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.

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.

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.

The genome sequence of Atlantic cod reveals a unique immune system.

Atlantic cod (Gadus morhua) is a large, cold-adapted teleost that sustains long-standing commercial fisheries and incipient aquaculture. Here we present the genome sequence of Atlantic cod, showing evidence for complex thermal adaptations in its haemoglobin gene cluster and an unusual immune architecture compared to other sequenced vertebrates. The genome assembly was obtained exclusively by 454 sequencing of shotgun and paired-end libraries, and automated annotation identified 22,154 genes. The major histocompatibility complex (MHC) II is a conserved feature of the adaptive immune system of jawed vertebrates, but we show that Atlantic cod has lost the genes for MHC II, CD4 and invariant chain (Ii) that are essential for the function of this pathway. Nevertheless, Atlantic cod is not exceptionally susceptible to disease under natural conditions. We find a highly expanded number of MHC I genes and a unique composition of its Toll-like receptor (TLR) families. This indicates how the Atlantic cod immune system has evolved compensatory mechanisms in both adaptive and innate immunity in the absence of MHC II. These observations affect fundamental assumptions about the evolution of the adaptive immune system and its components in vertebrates.

Speciation of a group I intron into a lariat capping ribozyme.

The lariat-capping (LC) ribozyme is a natural ribozyme isolated from eukaryotic microorganisms. Despite apparent structural similarity to group I introns, the LC ribozyme catalyzes cleavage by a 2',5' branching reaction, leaving the 3' product with a 3-nt lariat cap that functionally substitutes for a conventional mRNA cap in the downstream pre-mRNA encoding a homing endonuclease. We describe the crystal structures of the precleavage and postcleavage LC ribozymes, which suggest that structural features inherited from group I ribozymes have undergone speciation due to profound changes in molecular selection pressure, ultimately giving rise to an original branching ribozyme family. The structures elucidate the role of key elements that regulate the activity of the LC ribozyme by conformational switching and suggest a mechanism by which the signal for branching is transmitted to the catalytic core. The structures also show how conserved interactions twist residues, forming the lariat to join chemical groups involved in branching.

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.

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.

Temperature during early development has long-term effects on microRNA expression in Atlantic cod.

BACKGROUND: Environmental temperature has serious implications in life cycle of aquatic ectotherms. Understanding the molecular mechanisms of temperature acclimation and adaptation of marine organisms is of the uttermost importance for ecology, fisheries, and aquaculture, as it allows modeling the effects of global warming on population dynamics. Regulatory molecules are major modulators of acclimation and adaptation; among them, microRNAs (miRNAs) are versatile and substantial contributors to regulatory networks of development and adaptive plasticity. However, their role in thermal plasticity is poorly known. We have asked whether the temperature and its shift during the early ontogeny (embryonic and larval development) affect the miRNA repertoire of Atlantic cod (Gadus morhua), and if thermal experience has long-term consequences in the miRNA profile. RESULTS: We characterized miRNA during different developmental stages and in juvenile tissues using next generation sequencing. We identified 389 putative miRNA precursor loci, 120 novel precursor miRNAs, and 281 mature miRNAs. Some miRNAs showed stage- or tissue-enriched expression and miRNAs, such as the miR-17 ~ 92 cluster, myomiRs (miR-206), neuromiRs (miR-9, miR-124), miR-130b, and miR-430 showed differential expression in different temperature regimes. Long-term effect of embryonic incubation temperature was revealed on expression of some miRNAs in juvenile pituitary (miR-449), gonad (miR-27c, miR-30c, and miR-200a), and liver (let-7 h, miR-7a, miR-22, miR-34c, miR-132a, miR-192, miR-221, miR-451, miR-2188, and miR-7550), but not in brain. Some of differentially expressed miRNAs in the liver were confirmed using LNA-based rt-qPCR. The effect of temperature on methylation status of selected miRNA promoter regions was mostly inconclusive. CONCLUSIONS: Temperature elevation by several degrees during embryonic and larval developmental stages significantly alters the miRNA profile, both short-term and long-term. Our results suggest that a further rise in seas temperature might affect life history of Atlantic cod.

An evolutionary preserved intergenic spacer in gadiform mitogenomes generates a long noncoding RNA.

