitochondrial genome including those that lack any annotated genes. In some cases, these transcribed regions exhibited higher expression levels than some core mitochondrial protein-coding genes. We also identified RNA editing sites throughout the genome, including 97 sites that were outside of protein-coding gene sequences and found in pseudogenes, introns, UTRs, and transcribed intergenic regions. Unlike in protein-coding sequences, however, most of these RNA editing sites were only edited at intermediate frequencies. Finally, analysis of mitochondrial small RNAs indicated that most were likely degradation products from longer transcripts, but we did identify candidates for functional small RNAs that mapped to intergenic regions and were not associated with longer RNA transcripts. CONCLUSIONS: Our findings demonstrate transcriptional activity in many localized regions within the extensive intergenic sequence content in the S. noctiflora mitochondrial genome, supporting the possibility that the genome contains previously unidentified functional elements. However, transcription by itself is not proof of functional importance, and we discuss evidence that some of the observed transcription and post-transcriptional modifications are non-adaptive. Therefore, further investigations are required to determine whether any of the identified transcribed regions have played a functional role in the proliferation and maintenance of the enormous non-coding regions in Silene mitochondrial genomes.
- MeSH
- editace RNA MeSH
- genom mitochondriální * MeSH
- genom rostlinný * MeSH
- intergenová DNA MeSH
- messenger RNA MeSH
- otevřené čtecí rámce MeSH
- pseudogeny MeSH
- RNA MeSH
- rostlinné geny MeSH
- sekvenční analýza RNA MeSH
- Silene genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
A dozen mRNAs are edited by multiple insertions and/or deletions of uridine residues in the mitochondrion of Trypanosoma brucei Several protein complexes have been implicated in performing this type of RNA editing, including the mitochondrial RNA-binding complex 1 (MRB1). Two paralogous novel RNA-binding proteins, MRB8170 and MRB4160, are loosely associated with the core MRB1 complex. Their roles in RNA editing and effects on target mRNAs are so far not well understood. In this study, individual-nucleotide-resolution UV-cross-linking and affinity purification (iCLAP) revealed a preferential binding of both proteins to mitochondrial mRNAs, which was positively correlated with their extent of editing. Integrating additional in vivo and in vitro data, we propose that binding of MRB8170 and/or MRB4160 onto pre-mRNA marks it for the initiation of editing and that initial binding of both proteins may facilitate the recruitment of other components of the RNA editing/processing machinery to ensure efficient editing. Surprisingly, MRB8170 also binds never-edited mRNAs, suggesting that at least this paralog has an additional role outside RNA editing to shape the mitochondrial transcriptome. IMPORTANCE: Trypanosoma brucei mitochondrial mRNAs undergo maturation by RNA editing, a unique process involving decrypting open reading frames by the precise deletion and/or insertion of uridine (U) residues at specific positions on an mRNA. This process is catalyzed by multiprotein complexes, such as the RNA editing core complex, which provides the enzymatic activities needed for U insertion/deletion at a single editing site. Less well understood is how RNA editing occurs throughout an mRNA bearing multiple sites. To address this question, we mapped at single-nucleotide resolution the RNA interactions of two unique RNA-binding proteins (RBPs). These RBPs are part of the mitochondrial RNA-binding complex 1, hypothesized to mediate multiple rounds of RNA editing. Both RBPs were shown to mark mRNAs for the process in correlation with the number of editing sites on the transcript. Surprisingly, one also binds mRNAs that bypass RNA editing, indicating that it may have an additional role outside RNA editing.
