Mitochondrial Translation Occurs Preferentially in the Peri-Nuclear Mitochondrial Network of Cultured Human Cells

Human mitochondria are highly dynamic organelles, fusing and budding to maintain reticular networks throughout many cell types. Although extending to the extremities of the cell, the majority of the network is concentrated around the nucleus in most of the commonly cultured cell lines. This organelle harbours its own genome, mtDNA, with a different gene content to the nucleus, but the expression of which is critical for maintaining oxidative phosphorylation. Recent advances in click chemistry have allowed us to visualise sites of mitochondrial protein synthesis in intact cultured cells. We show that the majority of translation occurs in the peri-nuclear region of the network. Further analysis reveals that whilst there is a slight peri-nuclear enrichment in the levels of mitoribosomal protein and mitochondrial rRNA, it is not sufficient to explain this substantial heterogeneity in the distribution of translation. Finally, we also show that in contrast, a mitochondrial mRNA does not show such a distinct gradient in distribution. These data suggest that the relative lack of translation in the peripheral mitochondrial network is not due to an absence of mitoribosomes or an insufficient supply of the mt-mRNA transcripts.

Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore sequencing

RNA polyadenylation plays a central role in RNA maturation, fate and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translatability and stability of mRNAs. Here we develop Nano3P-seq, a novel method that relies on nanopore sequencing to simultaneously quantify RNA abundance and tail length dynamics at per-read resolution. By employing a template switching-based sequencing protocol, Nano3P-seq can sequence any given RNA molecule from its 3'end, regardless of its polyadenylation status, without the need of PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq captures a wide diversity of RNA biotypes, providing quantitative estimates of RNA abundance and tail lengths in mRNAs, lncRNAs, sn/snoRNAs, scaRNAs and rRNAs. We find that, in addition to mRNAs and lncRNAs, polyA tails can be identified in 16S mitochondrial rRNA in both mouse and zebrafish. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at the isoform-specific level, correlating with mRNA decay. Overall, Nano3P-seq is a simple and robust method to accurately estimate transcript levels and tail lengths in full-length individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

Mitochondrial translation occurs preferentially in the peri-nuclear mitochondrial network of cultured human cells.

Human mitochondria are highly dynamic organelles, fusing and budding to maintain reticular networks throughout many cell types. Although extending to the extremities of the cell, the majority of the network is concentrated around the nucleus in most of the commonly cultured cell lines. This organelle harbours its own genome, mtDNA, with a different gene content to the nucleus, but the expression of which is critical for maintaining oxidative phosphorylation. Recent advances in click chemistry have allowed us to visualise sites of mitochondrial protein synthesis in intact cultured cells. We show that the majority of translation occurs in the peri-nuclear region of the network. Further analysis reveals that whilst there is a slight peri-nuclear enrichment in the levels of mitoribosomal protein and mitochondrial rRNA, it is not sufficient to explain this substantial heterogeneity in distribution of translation. Finally, we also show that in contrast, a mitochondrial mRNA does not show such a distinct gradient in distribution. These data suggest that the relative lack of translation in the peripheral mitochondrial network is not due to an absence of mitoribosomes or an insufficient supply of the mt-mRNA transcripts.

Comparative analysis of RNA enrichment methods for preparation of Cryptococcus neoformans RNA sequencing libraries

RNA sequencing (RNA-Seq) experiments focused on gene expression involve removal of ribosomal RNA (rRNA) because it is the major RNA constituent of cells. This process, called RNA enrichment, is done primarily to reduce cost: without rRNA removal, deeper sequencing must be performed to compensate for the sequencing reads wasted on rRNA. The ideal RNA enrichment method removes all rRNA without affecting other RNA in the sample. We tested the performance of three RNA enrichment methods on RNA isolated from Cryptococcus neoformans, a fungal pathogen of humans. We find that the RNase H depletion method is more efficient in depleting rRNA and more specific in recapitulating non-rRNA levels present in unenriched controls than the commonly-used Poly(A) isolation method. The RNase H depletion method is also more effective than the Ribo-Zero depletion method as measured by rRNA depletion efficiency and recapitulation of protein-coding RNA levels present in unenriched controls, while the Ribo-Zero depletion method more closely recapitulates annotated non-coding RNA (ncRNA) levels. Finally, we leverage these data to accurately map the C. neoformans mitochondrial rRNA genes, and also demonstrate that RNA-Seq data generated with the RNase H and Ribo-Zero depletion methods can be used to explore novel C. neoformans long non-coding RNA genes.

