Context-specific inhibition of mitochondrial ribosomes by phenicol and oxazolidinone antibiotics.

The antibiotics chloramphenicol (CHL) and oxazolidinones including linezolid (LZD) are known to inhibit mitochondrial translation. This can result in serious, potentially deadly, side effects when used therapeutically. Although the mechanism by which CHL and LZD inhibit bacterial ribosomes has been elucidated in detail, their mechanism of action against mitochondrial ribosomes has yet to be explored.

Structural basis of LRPPRC-SLIRP-dependent translation by the mitoribosome.

In mammalian mitochondria, mRNAs are cotranscriptionally stabilized by the protein factor LRPPRC (leucine-rich pentatricopeptide repeat-containing protein). Here, we characterize LRPPRC as an mRNA delivery factor and report its cryo-electron microscopy structure in complex with SLIRP (SRA stem-loop-interacting RNA-binding protein), mRNA and the mitoribosome. The structure shows that LRPPRC associates with the mitoribosomal proteins mS39 and the N terminus of mS31 through recognition of the LRPPRC helical repeats.

A kinetic dichotomy between mitochondrial and nuclear gene expression processes.

Oxidative phosphorylation (OXPHOS) complexes, encoded by both mitochondrial and nuclear DNA, are essential producers of cellular ATP, but how nuclear and mitochondrial gene expression steps are coordinated to achieve balanced OXPHOS subunit biogenesis remains unresolved. Here, we present a parallel quantitative analysis of the human nuclear and mitochondrial messenger RNA (mt-mRNA) life cycles, including transcript production, processing, ribosome association, and degradation. The kinetic rates of nearly every stage of gene expression differed starkly across compartments.

Proteome-scale movements and compartment connectivity during the eukaryotic cell cycle.

Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of ∼20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both.

Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA.

Cellular metabolism relies on the regulation and maintenance of mitochondrial DNA (mtDNA). Hundreds to thousands of copies of mtDNA exist in each cell, yet because mitochondria lack histones or other machinery important for nuclear genome compaction, it remains unresolved how mtDNA is packaged into individual nucleoids. In this study, we used long-read single-molecule accessibility mapping to measure the compaction of individual full-length mtDNA molecules at near single-nucleotide resolution.

Human Mitoribosome Profiling: A Re-engineered Approach Tailored to Study Mitochondrial Translation.

To understand the human mitochondrial translation process, tools are required to dissect this system at a global scale. The mechanisms and regulation of translation in mitochondria are different from those in the cytosol, and mitochondrial ribosomes have distinct biochemical properties. In this chapter, we describe in detail the modifications we have made to the ribosome profiling approach to adapt it to the unique characteristics of the human mitochondrial ribosome.

A kinetic dichotomy between mitochondrial and nuclear gene expression drives OXPHOS biogenesis.

Oxidative phosphorylation (OXPHOS) complexes, encoded by both mitochondrial and nuclear DNA, are essential producers of cellular ATP, but how nuclear and mitochondrial gene expression steps are coordinated to achieve balanced OXPHOS biogenesis remains unresolved. Here, we present a parallel quantitative analysis of the human nuclear and mitochondrial messenger RNA (mt-mRNA) life cycles, including transcript production, processing, ribosome association, and degradation. The kinetic rates of nearly every stage of gene expression differed starkly across compartments.

Balanced mitochondrial and cytosolic translatomes underlie the biogenesis of human respiratory complexes.

Background: Oxidative phosphorylation (OXPHOS) complexes consist of nuclear and mitochondrial DNA-encoded subunits. Their biogenesis requires cross-compartment gene regulation to mitigate the accumulation of disproportionate subunits. To determine how human cells coordinate mitochondrial and nuclear gene expression processes, we tailored ribosome profiling for the unique features of the human mitoribosome.

Transcription elongation is finely tuned by dozens of regulatory factors.

Understanding the complex network that regulates transcription elongation requires the quantitative analysis of RNA polymerase II (Pol II) activity in a wide variety of regulatory environments. We performed native elongating transcript sequencing (NET-seq) in 41 strains of lacking known elongation regulators, including RNA processing factors, transcription elongation factors, chromatin modifiers, and remodelers. We found that the opposing effects of these factors balance transcription elongation and antisense transcription.

