mtR_find: A Parallel Processing Tool to Identify and Annotate RNAs Derived from the Mitochondrial Genome.

RNAs originating from mitochondrial genomes are abundant in transcriptomic datasets produced by high-throughput sequencing technologies, primarily in short-read outputs. Specific features of mitochondrial small RNAs (mt-sRNAs), such as non-templated additions, presence of length variants, sequence variants, and other modifications, necessitate the need for the development of an appropriate tool for their effective identification and annotation. We have developed mtR_find, a tool to detect and annotate mitochondrial RNAs, including mt-sRNAs and mitochondria-derived long non-coding RNAs (mt-lncRNA). mtR_find uses a novel method to compute the count of RNA sequences from adapter-trimmed reads. When analyzing the published datasets with mtR_find, we identified mt-sRNAs significantly associated with the health conditions, such as hepatocellular carcinoma and obesity, and we discovered novel mt-sRNAs. Furthermore, we identified mt-lncRNAs in early development in mice. These examples show the immediate impact of miR_find in extracting a novel biological information from the existing sequencing datasets. For benchmarking, the tool has been tested on a simulated dataset and the results were concordant. For accurate annotation of mitochondria-derived RNA, particularly mt-sRNA, we developed an appropriate nomenclature. mtR_find encompasses the mt-ncRNA transcriptomes in unpreceded resolution and simplicity, allowing re-analysis of the existing transcriptomic databases and the use of mt-ncRNAs as diagnostic or prognostic markers in the field of medicine.

Mobile group I introns at nuclear rDNA position L2066 harbor sense and antisense homing endonuclease genes intervened by spliceosomal introns.

BACKGROUND: Mobile group I introns encode homing endonucleases that confer intron mobility initiated by a double-strand break in the intron-lacking allele at the site of insertion. Nuclear ribosomal DNA of some fungi and protists contain mobile group I introns harboring His-Cys homing endonuclease genes (HEGs). An intriguing question is how protein-coding genes embedded in nuclear ribosomal DNA become expressed. To address this gap of knowledge we analyzed nuclear L2066 group I introns from myxomycetes and ascomycetes. RESULTS: A total of 34 introns were investigated, including two identified mobile-type introns in myxomycetes with HEGs oriented in sense or antisense directions. Intriguingly, both HEGs are interrupted by spliceosomal introns. The intron in Didymium squamulosum, which harbors an antisense oriented HEG, was investigated in more detail. The group I intron RNA self-splices in vitro, thus generating ligated exons and full-length intron circles. The intron HEG is expressed in vivo in Didymium cells, which involves removal of a 47-nt spliceosomal intron (I-47) and 3' polyadenylation of the mRNA. The D. squamulosum HEG (lacking the I-47 intron) was over-expressed in E. coli, and the corresponding protein was purified and shown to confer endonuclease activity. The homing endonuclease was shown to cleave an intron-lacking DNA and to produce a pentanucleotide 3' overhang at the intron insertion site. CONCLUSIONS: The L2066 family of nuclear group I introns all belong to the group IE subclass. The D. squamulosum L2066 intron contains major hallmarks of a true mobile group I intron by encoding a His-Cys homing endonuclease that generates a double-strand break at the DNA insertion site. We propose a potential model to explain how an antisense HEG becomes expressed from a nuclear ribosomal DNA locus.

Structural Organization of S516 Group I Introns in Myxomycetes.

Group I introns are mobile genetic elements encoding self-splicing ribozymes. Group I introns in nuclear genes are restricted to ribosomal DNA of eukaryotic microorganisms. For example, the myxomycetes, which represent a distinct protist phylum with a unique life strategy, are rich in nucleolar group I introns. We analyzed and compared 75 group I introns at position 516 in the small subunit ribosomal DNA from diverse and distantly related myxomycete taxa. A consensus secondary structure revealed a conserved group IC1 ribozyme core, but with a surprising RNA sequence complexity in the peripheral regions. Five S516 group I introns possess a twintron organization, where a His-Cys homing endonuclease gene insertion was interrupted by a small spliceosomal intron. Eleven S516 introns contained direct repeat arrays with varying lengths of the repeated motif, a varying copy number, and different structural organizations. Phylogenetic analyses of S516 introns and the corresponding host genes revealed a complex inheritance pattern, with both vertical and horizontal transfers. Finally, we reconstructed the evolutionary history of S516 nucleolar group I introns from insertion of mobile-type introns at unoccupied cognate sites, through homing endonuclease gene degradation and loss, and finally to the complete loss of introns. We conclude that myxomycete S516 introns represent a family of genetic elements with surprisingly dynamic structures despite a common function in RNA self-splicing.