Molecular Phylogeny and Global Diversity of the Genus Haploporus (Polyporales, Basidiomycota)

Phylogeny and taxonomy of the genus Haploporus were carried out based on a larger number of samples covering a wider geographic range including East Asia, South Asia, Europe, and America, and the species diversity of the genus is updated. Four species, Haploporus bicolor, H. longisporus, H. punctatus and H. srilankensis, are described as new species based on morphology and molecular phylogenetic analyses inferred from the internal transcribed spacer (ITS), the large subunit nuclear ribosomal RNA gene (nLSU), and the small subunit mitochondrial rRNA gene (mtSSU). Haploporus bicolor is characterized by the distinctly different colors between the pore surface and the tubes, small pores measuring 5–7 per mm, and narrow basidiospores measuring 10.5–11.9 × 4.5–5 µm; H. longisporus differs from other species in the genus by its large pores measuring 2–3 per mm, hyphae at dissepiment edge with simple septum, and the long basidiospores (up to 22 µm); H. punctatus is distinguished by its cushion-shaped basidiocarps, wide fusiform cystidioles with a simple septum at the tips, the absence of dendrohyphidia and the cylindrical to slightly allantoid basidiospores measuring 9–10.8 × 3.8–5 µm; H. srilankensis is characterized by its perennial habit, small pores measuring 4–5 per mm, dextrinoid skeletal hyphae, the presence of cystidioles and dendrohyphidia. An identification key to accepted species of Haploporus is provided.

Epitranscriptomics of Mammalian Mitochondrial Ribosomal RNA

Modified nucleotides are present in all ribosomal RNA molecules. Mitochondrial ribosomes are unique to have a set of methylated residues that includes universally conserved ones, those that could be found either in bacterial or in archaeal/eukaryotic cytosolic ribosomes and those that are present exclusively in mitochondria. A single pseudouridine within the mt-rRNA is located in the peptidyltransferase center at a position similar to that in bacteria. After recent completion of the list of enzymes responsible for the modification of mammalian mitochondrial rRNA it became possible to summarize an evolutionary history, functional role of mt-rRNA modification enzymes and an interplay of the mt-rRNA modification and mitoribosome assembly process, which is a goal of this review.

Functional conservation of mitochondrial RNA levels despite divergent mtDNA organization

Objective: Mitochondria-encoded ribosomal RNA (rRNA) genes in humans are expressed at a higher rate than protein coding genes of the mitochondria. The organization of the human mitochondrial genome (mtDNA) is amenable to differential expression of rRNAs as the rRNA encoding genes lie in tandem immediately downstream of the promoter-containing region. However, mtDNA is not organized in the same way as humans in all metazoans. In the nematode, Caenorhabditis elegans, the rRNA genes are on opposite sides of the mtDNA molecule and there are no obvious promoter sequences specific to the rRNA genes. Thus, we asked whether rRNA levels are higher relative to mRNAs in mitochondria of C. elegans as they are in humans.Results: Using droplet digital PCR, we discovered that steady-state mitochondrial rRNA transcript levels are approximately 120 times higher than the levels of mitochondrial mRNAs. These data demonstrate that despite the lack of conservation in mitochondrial genome organization, a high mitochondrial rRNA-to-mRNA ratio is a conserved feature of metazoans.

A new large canopy-dwelling species of Phyllodytes Wagler, 1930 (Anura, Hylidae) from the Atlantic Forest of the state of Bahia, Northeastern Brazil

The known diversity of treefrogs of the genus Phyllodytes has rapidly increased in recent years, currently comprising 14 species. Recent field work in the Atlantic Rainforest of the state of Bahia lead to the discovery of a new large species of Phyllodytes which is herein described based on multiple evidence including morphological, acoustical and genetic data. Phyllodytes sp. nov. is one of the largest species within the genus and presents immaculate yellowish dorsum and limbs. The advertisement call of the species is composed of 7–31 notes (half pulsed/pulsatile-half harmonic) with frequency-modulated harmonics. Phyllodytes sp. nov. has a karyotype of 2n = 22 chromosomes, as also found in other species of the genus. Genetic distance values of the 16S mitochondrial rRNA among Phyllodytes sp. nov. and its congeners range between 6.4 to 10.2%. The description of another new species for this state reinforces the need for further taxonomic work with Phyllodytes in this region that has been revealed as a priority area for research and conservation of this genus.

Functional Conservation of Mitochondrial RNA Levels Despite Divergent mtDNA Organization

Objective: Mitochondria-encoded ribosomal RNA (rRNA) genes in humans are expressed at a higher rate than protein coding genes of the mitochondria. The organization of the human mitochondrial genome (mtDNA) is amenable to differential expression of rRNAs as it contains a promoter specific to the transcription of the two rRNAs. However, mtDNA is not organized in the same way as humans in all metazoans. In the nematode, Caenorhabditis elegans, the rRNA genes are on opposite sides of the mtDNA molecule and there are no obvious promoter sequences specific to the rRNA genes. Thus, we asked whether rRNA levels are higher relative to mRNAs in mitochondria of C. elegans as they are in humans.Results: Using droplet digital PCR, we discovered that steady-state mitochondrial rRNA transcript levels are approximately 120 times higher than the levels of mitochondrial mRNAs. These data demonstrate that despite the lack of conservation in mitochondrial genome organization, a high mitochondrial rRNA-to-mRNA ratio is a conserved feature of metazoans.