Mitochondrial Ribosome (Mitoribosome) Profiling for Monitoring Mitochondrial Translation In Vivo.

Translation in the mitochondria is regulated by mechanisms distinct from those acting in the cytosol and in bacteria, yet precise methods for investigating it have lagged behind. This unit describes an approach, mitochondrial ribosome (mitoribosome) profiling, to quantitatively monitor mitochondrial translation with high temporal and spatial resolution in Saccharomyces cerevisiae. Mitoribosomes are immunoprecipitated from whole-cell lysate and the protected mRNA fragments are isolated.

Synchronized mitochondrial and cytosolic translation programs.

Oxidative phosphorylation (OXPHOS) is a vital process for energy generation, and is carried out by complexes within the mitochondria. OXPHOS complexes pose a unique challenge for cells because their subunits are encoded on both the nuclear and the mitochondrial genomes. Genomic approaches designed to study nuclear/cytosolic and bacterial gene expression have not been broadly applied to mitochondria, so the co-regulation of OXPHOS genes remains largely unexplored.

A Tetrahymena Piwi bound to mature tRNA 3' fragments activates the exonuclease Xrn2 for RNA processing in the nucleus.

Emerging evidence suggests that Argonaute (Ago)/Piwi proteins have diverse functions in the nucleus and cytoplasm, but the molecular mechanisms employed in the nucleus remain poorly defined. The Tetrahymena thermophila Ago/Piwi protein Twi12 is essential for growth and functions in the nucleus. Twi12-bound small RNAs (sRNAs) are 3' tRNA fragments that contain modified bases and thus are attenuated for base pairing to targets.

Biochemical approaches including the design and use of strains expressing epitope-tagged proteins.

Epitope tagging is a powerful approach used to enable investigations of a cellular component by elucidating its localization, interaction partners, and/or activity targets. Successful tag-based affinity purification yields a mixture of the molecule of interest, associated proteins and nucleic acids, and nonspecific background proteins and nucleic acids, many of which can depend on details of the protocol for enrichment. This chapter provides guidelines and considerations for designing an affinity purification experiment, beginning with construction of a strain expressing a tagged subunit.

Genome-Scale Analysis of Programmed DNA Elimination Sites in Tetrahymena thermophila.

Genetically programmed DNA rearrangements can regulate mRNA expression at an individual locus or, for some organisms, on a genome-wide scale. Ciliates rely on a remarkable process of whole-genome remodeling by DNA elimination to differentiate an expressed macronucleus (MAC) from a copy of the germline micronucleus (MIC) in each cycle of sexual reproduction. Here we describe results from the first high-throughput sequencing effort to investigate ciliate genome restructuring, comparing Sanger long-read sequences from a Tetrahymena thermophila MIC genome library to the MAC genome assembly.

A growth-essential Tetrahymena Piwi protein carries tRNA fragment cargo.

Argonaute/Piwi proteins associate with small RNAs that typically provide sequence specificity for RNP function in gene and genome regulation. Here we show that Twi12, a Tetrahymena Piwi protein essential for growth, is loaded with mature tRNA fragments. The tightly bound ~18- to 22-nucleotide tRNA 3' fragments are biochemically distinct from the tRNA halves produced transiently in response to stress.

Sequence, biogenesis, and function of diverse small RNA classes bound to the Piwi family proteins of Tetrahymena thermophila.

PAZ/PIWI domain (PPD) proteins carrying small RNAs (sRNAs) function in gene and genome regulation. The ciliate Tetrahymena thermophila encodes numerous PPD proteins exclusively of the Piwi clade. We show that the three Tetrahymena Piwi family proteins (Twis) preferentially expressed in growing cells differ in their genetic essentiality and subcellular localization.

Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure.

Background: Tetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a germline micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly.

ATM and ATR make distinct contributions to chromosome end protection and the maintenance of telomeric DNA in Arabidopsis.

Here we examine the function of ATM and ATR at telomeres in Arabidopsis. Although plants lacking ATM or ATR display wild-type telomere length homeostasis, chromosome end protection is compromised in atm atr mutants. Moreover, atm tert Arabidopsis experience an abrupt, early onset of genome instability, arguing that ATM is required for protection of short telomeres.