BACKGROUND: Vertebrate mitogenomes are economically organized and usually lack intergenic sequences other than the control region. Intergenic spacers located between the tRNA(Thr) and tRNA(Pro) genes ("T-P spacers") have been observed in several taxa, including gadiform species, but information about their biological roles and putative functions is still lacking. RESULTS: Sequence characterization of the complete European hake Merluccius merluccius mitogenome identified a complex T-P spacer ranging in size from 223-532 bp. Further analyses of 32 gadiform species, representing 8 families and 28 genera, revealed the evolutionary preserved presence of T-P spacers across all taxa. Molecular complexity of the T-P spacers was found to be coherent with the phylogenetic relationships, supporting a common ancestral origin and gain of function during codfish evolution. Intraspecific variation of T-P spacer sequences was assessed in 225 Atlantic cod specimens and revealed 26 haplotypes. Pyrosequencing data representing the mito-transcriptome poly (A) fraction in Atlantic cod identified an abundant H-strand specific long noncoding RNA of about 375 nt. The T-P spacer corresponded to the 5' part of this transcript, which terminated within the control region in a tail-to-tail configuration with the L-strand specific transcript (the 7S RNA). CONCLUSIONS: The T-P spacer is inferred to be evolutionary preserved in gadiform mitogenomes due to gain of function through a long noncoding RNA. We suggest that the T-P spacer adds stability to the H-strand specific long noncoding RNA by forming stable hairpin structures and additional protein binding sites.

Impact of the tumor necrosis factor receptor-associated protein 1 (Trap1) on renal DNaseI shutdown and on progression of murine and human lupus nephritis.

Recent findings show that transformation of mild glomerulonephritis into end-stage disease coincides with shutdown of renal DNaseI expression in (NZBxNZW)F1 mice. Down-regulation of DNaseI results in reduced chromatin fragmentation and deposition of extracellular chromatin fragments in glomerular basement membranes where they appear in complex with IgG antibodies. Here, we implicate the anti-apoptotic and survival protein, tumor necrosis factor receptor-associated protein 1 (Trap1) in the disease process, based on the observation that annotated transcripts from this gene overlap with transcripts from the DNaseI gene. Furthermore, we translate these observations to human lupus nephritis. In this study, mouse and human DNaseI and Trap1 mRNA levels were determined by real-time quantitative PCR and compared with protein expression levels and clinical data. Cellular localization was analyzed by immune electron microscopy, IHC, and in situ hybridization. Data indicate that silencing of DNaseI gene expression correlates inversely with expression of the Trap1 gene. Our observations suggest that the mouse model is relevant for the aspects of disease progression in human lupus nephritis. Acquired silencing of the renal DNaseI gene has been shown to be important for progression of disease in both the murine and human forms of lupus nephritis. Early mesangial nephritis initiates a cascade of inflammatory signals that lead to up-regulation of Trap1 and a consequent down-regulation of renal DNaseI by transcriptional interference.

Sea anemones possess dynamic mitogenome structures.

A notable feature of hexacoral mitogenomes is the presence of complex self-catalytic group I introns. We investigated mitogenome structural variations and evolutionary mechanisms in actiniarian sea anemones based on the complete mitogenome sequence of the cold-water sea anemone species Urticina eques, Bolocera tuediae, Hormathia digitata and Metridium senile, and two isolates of the sub-tropical Aiptasia pulchella. Whole genome sequencing at 50 times coverage of B. tuediae and H. digitata indicated low mtDNA copy number of per haploid nuclear genome and presence of rare haplotypes. A group I intron inserted in ND5 was found to host essential mitochondrial protein genes in all species, and an additional truncated copy of ND5 in B. tuediae. A second group I intron (inserted in COI) that contained a homing endonuclease gene (HEG) was present in all mtDNA examined. Different variants of HEGs were observed, and included expressed elements fused in-frame with upstream exons and free-standing HEGs embedded within the intron. A notable hallmark of HEGs was a high extent of overlap with ribozyme structural elements; the U. eques HEG overlapped with the entire intron. We reconstructed the evolutionary history of the COI intron from insertion at unoccupied cognate sites, through HEG degradation, to intron loss. We also identified a novel insertion element in U. eques that contained two expressed protein-coding genes. An evolutionary analysis of the sea anemone mtDNA genes revealed higher substitution rates in the HEG and the insertion sequence as compared to the other loci, indicating relaxed selective pressures in these elements. We conclude that sea anemone mitogenomes are surprisingly dynamic in structure despite the economical organization and low sequence mutation rate.