A Phylogenetic Approach to Structural Variation in Organization of Nuclear Group I Introns and Their Ribozymes.

Nuclear group I introns are restricted to the ribosomal DNA locus where they interrupt genes for small subunit and large subunit ribosomal RNAs at conserved sites in some eukaryotic microorganisms. Here, the myxomycete protists are a frequent source of nuclear group I introns due to their unique life strategy and a billion years of separate evolution. The ribosomal DNA of the myxomycete Mucilago crustacea was investigated and found to contain seven group I introns, including a direct repeat-containing intron at insertion site S1389 in the small subunit ribosomal RNA gene. We collected, analyzed, and compared 72 S1389 group IC1 introns representing diverse myxomycete taxa. The consensus secondary structure revealed a conserved ribozyme core, but with surprising sequence variations in the guanosine binding site in segment P7. Some S1389 introns harbored large extension sequences in the peripheral region of segment P9 containing direct repeat arrays. These repeats contained up to 52 copies of a putative internal guide sequence motif. Other S1389 introns harbored homing endonuclease genes in segment P1 encoding His-Cys proteins. Homing endonuclease genes were further interrupted by small spliceosomal introns that have to be removed in order to generate the open reading frames. Phylogenetic analyses of S1389 intron and host gene indicated both vertical and horizontal intron transfer during evolution, and revealed sporadic appearances of direct repeats, homing endonuclease genes, and guanosine binding site variants among the myxomycete taxa.

High-Throughput Identification of Adapters in Single-Read Sequencing Data.

Sequencing datasets available in public repositories are already high in number, and their growth is exponential. Raw sequencing data files constitute a substantial portion of these data, and they need to be pre-processed for any downstream analyses. The removal of adapter sequences is the first essential step. Tools available for the automated detection of adapters in single-read sequencing protocol datasets have certain limitations. To explore these datasets, one needs to retrieve the information on adapter sequences from the methods sections of appropriate research articles. This can be time-consuming in metadata analyses. Moreover, not all research articles provide the information on adapter sequences. We have developed adapt_find, a tool that automates the process of adapter sequences identification in raw single-read sequencing datasets. We have verified adapt_find through testing a number of publicly available datasets. adapt_find secures a robust, reliable and high-throughput process across different sequencing technologies and various adapter designs. It does not need prior knowledge of the adapter sequences. We also produced associated tools: random_mer, for the detection of random N bases either on one or both termini of the reads, and fastqc_parser, for consolidating the results from FASTQC outputs. Together, this is a valuable tool set for metadata analyses on multiple sequencing datasets.

Proteomics Analysis of Early Developmental Stages of Zebrafish Embryos.

Zebrafish is a well-recognized organism for investigating vertebrate development and human diseases. However, the data on zebrafish proteome are scarce, particularly during embryogenesis. This is mostly due to the overwhelming abundance of egg yolk proteins, which tend to mask the detectable presence of less abundant proteins. We developed an efficient procedure to reduce the amount of yolk in zebrafish early embryos to improve the Liquid chromatography-tandem mass spectrometry (LC-MS)-based shotgun proteomics analysis. We demonstrated that the deyolking procedure resulted in a greater number of proteins being identified. This protocol resulted in approximately 2-fold increase in the number of proteins identified in deyolked samples at cleavage stages, and the number of identified proteins increased greatly by 3-4 times compared to non-deyolked samples in both oblong and bud stages. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed a high number of functional proteins differentially accumulated in the deyolked versus non-deyolked samples. The most prominent enrichments after the deyolking procedure included processes, functions, and components related to cellular organization, cell cycle, control of replication and translation, and mitochondrial functions. This deyolking procedure improves both qualitative and quantitative proteome analyses and provides an innovative tool in molecular embryogenesis of polylecithal animals, such as fish, amphibians, reptiles, or birds.

