A single pseudouridine on rRNA regulates ribosome structure and function in the mammalian parasite Trypanosoma brucei.

Trypanosomes are protozoan parasites that cycle between insect and mammalian hosts and are the causative agent of sleeping sickness. Here, we describe the changes of pseudouridine (Ψ) modification on rRNA in the two life stages of the parasite using four different genome-wide approaches. CRISPR-Cas9 knock-outs of all four snoRNAs guiding Ψ on helix 69 (H69) of the large rRNA subunit were lethal. A single knock-out of a snoRNA guiding Ψ530 on H69 altered the composition of the 80S monosome. These changes specifically affected the translation of only a subset of proteins. This study correlates a single site Ψ modification with changes in ribosomal protein stoichiometry, supported by a high-resolution cryo-EM structure. We propose that alteration in rRNA modifications could generate ribosomes preferentially translating state-beneficial proteins.

Characterizing RNA stability genome-wide through combined analysis of PRO-seq and RNA-seq data.

BACKGROUND: The concentrations of distinct types of RNA in cells result from a dynamic equilibrium between RNA synthesis and decay. Despite the critical importance of RNA decay rates, current approaches for measuring them are generally labor-intensive, limited in sensitivity, and/or disruptive to normal cellular processes. Here, we introduce a simple method for estimating relative RNA half-lives that is based on two standard and widely available high-throughput assays: Precision Run-On sequencing (PRO-seq) and RNA sequencing (RNA-seq). RESULTS: Our method treats PRO-seq as a measure of transcription rate and RNA-seq as a measure of RNA concentration, and estimates the rate of RNA decay required for a steady-state equilibrium. We show that this approach can be used to assay relative RNA half-lives genome-wide, with good accuracy and sensitivity for both coding and noncoding transcription units. Using a structural equation model (SEM), we test several features of transcription units, nearby DNA sequences, and nearby epigenomic marks for associations with RNA stability after controlling for their effects on transcription. We find that RNA splicing-related features are positively correlated with RNA stability, whereas features related to miRNA binding and DNA methylation are negatively correlated with RNA stability. Furthermore, we find that a measure based on U1 binding and polyadenylation sites distinguishes between unstable noncoding and stable coding transcripts but is not predictive of relative stability within the mRNA or lincRNA classes. We also identify several histone modifications that are associated with RNA stability. CONCLUSION: We introduce an approach for estimating the relative half-lives of individual RNAs. Together, our estimation method and systematic analysis shed light on the pervasive impacts of RNA stability on cellular RNA concentrations.

mtDNA Chromatin-like Organization Is Gradually Established during Mammalian Embryogenesis.

Unlike the nuclear genome, the mammalian mitochondrial genome (mtDNA) is thought to be coated solely by mitochondrial transcription factor A (TFAM), whose binding sequence preferences are debated. Therefore, higher-order mtDNA organization is considered much less regulated than both the bacterial nucleoid and the nuclear chromatin. However, our recently identified conserved DNase footprinting pattern in human mtDNA, which co-localizes with regulatory elements and responds to physiological conditions, likely reflects a structured higher-order mtDNA organization. We hypothesized that this pattern emerges during embryogenesis. To test this hypothesis, we analyzed assay for transposase-accessible chromatin sequencing (ATAC-seq) results collected during the course of mouse and human early embryogenesis. Our results reveal, for the first time, a gradual and dynamic emergence of the adult mtDNA footprinting pattern during embryogenesis of both mammals. Taken together, our findings suggest that the structured adult chromatin-like mtDNA organization is gradually formed during mammalian embryogenesis.

A common pattern of DNase I footprinting throughout the human mtDNA unveils clues for a chromatin-like organization.

Human mitochondrial DNA (mtDNA) is believed to lack chromatin and histones. Instead, it is coated solely by the transcription factor TFAM. We asked whether mtDNA packaging is more regulated than once thought. To address this, we analyzed DNase-seq experiments in 324 human cell types and found, for the first time, a pattern of 29 mtDNA Genomic footprinting (mt-DGF) sites shared by ∼90% of the samples. Their syntenic conservation in mouse DNase-seq experiments reflect selective constraints. Colocalization with known mtDNA regulatory elements, with G-quadruplex structures, in TFAM-poor sites (in HeLa cells) and with transcription pausing sites, suggest a functional regulatory role for such mt-DGFs. Altered mt-DGF pattern in interleukin 3-treated CD34+ cells, certain tissue differences, and significant prevalence change in fetal versus nonfetal samples, offer first clues to their physiological importance. Taken together, human mtDNA has a conserved protein-DNA organization, which is likely involved in mtDNA regulation.

