Genome-wide allele- and strand-specific expression profiling

Recent reports have shown that most of the genome is transcribed and that transcription frequently occurs concurrently on both DNA strands. In diploid genomes, the expression level of each allele conditions the degree to which sequence polymorphisms affect the phenotype. It is thus essential to quantify expression in an allele- and strand-specific manner. Using a custom-designed tiling array and a new computational approach, we piloted measuring allele- and strand-specific expression in yeast. Confident quantitative estimates of allele-specific expression were obtained for about half of the coding and non-coding transcripts of a heterozygous yeast strain, of which 371 transcripts (13%) showed significant allelic differential expression greater than 1.5-fold. The data revealed complex allelic differential expression on opposite strands. Furthermore, combining allele-specific expression with linkage mapping enabled identifying allelic variants that act in cis and in trans to regulate allelic expression in the heterozygous strain. Our results provide the first high-resolution analysis of differential expression on all four strands of an eukaryotic genome

Assessing Systems Properties of Yeast Mitochondria through an Interaction Map of the Organelle

Mitochondria carry out specialized functions; compartmentalized, yet integrated into the metabolic and signaling processes of the cell. Although many mitochondrial proteins have been identified, understanding their functional interrelationships has been a challenge. Here we construct a comprehensive network of the mitochondrial system. We integrated genome-wide datasets to generate an accurate and inclusive mitochondrial parts list. Together with benchmarked measures of protein interactions, a network of mitochondria was constructed in their cellular context, including extra-mitochondrial proteins. This network also integrates data from different organisms to expand the known mitochondrial biology beyond the information in the existing databases. Our network brings together annotated and predicted functions into a single framework. This enabled, for the entire system, a survey of mutant phenotypes, gene regulation, evolution, and disease susceptibility. Furthermore, we experimentally validated the localization of several candidate proteins and derived novel functional contexts for hundreds of uncharacterized proteins. Our network thus advances the understanding of the mitochondrial system in yeast and identifies properties of genes underlying human mitochondrial disorders

MRM2 and MRM3 are involved in biogenesis of the large subunit of the mitochondrial ribosome

Defects of the translation apparatus in human mitochondria are known to cause disease, yet details of how protein synthesis is regulated in this organelle remain to be unveiled. Ribosome production in all organisms studied thus far entails a complex, multistep pathway involving a number of auxiliary factors. This includes several RNA processing and modification steps required for correct rRNA maturation. Little is known about the maturation of human mitochondrial 16S rRNA and its role in biogenesis of the mitoribosome. Here we investigate two methyltransferases, MRM2 (also known as RRMJ2, encoded by FTSJ2) and MRM3 (also known as RMTL1, encoded by RNMTL1), that are responsible for modification of nucleotides of the 16S rRNA A-loop, an essential component of the peptidyl transferase center. Our studies show that inactivation of MRM2 or MRM3 in human cells by RNA interference results in respiratory incompetence as a consequence of diminished mitochondrial translation. Ineffective translation in MRM2- and MRM3-depleted cells results from aberrant assembly of the large subunit of the mitochondrial ribosome (mt-LSU). Our findings show that MRM2 and MRM3 are human mitochondrial methyltransferases involved in the modification of 16S rRNA and are important factors for the biogenesis and function of the large subunit of the mitochondrial ribosome

Prediction of Adipose Browning Capacity by Systematic Integration of Transcriptional Profiles

Activation and recruitment of thermogenic cells in human white adipose tissues ("browning'') can counteract obesity and associated metabolic disorders. However, quantifying the effects of therapeutic interventions on browning remains enigmatic. Here, we devise a computational tool, named ProFAT (profiling of fat tissue types), for quantifying the thermogenic potential of heterogeneous fat biopsies based on prediction of white and brown adipocyte content from raw gene expression datasets. ProFAT systematically integrates 103 mouse-fat-derived transcriptomes to identify unbiased and robust gene signatures of brown and white adipocytes. We validate ProFAT on 80 mouse and 97 human transcriptional profiles from 14 independent studies and correctly predict browning capacity upon various physiological and pharmacological stimuli. Our study represents the most exhaustive comparative analysis of public data on adipose biology toward quantification of browning after personalized medical intervention. ProFAT is freely available and should become increasingly powerful with the growing wealth of transcriptomics data

MICU1 Controls Both the Threshold and Cooperative Activation of the Mitochondrial Ca(2+) Uniporter.