Giant group I intron in a mitochondrial genome is removed by RNA back-splicing.

BACKGROUND: The mitochondrial genomes of mushroom corals (Corallimorpharia) are remarkable for harboring two complex group I introns; ND5-717 and COI-884. How these autocatalytic RNA elements interfere with mitochondrial RNA processing is currently not known. Here, we report experimental support for unconventional processing events of ND5-717 containing RNA. RESULTS: We obtained the complete mitochondrial genome sequences and corresponding mitochondrial transcriptomes of the two distantly related corallimorpharian species Ricordea yuma and Amplexidiscus fenestrafer. All mitochondrial genes were found to be expressed at the RNA-level. Both introns were perfectly removed by autocatalytic splicing, but COI-884 excision appeared more efficient than ND5-717. ND5-717 was organized into giant group I intron elements of 18.1 kb and 19.3 kb in A. fenestrafer and R. yuma, respectively. The intron harbored almost the entire mitochondrial genome embedded within the P8 peripheral segment. CONCLUSION: ND5-717 was removed by group I intron splicing from a small primary transcript that contained a permutated intron-exon arrangement. The splicing pathway involved a circular exon-containing RNA intermediate, which is a hallmark of RNA back-splicing. ND5-717 represents the first reported natural group I intron that becomes excised by back-splicing from a permuted precursor RNA. Back-splicing may explain why Corallimorpharia mitochondrial genomes tolerate giant group I introns.

Ocean acidification at a coastal CO2 vent induces expression of stress-related transcripts and transposable elements in the sea anemone Anemonia viridis.

Ocean acidification threatens to disrupt interactions between organisms throughout marine ecosystems. The diversity of reef-building organisms decreases as seawater CO2 increases along natural gradients, yet soft-bodied animals, such as sea anemones, are often resilient. We sequenced the polyA-enriched transcriptome of adult sea anemone Anemonia viridis and its dinoflagellate symbiont sampled along a natural CO2 gradient in Italy to assess stress levels in these organisms. We found that about 3.1% of the anemone transcripts, but <1% of the Symbiodinium sp. transcripts were differentially expressed. Processes enriched at high seawater CO2 were linked to cellular stress and inflammation, including significant up-regulation of protective cellular functions and down-regulation of metabolic pathways. Transposable elements were differentially expressed at high seawater CO2, with an extreme up-regulation (> 100-fold) of the BEL-family of long terminal repeat retrotransposons. Seawater acidified by CO2 generated a significant stress reaction in A. viridis, but no bleaching was observed and Symbiodinium sp. appeared to be less affected. These observed changes indicate the mechanisms by which A. viridis acclimate to survive chronic exposure to ocean acidification conditions. We conclude that many organisms that are common in acidified conditions may nevertheless incur costs due to hypercapnia and/or lowered carbonate saturation states.

Deep-water sea anemone with a two-chromosome mitochondrial genome.

Mitochondrial genome organization of sea anemones appears conserved among species and families, and is represented by a single circular DNA molecule of 17 to 21 kb. The mitochondrial gene content corresponds to the same 13 protein components of the oxidative phosphorylation (OxPhos) system as in vertebrates. Hallmarks, however, include a highly reduced tRNA gene repertoire and the presence of autocatalytic group I introns. Here we demonstrate that the mitochondrial genome of the deep-water sea anemone Protanthea simplex deviates significantly from that of other known sea anemones. The P. simplex mitochondrial genome contains a heavily scrambled order of genes that are coded on both DNA strands and organized along two circular mito-chromosomes, MCh-I and MCh-II. We found MCh-I to be representative of the prototypic sea anemone mitochondrial genome, encoding 12 OxPhos proteins, two ribosomal RNAs, two transfer RNAs, and a group I intron. In contrast, MCh-II was found to be a laterally transferred plasmid-like DNA carrying the conserved cytochrome oxidase II gene and a second allele of the small subunit ribosomal RNA gene.

