ANGEL2 phosphatase activity is required for non-canonical mitochondrial RNA processing.

Canonical RNA processing in mammalian mitochondria is defined by tRNAs acting as recognition sites for nucleases to release flanking transcripts. The relevant factors, their structures, and mechanism are well described, but not all mitochondrial transcripts are punctuated by tRNAs, and their mode of processing has remained unsolved. Using Drosophila and mouse models, we demonstrate that non-canonical processing results in the formation of 3\u27 phosphates, and that phosphatase activity by the carbon catabolite repressor 4 domain-containing family member ANGEL2 is required for their hydrolysis. Furthermore, our data suggest that members of the FAST kinase domain-containing protein family are responsible for these 3\u27 phosphates. Our results therefore propose a mechanism for non-canonical RNA processing in metazoan mitochondria, by identifying the role of ANGEL2

Using XML for long-term preservation

One of the objectives of the project is to explore the possibility of using XML as a format for long-term preservation. For this reason, we evaluated the practical use of XML in different parts of the system before deciding on the design. An XML schema, DiVA XML schema, has been developed to describe the inter-relationships amongst the various data elements and processes, and to support long-term preservation of the actual documents. XML Schema provides a means for defining the structure, content and semantics of XML documents. It is an XML based alternative to the XML Document Type Definition (DTD). Because one of the primary reasons for using XML was to support long-term preservation, the most popular DTDs for documents: DocBook and TEI were reviewed. Limitations regarding metadata descriptions were found in both of these DTDs, so the decision to develop a new structure for DiVA, using XML schema, was made. This schema combines the DocBook DTD for the textual parts of the document with the internal schema for all metadata (bibliographic and administrative data). Several applications, which implement the DiVA XML schema for content managing and communication between applications, were developed. Some of their purposes are essential for long-term preservation: - Make persistent National Bibliographic Numbers (NBN) available for the URN resolver (http://urn.kb.se/resolve) at the Royal Library in Stockholm available. - Send MARC21 records in MARC-XML to the National Library. - Create file archives for the long-term preservation, checksum them, archive them in the DiVA archive and send them to the Royal Library. Currently the file archives for long-term preservation contain the original full-text file in various formats and the DiVA XML file, which contains all the metadata about the document. Furthermore the DiVA XML file contains all parts of the full-text file, which can be converted into XML. In the future it might be possible to transfer the whole full-text into XML, so the file-archives could contain only DiVA XML files

CirGO: an alternative circular way of visualising gene ontology terms

Abstract Background Prioritisation of gene ontology terms from differential gene expression analyses in a two-dimensional format remains a challenge with exponentially growing data volumes. Typically, gene ontology terms are represented as tree-maps that enclose all data into defined space. However, large datasets make this type of visualisation appear cluttered and busy, and often not informative as some labels are omitted due space limits, especially when published in two-dimensional (2D) figures. Results Here we present an open source CirGO (Circular Gene Ontology) software that visualises non-redundant two-level hierarchically structured ontology terms from gene expression data in a 2D space. Gene ontology terms based on statistical significance were summarised with a semantic similarity algorithm and grouped by hierarchical clustering. This software visualises the most enriched gene ontology terms in an informative, comprehensive and intuitive format that is achieved by organising data from the most relevant to the least, as well as the appropriate use of colours and supporting information. Additionally, CirGO is an easy to use software that supports researchers with little computational background to present their gene ontology data in a publication ready format. Conclusions Our easy to use open source CirGO Python software package provides biologists with a succinct presentation of terms and functions that are most represented in a specific gene expression data set in a visually appealing 2D format (e.g. for reporting research results in scientific articles). CirGO is freely available at https://github.com/IrinaVKuznetsova/CirGO.git

LRPPRC-mediated folding of the mitochondrial transcriptome

The mitochondrial genome, being compressed to 16 kb, is an attractive model system to investigate how RNA-binding proteins chaperone mRNA lifecycles. Here the authors use RNase footprinting and PAR-CLIP to show that the LRPPRC–SLIRP complex stabilizes mRNA structures to expose sites required for translation and polyadenylation

Mitochondrial gene expression is required for platelet function and blood clotting

Summary: Platelets are anucleate blood cells that contain mitochondria and regulate blood clotting in response to injury. Mitochondria contain their own gene expression machinery that relies on nuclear-encoded factors for the biogenesis of the oxidative phosphorylation system to produce energy required for thrombosis. The autonomy of the mitochondrial gene expression machinery from the nucleus is unclear, and platelets provide a valuable model to understand its importance in anucleate cells. Here, we conditionally delete Elac2, Ptcd1, or Mtif3 in platelets, which are essential for mitochondrial gene expression at the level of RNA processing, stability, or translation, respectively. Loss of ELAC2, PTCD1, or MTIF3 leads to increased megakaryocyte ploidy, elevated circulating levels of reticulated platelets, thrombocytopenia, and consequent extended bleeding time. Impaired mitochondrial gene expression reduces agonist-induced platelet activation. Transcriptomic and proteomic analyses show that mitochondrial gene expression is required for fibrinolysis, hemostasis, and blood coagulation in response to injury