Human primitive brain displays negative mitochondrial-nuclear expression correlation of respiratory genes.

Oxidative phosphorylation (OXPHOS), a fundamental energy source in all human tissues, requires interactions between mitochondrial (mtDNA)- and nuclear (nDNA)-encoded protein subunits. Although such interactions are fundamental to OXPHOS, bi-genomic coregulation is poorly understood. To address this question, we analyzed ∼8500 RNA-seq experiments from 48 human body sites. Despite well-known variation in mitochondrial activity, quantity, and morphology, we found overall positive mtDNA-nDNA OXPHOS genes' co-expression across human tissues. Nevertheless, negative mtDNA-nDNA gene expression correlation was identified in the hypothalamus, basal ganglia, and amygdala (subcortical brain regions, collectively termed the "primitive" brain). Single-cell RNA-seq analysis of mouse and human brains revealed that this phenomenon is evolutionarily conserved, and both are influenced by brain cell types (involving excitatory/inhibitory neurons and nonneuronal cells) and by their spatial brain location. As the "primitive" brain is highly oxidative, we hypothesized that such negative mtDNA-nDNA co-expression likely controls for the high mtDNA transcript levels, which enforce tight OXPHOS regulation, rather than rewiring toward glycolysis. Accordingly, we found "primitive" brain-specific up-regulation of lactate dehydrogenase B (LDHB), which associates with high OXPHOS activity, at the expense of LDHA, which promotes glycolysis. Analyses of co-expression, DNase-seq, and ChIP-seq experiments revealed candidate RNA-binding proteins and CEBPB as the best regulatory candidates to explain these phenomena. Finally, cross-tissue expression analysis unearthed tissue-dependent splice variants and OXPHOS subunit paralogs and allowed revising the list of canonical OXPHOS transcripts. Taken together, our analysis provides a comprehensive view of mito-nuclear gene co-expression across human tissues and provides overall insights into the bi-genomic regulation of mitochondrial activities.

Initiation of mtDNA transcription is followed by pausing, and diverges across human cell types and during evolution.

Mitochondrial DNA (mtDNA) genes are long known to be cotranscribed in polycistrones, yet it remains impossible to study nascent mtDNA transcripts quantitatively in vivo using existing tools. To this end, we used deep sequencing (GRO-seq and PRO-seq) and analyzed nascent mtDNA-encoded RNA transcripts in diverse human cell lines and metazoan organisms. Surprisingly, accurate detection of human mtDNA transcription initiation sites (TISs) in the heavy and light strands revealed a novel conserved transcription pausing site near the light-strand TIS. This pausing site correlated with the presence of a bacterial pausing sequence motif, with reduced SNP density, and with a DNase footprinting signal in all tested cells. Its location within conserved sequence block 3 (CSBIII), just upstream of the known transcription-replication transition point, suggests involvement in such transition. Analysis of nonhuman organisms enabled de novo mtDNA sequence assembly, as well as detection of previously unknown mtDNA TIS, pausing, and transcription termination sites with unprecedented accuracy. Whereas mammals (Pan troglodytes, Macaca mulatta, Rattus norvegicus, and Mus musculus) showed a human-like mtDNA transcription pattern, the invertebrate pattern (Drosophila melanogaster and Caenorhabditis elegans) profoundly diverged. Our approach paves the path toward in vivo, quantitative, reference sequence-free analysis of mtDNA transcription in all eukaryotes.

Correction: Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates.

[This corrects the article DOI: 10.1371/journal.pbio.1002557.].

Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates.

The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m1A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m1A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m1A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited Escherichia coli showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function.

Transcription factors bind negatively selected sites within human mtDNA genes.