Cancer-specific SNPs originate from low-level heteroplasmic variants in human mitochondrial genomes of a matched cell line pair.

Low-level mitochondrial heteroplasmy is a common phenomenon in both normal and cancer cells. Here, we investigate the link between low-level heteroplasmy and mitogenome mutations in a human breast cancer matched cell line by high-throughput sequencing. We identified 23 heteroplasmic sites, of which 15 were common between normal cells (Hs578Bst) and cancer cells (Hs578T). Most sites were clustered within the highly conserved Complex IV and ribosomal RNA genes. Two heteroplasmic variants in normal cells were found as fixed mutations in cancer cells. This indicates a positive selection of these variants in cancer cells. RNA-Seq analysis identified upregulated L-strand specific transcripts in cancer cells, which include three mitochondrial long non-coding RNA molecules. We hypothesize that this is due to two cancer cell-specific mutations in the control region.

A mitochondrial long noncoding RNA in atlantic cod harbors complex heteroplasmic tandem repeat motifs.

A heteroplasmic tandem repeat (HTR) array occupies 100 to 300 bp of the mitochondrial DNA control region in the Atlantic cod, and recently we noted that the repeat appeared integrated in a polyadenylated mitochondrial long noncoding RNA. Here we provide a more detailed analysis of the mitochondrial HTR in the mitochondrial genome of 134 Atlantic cod specimens. We report all specimens to harbor mitochondrial HTRs in the control region, and identified 26 distinct variants among the 402 repeat motifs assessed. Whereas most specimens contained HTR profiles of 2-5 copies consisting of the same 40-bp motif, 22 specimens showed compound HTR arrays of at least two types of motifs present in the same mitochondrial DNA molecule. We found HTR profiles to be highly conserved between different tissue types of a single individual, and strictly maternally inherited in a mating experiment between parental Atlantic cod expressing different HTR profiles and array motifs. We conclude that mitochondrial heteroplasmy in the control region is very common in Atlantic cod, and results in length heterogenity of the long noncoding RNA lncCR-H.

Mitochondrial genome variation of Atlantic cod.

OBJECTIVE: The objective of this study was to analyse intraspecific sequence variation of Atlantic cod mitochondrial DNA, based on a comprehensive collection of completely sequenced mitochondrial genomes. RESULTS: We determined the complete mitochondrial DNA sequence of 124 cod specimens from the eastern and western part of the species' distribution range in the North Atlantic Ocean. All specimens harboured a unique mitochondrial DNA haplotype. Nine hundred and fifty-two polymorphic sites were identified, including 109 non-synonymous sites within protein coding regions. Eighteen variable sites were identified as indels, exclusively distributed in structural RNA genes and non-coding regions. Phylogeographic analyses based on 156 available cod mitochondrial genomes did not reveal a clear structure. There was a lack of mitochondrial genetic differentiation between two ecotypes of cod in the eastern North Atlantic, but eastern and western cod were differentiated and mitochondrial genome diversity was higher in the eastern than the western Atlantic, suggesting deviating population histories. The geographic distribution of mitochondrial genome variation seems to be governed by demographic processes and gene flow among ecotypes that are otherwise characterized by localized genomic divergence associated with chromosomal inversions.

Elucidating the Small Regulatory RNA Repertoire of the Sea Anemone Anemonia viridis Based on Whole Genome and Small RNA Sequencing.