Genomic divergence between the migratory and stationary ecotypes of Atlantic cod.

Atlantic cod displays a range of phenotypic and genotypic variations, which includes the differentiation into coastal stationary and offshore migratory types of cod that co-occur in several parts of its distribution range and are often sympatric on the spawning grounds. Differentiation of these ecotypes may involve both historical separation and adaptation to ecologically distinct environments, the genetic basis of which is now beginning to be unravelled. Genomic analyses based on recent sequencing advances are able to document genomic divergence in more detail and may facilitate the exploration of causes and consequences of genome-wide patterns. We examined genomic divergence between the stationary and migratory types of cod in the Northeast Atlantic, using next-generation sequencing of pooled DNA from each of two population samples. Sequence data was mapped to the published cod genome sequence, arranged in more than 6000 scaffolds (611 Mb). We identified 25 divergent scaffolds (26 Mb) with a higher than average gene density, against a backdrop of overall moderate genomic differentiation. Previous findings of localized genomic divergence in three linkage groups were confirmed, including a large (15 Mb) genomic region, which seems to be uniquely involved in the divergence of migratory and stationary cod. The results of the pooled sequencing approach support and extend recent findings based on single-nucleotide polymorphism markers and suggest a high degree of reproductive isolation between stationary and migratory cod in the North-east Atlantic.

Polyphyletic origin of the genus Physarum (Physarales, Myxomycetes) revealed by nuclear rDNA mini-chromosome analysis and group I intron synapomorphy.

BACKGROUND: Physarales represents the largest taxonomic order among the plasmodial slime molds (myxomycetes). Physarales is of particular interest since the two best-studied myxomycete species, Physarum polycephalum and Didymium iridis, belong to this order and are currently subjected to whole genome and transcriptome analyses. Here we report molecular phylogeny based on ribosomal DNA (rDNA) sequences that includes 57 Physarales isolates. RESULTS: The Physarales nuclear rDNA sequences were found to be loaded with 222 autocatalytic group I introns, which may complicate correct alignments and subsequent phylogenetic tree constructions. Phylogenetic analysis of rDNA sequences depleted of introns confirmed monophyly of the Physarales families Didymiaceae and Physaraceae. Whereas good correlation was noted between phylogeny and taxonomy among the Didymiaceae isolates, significant deviations were seen in Physaraceae. The largest genus, Physarum, was found to be polyphyletic consisting of at least three well supported clades. A synapomorphy, located at the highly conserved G-binding site of L2449 group I intron ribozymes further supported the Physarum clades. CONCLUSIONS: Our results provide molecular relationship of Physarales genera, species, and isolates. This information is important in further interpretations of comparative genomics nd transcriptomics. In addition, the result supports a polyphyletic origin of the genus Physarum and calls for a reevaluation of current taxonomy.

Differential expression patterns of conserved miRNAs and isomiRs during Atlantic halibut development.

BACKGROUND: MicroRNAs (miRNAs) play a major role in animal ontogenesis. Size variants of miRNAs, isomiRs, are observed along with the main miRNA types, but their origin and possible biological role are uncovered yet. Developmental profiles of miRNAs have been reported in few fish species only and, to our knowledge, differential expressions of isomiRs have not yet been shown during fish development. Atlantic halibut, Hippoglossus hippoglossus L., undergoes dramatic metamorphosis during early development from symmetrical pelagic larval stage to unsymmetrical flatfish. No data exist on role of miRNAs in halibut metamorphosis. RESULTS: miRNA profiling using SOLiD deep sequencing technology revealed a total of 199 conserved, one novel antisense, and one miRNA* mature form. Digital expression profiles of selected miRNAs were validated using reverse transcription quantitative PCR. We found developmental transition-specific miRNA expression. Expression of some miRNA* exceeded the guide strand miRNA. We revealed that nucleotide truncations and/or additions at the 3' end of mature miRNAs resulted in size variants showing differential expression patterns during the development in a number of miRNA families. We confirmed the presence of isomiRs by cloning and Sanger sequencing. Also, we found inverse relationship between expression levels of sense/antisense miRNAs during halibut development. CONCLUSION: Developmental transitions during early development of Atlantic halibut are associated with expression of certain miRNA types. IsomiRs are abundant and often show differential expression during the development.

Molecular modelling of the GIR1 branching ribozyme gives new insight into evolution of structurally related ribozymes.