Mitochondrial Ca(2+) uptake via the uniporter is central to cell metabolism, signaling, and survival. Recent studies identified MCU as the uniporter\u27s likely pore and MICU1, an EF-hand protein, as its critical regulator. How this complex decodes dynamic cytoplasmic [Ca(2+)] ([Ca(2+)]c) signals, to tune out small [Ca(2+)]c increases yet permit pulse transmission, remains unknown. We report that loss of MICU1 in mouse liver and cultured cells causes mitochondrial Ca(2+) accumulation during small [Ca(2+)]c elevations but an attenuated response to agonist-induced [Ca(2+)]c pulses. The latter reflects loss of positive cooperativity, likely via the EF-hands. MICU1 faces the intermembrane space and responds to [Ca(2+)]c changes. Prolonged MICU1 loss leads to an adaptive increase in matrix Ca(2+) binding, yet cells show impaired oxidative metabolism and sensitization to Ca(2+) overload. Collectively, the data indicate that MICU1 senses the [Ca(2+)]c to establish the uniporter\u27s threshold and gain, thereby allowing mitochondria to properly decode different inputs

Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D

Recent transcription profiling studies have revealed an unanticipatedly large proportion of antisense transcription across eukaryotic and bacterial genomes. However, the extent and significance of antisense transcripts is controversial partly because experimental artifacts are suspected. Here, we present a method to generate clean genome-wide transcriptome profiles, using actinomycin D (ActD) during reverse transcription. We show that antisense artifacts appear to be triggered by spurious synthesis of second-strand cDNA during reverse transcription reactions. Strand-specific hybridization signals obtained from Saccharomyces cerevisiae tiling arrays were compared between samples prepared with and without ActD. Use of ActD removed about half of the detectable antisense transcripts, consistent with their being artifacts, while sense expression levels and about 200 antisense transcripts were not affected. Our findings thus facilitate a more accurate assessment of the extent and position of antisense transcription, towards a better understanding of its role in cells

Targeting the TCA cycle can ameliorate widespread axonal energy deficiency in neuroinflammatory lesions

Inflammation in the central nervous system can impair the function of neuronal mitochondria and contributes to axon degeneration in the common neuroinflammatory disease multiple sclerosis (MS). Here we combine cell-type-specific mitochondrial proteomics with in vivo biosensor imaging to dissect how inflammation alters the molecular composition and functional capacity of neuronal mitochondria. We show that neuroinflammatory lesions in the mouse spinal cord cause widespread and persisting axonal ATP deficiency, which precedes mitochondrial oxidation and calcium overload. This axonal energy deficiency is associated with impaired electron transport chain function, but also an upstream imbalance of tricarboxylic acid (TCA) cycle enzymes, with several, including key rate-limiting, enzymes being depleted in neuronal mitochondria in experimental models and in MS lesions. Notably, viral overexpression of individual TCA enzymes can ameliorate the axonal energy deficits in neuroinflammatory lesions, suggesting that TCA cycle dysfunction in MS may be amendable to therapy

Targeting the TCA cycle can ameliorate widespread axonal energy deficiency in neuroinflammatory lesions

In this study, Tai et al. provide insights into the metabolic and bioenergetic responses in the axonal compartment in the context of multiple sclerosis. Moreover, they show how upregulating the tricarboxylic acid cycle confers protection against neuroinflammation-induced energy deprivation. Inflammation in the central nervous system can impair the function of neuronal mitochondria and contributes to axon degeneration in the common neuroinflammatory disease multiple sclerosis (MS). Here we combine cell-type-specific mitochondrial proteomics with in vivo biosensor imaging to dissect how inflammation alters the molecular composition and functional capacity of neuronal mitochondria. We show that neuroinflammatory lesions in the mouse spinal cord cause widespread and persisting axonal ATP deficiency, which precedes mitochondrial oxidation and calcium overload. This axonal energy deficiency is associated with impaired electron transport chain function, but also an upstream imbalance of tricarboxylic acid (TCA) cycle enzymes, with several, including key rate-limiting, enzymes being depleted in neuronal mitochondria in experimental models and in MS lesions. Notably, viral overexpression of individual TCA enzymes can ameliorate the axonal energy deficits in neuroinflammatory lesions, suggesting that TCA cycle dysfunction in MS may be amendable to therapy

Crosslink of calcium and sodium signalling in health and disease

Transmembrane ionic gradients, which are an indispensable feature of life, are used for generation of cytosolic ionic signals that regulate a host of cellular functions. Intracellular signalling mediated by Ca2+ and Na+ is tightly linked through several molecular pathways that generate Ca2+ and Na+ fluxes and are in turn regulated by both ions. Transient receptor potential (TRP) channels bridge endoplasmic reticulum Ca2+ release with generation of Na+ and Ca2+ currents. The plasmalemmal Na+-Ca2+ exchanger (NCX) flickers between forward and reverse mode to co-ordinate the influx and efflux of both ions with membrane polarization and cytosolic ion concentrations. The mitochondrial calcium uniporter channel (MCU) and mitochondrial Na+-Ca2+ exchanger (NCLX) mediate Ca2+ entry into and release from this organelle and couple cytosolic Ca2+ and Na+ fluctuations with cellular energetics. Cellular Ca2+ and Na+ signalling controls numerous functional responses and, in the CNS, provides for fast regulation of astroglial homeostatic cascades that are crucial for maintenance of synaptic transmission