Cnidarians harbor a variety of small regulatory RNAs that include microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), but detailed information is limited. Here, we report the identification and expression of novel miRNAs and putative piRNAs, as well as their genomic loci, in the symbiotic sea anemone Anemonia viridis. We generated a draft assembly of the A. viridis genome with putative size of 313 Mb that appeared to be composed of about 36% repeats, including known transposable elements. We detected approximately equal fractions of DNA transposons and retrotransposons. Deep sequencing of small RNA libraries constructed from A. viridis adults sampled at a natural CO2 gradient off Vulcano Island, Italy, identified 70 distinct miRNAs. Eight were homologous to previously reported miRNAs in cnidarians, whereas 62 appeared novel. Nine miRNAs were recognized as differentially expressed along the natural seawater pH gradient. We found a highly abundant and diverse population of piRNAs, with a substantial fraction showing ping-pong signatures. We identified nearly 22% putative piRNAs potentially targeting transposable elements within the A. viridis genome. The A. viridis genome appeared similar in size to that of other hexacorals with a very high divergence of transposable elements resembling that of the sea anemone genus Exaiptasia. The genome encodes and expresses a high number of small regulatory RNAs, which include novel miRNAs and piRNAs. Differentially expressed small RNAs along the seawater pH gradient indicated regulatory gene responses to environmental stressors.

Expression of homing endonuclease gene and insertion-like element in sea anemone mitochondrial genomes: Lesson learned from Anemonia viridis.

The mitochondrial genomes of sea anemones are dynamic in structure. Invasion by genetic elements, such as self-catalytic group I introns or insertion-like sequences, contribute to sea anemone mitochondrial genome expansion and complexity. By using next generation sequencing we investigated the complete mtDNAs and corresponding transcriptomes of the temperate sea anemone Anemonia viridis and its closer tropical relative Anemonia majano. Two versions of fused homing endonuclease gene (HEG) organization were observed among the Actiniidae sea anemones; in-frame gene fusion and pseudo-gene fusion. We provided support for the pseudo-gene fusion organization in Anemonia species, resulting in a repressed HEG from the COI-884 group I intron. orfA, a putative protein-coding gene with insertion-like features, was present in both Anemonia species. Interestingly, orfA and COI expression were significantly up-regulated upon long-term environmental stress corresponding to low seawater pH conditions. This study provides new insights to the dynamics of sea anemone mitochondrial genome structure and function.

Zoantharian mitochondrial genomes contain unique complex group I introns and highly conserved intergenic regions.

Complex group I introns represent hallmarks of hexacoral mitochondrial genomes (mtDNAs). These intron elements are expected to influence the gene organization and gene expression. We sequenced the mitochondrial genome and transcriptome of Zoanthus sansibariscus and Palythoa heliodiscus, two zoantharian species (colonial anemones) representing different families within the suborder Brachycnemina. The circular and approximately 21kb mtDNAs contained two group I introns, one in ND5 and another in COI. The ND5-717 intron harbored two conventional mitochondrial genes (ND1 and ND3) within its structure and revealed several conserved features compared to ND5-717 in sea anemones. The COI intron, however, was inserted at a unique location (after position 867), which was different from that in sea anemones (position 884) and stony corals (position 720). COI-867 contained a homing endonuclease gene (HEG) with remarkable features, including species-specific length variations and only one copy of the essential LAGLIDADG motif. Whereas transcriptome analysis indicated that all conventional mtDNA genes were expressed, HEG expression appeared significantly repressed. Finally, we identified absolutely conserved non-coding repeat motifs with antisense features and potential regulatory functions.

Accumulation of Stable Full-Length Circular Group I Intron RNAs during Heat-Shock.

Group I introns in nuclear ribosomal RNA of eukaryotic microorganisms are processed by splicing or circularization. The latter results in formation of full-length circular introns without ligation of the exons and has been proposed to be active in intron mobility. We applied qRT-PCR to estimate the copy number of circular intron RNA from the myxomycete Didymium iridis. In exponentially growing amoebae, the circular introns are nuclear and found in 70 copies per cell. During heat-shock, the circular form is up-regulated to more than 500 copies per cell. The intron harbours two ribozymes that have the potential to linearize the circle. To understand the structural features that maintain circle integrity, we performed chemical and enzymatic probing of the splicing ribozyme combined with molecular modeling to arrive at models of the inactive circular form and its active linear counterpart. We show that the two forms have the same overall structure but differ in key parts, including the catalytic core element P7 and the junctions at which reactions take place. These differences explain the relative stability of the circular species, demonstrate how it is prone to react with a target molecule for circle integration and thus supports the notion that the circular form is a biologically significant molecule possibly with a role in intron mobility.