Twin-ribozyme introns contain a branching ribozyme (GIR1) followed by a homing endonuclease (HE) encoding sequence embedded in a peripheral domain of a group I splicing ribozyme (GIR2). GIR1 catalyses the formation of a lariat with 3 nt in the loop, which caps the HE mRNA. GIR1 is structurally related to group I ribozymes raising the question about how two closely related ribozymes can carry out very different reactions. Modelling of GIR1 based on new biochemical and mutational data shows an extended substrate domain containing a GoU pair distinct from the nucleophilic residue that dock onto a catalytic core showing a different topology from that of group I ribozymes. The differences include a core J8/7 region that has been reduced and is complemented by residues from the pre-lariat fold. These findings provide the basis for an evolutionary mechanism that accounts for the change from group I splicing ribozyme to the branching GIR1 architecture. Such an evolutionary mechanism can be applied to other large RNAs such as the ribonuclease P.

Digital marine bioprospecting: mining new neurotoxin drug candidates from the transcriptomes of cold-water sea anemones.

Marine bioprospecting is the search for new marine bioactive compounds and large-scale screening in extracts represents the traditional approach. Here, we report an alternative complementary protocol, called digital marine bioprospecting, based on deep sequencing of transcriptomes. We sequenced the transcriptomes from the adult polyp stage of two cold-water sea anemones, Bolocera tuediae and Hormathia digitata. We generated approximately 1.1 million quality-filtered sequencing reads by 454 pyrosequencing, which were assembled into approximately 120,000 contigs and 220,000 single reads. Based on annotation and gene ontology analysis we profiled the expressed mRNA transcripts according to known biological processes. As a proof-of-concept we identified polypeptide toxins with a potential blocking activity on sodium and potassium voltage-gated channels from digital transcriptome libraries.

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.

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.

Characterization of mitochondrial mRNAs in codfish reveals unique features compared to mammals.

Expression and processing of mitochondrial gene transcripts are fundamental to mitochondrial function, but information from early vertebrates like teleost fishes is essentially lacking. We have analyzed mitogenome sequences of ten codfishes (family Gadidae), and provide complete sequences from three new species (Saithe, Pollack and Blue whiting). Characterization of the mitochondrial mRNAs in Saithe and Atlantic cod identified a set of ten poly(A) transcripts, and six UAA stop codons are generated by posttranscriptional polyadenylation. Structural assessment of poly(A) sites is consistent with an RNaseP cleavage activity 5' of tRNA acceptor-like stems. COI, ND5 and ND6 mRNAs were found to harbor 3' UTRs with antisense potential extending into neighboring gene regions. While the 3' UTR of COI mRNA is complementary to the tRNA(Ser UCN) and highly similar to that detected in human mitochondria, the ND5 and ND6 3' UTRs appear more heterogenic. Deep sequencing confirms expression of all mitochondrial mRNAs and rRNAs, and provides information about the precise 5' ends in mature transcripts. Our study supports an overall evolutionary conservation in mitochondrial RNA processing events among vertebrates, but reveals some unique 5' and 3' end characteristics in codfish mRNAs with implications to antisense regulation of gene expression.

Intermolecular interaction between a branching ribozyme and associated homing endonuclease mRNA.

RNA tertiary interactions involving docking of GNRA (N; any base; R; purine) hairpin loops into helical stem structures on other regions of the same RNA are one of the most common RNA tertiary interactions. In this study, we investigated a tertiary association between a GAAA hairpin tetraloop in a small branching ribozyme (DiGIR1) and a receptor motif (HEG P1 motif) present in a hairpin structure on a separate mRNA molecule. DiGIR1 generates a 2', 5' lariat cap at the 5' end of its downstream homing endonuclease mRNA by catalysing a self-cleavage branching reaction at an internal processing site. Upon release, the 5' end of the mRNA forms a distinct hairpin structure termed HEG P1. Our biochemical data, in concert with molecular 3D modelling, provide experimental support for an intermolecular tetraloop receptor interaction between the L9 GAAA in DiGIR1 and a GNRA tetraloop receptor-like motif (UCUAAG-CAAGA) found within the HEG P1. The biological role of this interaction appears to be linked to the homing endonuclease expression by promoting post-cleavage release of the lariat capped mRNA. These findings add to our understanding of how protein-coding genes embedded in nuclear ribosomal DNA are expressed in eukaryotes and controlled by ribozymes.