Molecular basis of translation termination at noncanonical stop codons in human mitochondria

The genetic code that specifies the identity of amino acids incorporated into proteins during protein synthesis is almost universally conserved. Mitochondrial genomes feature deviations from the standard genetic code, including the reassignment of two arginine codons to stop codons. The protein required for translation termination at these noncanonical stop codons to release the newly synthesized polypeptides is not currently known. In this study, we used gene editing and ribosomal profiling in combination with cryo-electron microscopy to establish that mitochondrial release factor 1 (mtRF1) detects noncanonical stop codons in human mitochondria by a previously unknown mechanism of codon recognition. We discovered that binding of mtRF1 to the decoding center of the ribosome stabilizes a highly unusual conformation in the messenger RNA in which the ribosomal RNA participates in specific recognition of the noncanonical stop codons.ISSN:0036-8075ISSN:1095-920

SLIRP Regulates the Rate of Mitochondrial Protein Synthesis and Protects LRPPRC from Degradation

We have studied the in vivo role of SLIRP in regulation of mitochondrial DNA (mtDNA) gene expression and show here that it stabilizes its interacting partner protein LRPPRC by protecting it from degradation. Although SLIRP is completely dependent on LRPPRC for its stability, reduced levels of LRPPRC persist in the absence of SLIRP in vivo. Surprisingly, Slirp knockout mice are apparently healthy and only display a minor weight loss, despite a 50-70% reduction in the steady-state levels of mtDNA-encoded mRNAs. In contrast to LRPPRC, SLIRP is dispensable for polyadenylation of mtDNA-encoded mRNAs. Instead, deep RNA sequencing (RNAseq) of mitochondrial ribosomal fractions and additional molecular analyses show that SLIRP is required for proper association of mRNAs to the mitochondrial ribosome and efficient translation. Our findings thus establish distinct functions for SLIRP and LRPPRC within the LRPPRC-SLIRP complex, with a novel role for SLIRP in mitochondrial translation. Very surprisingly, our results also demonstrate that mammalian mitochondria have a great excess of transcripts under basal physiological conditions in vivo

Hierarchical RNA Processing Is Required for Mitochondrial Ribosome Assembly

The regulation of mitochondrial RNA processing and its importance for ribosome biogenesis and energy metabolism are not clear. We generated conditional knockout mice of the endoribonuclease component of the RNase P complex, MRPP3, and report that it is essential for life and that heart and skeletal-muscle-specific knockout leads to severe cardiomyopathy, indicating that its activity is non-redundant. Transcriptome-wide parallel analyses of RNA ends (PARE) and RNA-seq enabled us to identify that in vivo 5′ tRNA cleavage precedes 3′ tRNA processing, and this is required for the correct biogenesis of the mitochondrial ribosomal subunits. We identify that mitoribosomal biogenesis proceeds co-transcriptionally because large mitoribosomal proteins can form a subcomplex on an unprocessed RNA containing the 16S rRNA. Taken together, our data show that RNA processing links transcription to translation via assembly of the mitoribosome

Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria

Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria

TEFM regulates both transcription elongation and RNA processing in mitochondria

International audienceRegulation of replication and expression of mitochondrial DNA (mtDNA) is essential for cellular energy conversion via oxidative phosphorylation. The mitochondrial transcription elongation factor (TEFM) has been proposed to regulate the switch between transcription termination for replication primer formation and processive, near genome-length transcription for mtDNA gene expression. Here, we report that Tefm is essential for mouse embryogenesis and that levels of promoter-distal mitochondrial transcripts are drastically reduced in conditional Tefm-knockout hearts. In contrast, the promoter-proximal transcripts are much increased in Tefm knockout mice, but they mostly terminate before the region where the switch from transcription to replication occurs, and consequently, de novo mtDNA replication is profoundly reduced. Unexpectedly, deep sequencing of RNA from Tefm knockouts revealed accumulation of unprocessed transcripts in addition to defective transcription elongation. Furthermore, a proximity-labeling (BioID) assay showed that TEFM interacts with multiple RNA processing factors. Our data demonstrate that TEFM acts as a general transcription elongation factor, necessary for both gene transcription and replication primer formation, and loss of TEFM affects RNA processing in mammalian mitochondria