Differentially Expressed MicroRNAs in Meningiomas Grades I and II Suggest Shared Biomarkers with Malignant Tumors.

Meningiomas represent the most common primary tumors of the central nervous system, but few microRNA (miRNA) profiling studies have been reported so far. Deep sequencing of small RNA libraries generated from two human meningioma biopsies WHO grades I (benign) and II (atypical) were compared to excess dura controls. Nineteen differentially expressed miRNAs were validated by RT-qPCR using tumor RNA from 15 patients and 5 meninges controls. Tumor suppressor miR-218 and miR-34a were upregulated relative to normal controls, however, miR-143, miR-193b, miR-451 and oncogenic miR-21 were all downregulated. From 10 selected putative mRNA targets tested by RT-qPCR only four were differentially expressed relative to normal controls. PTEN and E-cadherin (CDH1) were upregulated, but RUNX1T1 was downregulated. Proliferation biomarker p63 was upregulated with nuclear localization, but not detected in most normal arachnoid tissues. Immunoreactivity of E-cadherin was detected in the outermost layer of normal arachnoids, but was expressed throughout the tumors. Nuclear Cyclin D1 expression was positive in all studied meningiomas, while its expression in arachnoid was limited to a few trabecular cells. Meningiomas of grades I and II appear to share biomarkers with malignant tumors, but with some additional tumor suppressor biomarkers expression. Validation in more patients is of importance.

First feed affects the expressions of microRNA and their targets in Atlantic cod.

To our knowledge, there is no report on microRNA (miRNA) expression and their target analysis in relation to the type of the first feed and its effect on the further growth of fish. Atlantic cod (Gadus morhua) larvae have better growth and development performance when fed natural zooplankton as a start-feed, as compared with those fed typical aquaculture start-feeds. In our experiment, two groups of Atlantic cod larvae were fed reference feed (zooplankton, mostly copepods, filtered from a seawater pond) v. aquaculture feeds: enriched rotifers (Brachionus sp.) and later brine shrimp (Artemia salina). We examined the miRNA expressions of six defined developmental stages as determined and standardised by body length from first feeding for both diet groups. We found eight miRNA (miR-9, miR-19a, miR-130b, miR-146, miR-181a, miR-192, miR-206 and miR-11240) differentially expressed between the two feeding groups in at least one developmental stage. We verified the next-generation sequencing data using real-time RT-PCR. We found 397 putative targets (mRNA) to the differentially expressed miRNA; eighteen of these mRNA showed differential expression in at least one stage. The patterns of differentially expressed miRNA and their putative target mRNA were mostly inverse, but sometimes also concurrent. The predicted miRNA targets were involved in different pathways, including metabolic, phototransduction and signalling pathways. The results of this study provide new nutrigenomic information on the potential role of miRNA in mediating nutritional effects on growth during the start-feeding period in fish larvae.

Next generation sequencing of microRNAs from isogenic neuroblastoma cell lines isolated before and after treatment.

Neuroblastoma is a pediatric cancer of the developing sympathetic nervous system. High risk neuroblastoma patients typically undergo an initial remission in response to treatment, followed by recurrence of aggressive tumors that have become refractory to further treatment. Recent works have underlined the involvement of microRNAs (miRNAs) in neuroblastoma development and evolution of drug resistance. In this study we have used deep sequencing technology to identify miRNAs differentially expressed in neuroblastoma cell lines isolated from 6 patients at diagnosis and at relapse after intensive treatments. This approach revealed a panel of 42 differentially expressed miRNAs, 8 of which were upregulated and 34 were downregulated. Most strikingly, the 14q32 miRNA clusters encode 22 of the downregulated miRNAs. Reduced expression of 14q32 miRNAs in tumors associated with poor prognosis factors was confirmed in a cohort consisting of 226 primary neuroblastomas. In order to gain insight into the nature of the genes that may be affected by the differentially expressed miRNAs we utilized Ingenuity Pathway Analysis (IPA). This analysis revealed several biological functions and canonical pathways associated with cancer progression and drug resistance. The results of this study contribute to the identification of miRNAs involved in the complex processes of surviving therapeutic treatment and developing drug resistance in neuroblastoma.