Transcription of mitochondrial DNA (mtDNA)-encoded genes is thought to be regulated by a handful of dedicated transcription factors (TFs), suggesting that mtDNA genes are separately regulated from the nucleus. However, several TFs, with known nuclear activities, were found to bind mtDNA and regulate mitochondrial transcription. Additionally, mtDNA transcriptional regulatory elements, which were proved important in vitro, were harbored by a deletion that normally segregated among healthy individuals. Hence, mtDNA transcriptional regulation is more complex than once thought. Here, by analyzing ENCODE chromatin immunoprecipitation sequencing (ChIP-seq) data, we identified strong binding sites of three bona fide nuclear TFs (c-Jun, Jun-D, and CEBPb) within human mtDNA protein-coding genes. We validated the binding of two TFs by ChIP-quantitative polymerase chain reaction (c-Jun and Jun-D) and showed their mitochondrial localization by electron microscopy and subcellular fractionation. As a step toward investigating the functionality of these TF-binding sites (TFBS), we assessed signatures of selection. By analyzing 9,868 human mtDNA sequences encompassing all major global populations, we recorded genetic variants in tips and nodes of mtDNA phylogeny within the TFBS. We next calculated the effects of variants on binding motif prediction scores. Finally, the mtDNA variation pattern in predicted TFBS, occurring within ChIP-seq negative-binding sites, was compared with ChIP-seq positive-TFBS (CPR). Motifs within CPRs of c-Jun, Jun-D, and CEBPb harbored either only tip variants or their nodal variants retained high motif prediction scores. This reflects negative selection within mtDNA CPRs, thus supporting their functionality. Hence, human mtDNA-coding sequences may have dual roles, namely coding for genes yet possibly also possessing regulatory potential.

Mito-nuclear co-evolution: the positive and negative sides of functional ancient mutations.

Most cell functions are carried out by interacting factors, thus underlying the functional importance of genetic interactions between genes, termed epistasis. Epistasis could be under strong selective pressures especially in conditions where the mutation rate of one of the interacting partners notably differs from the other. Accordingly, the order of magnitude higher mitochondrial DNA (mtDNA) mutation rate as compared to the nuclear DNA (nDNA) of all tested animals, should influence systems involving mitochondrial-nuclear (mito-nuclear) interactions. Such is the case of the energy producing oxidative phosphorylation (OXPHOS) and mitochondrial translational machineries which are comprised of factors encoded by both the mtDNA and the nDNA. Additionally, the mitochondrial RNA transcription and mtDNA replication systems are operated by nDNA-encoded proteins that bind mtDNA regulatory elements. As these systems are central to cell life there is strong selection toward mito-nuclear co-evolution to maintain their function. However, it is unclear whether (A) mito-nuclear co-evolution befalls only to retain mitochondrial functions during evolution or, also, (B) serves as an adaptive tool to adjust for the evolving energetic demands as species' complexity increases. As the first step to answer these questions we discuss evidence of both negative and adaptive (positive) selection acting on the mtDNA and nDNA-encoded genes and the effect of both types of selection on mito-nuclear interacting factors. Emphasis is given to the crucial role of recurrent ancient (nodal) mutations in such selective events. We apply this point-of-view to the three available types of mito-nuclear co-evolution: protein-protein (within the OXPHOS system), protein-RNA (mainly within the mitochondrial ribosome), and protein-DNA (at the mitochondrial replication and transcription machineries).

Mitochondrial-nuclear co-evolution and its effects on OXPHOS activity and regulation.

Factors required for mitochondrial function are encoded both by the nuclear and mitochondrial genomes. The order of magnitude higher mutation rate of animal mitochondrial DNA (mtDNA) enforces tight co-evolution of mtDNA and nuclear DNA encoded factors. In this essay we argue that such co evolution exists at the population and inter-specific levels and affect disease susceptibility. We also argue for the existence of three modes of co-evolution in the mitochondrial genetic system, which include the interaction of mtDNA and nuclear DNA encoded proteins, nuclear protein - mtDNA-encoded RNA interaction within the mitochondrial translation machinery and nuclear DNA encoded proteins-mtDNA binging sites interaction in the frame of the mtDNA replication and transcription machineries. These modes of co evolution require co-regulation of the interacting factors encoded by the two genomes. Thus co evolution plays an important role in modulating mitochondrial activity. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.