Alternative translation initiation augments the human mitochondrial proteome

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License.-- et al.Alternative translation initiation (ATI) is a mechanism of producing multiple proteins from a single transcript, which in some cases regulates trafficking of proteins to different cellular compartments, including mitochondria. Application of a genome-wide computational screen predicts a cryptic mitochondrial targeting signal for 126 proteins in mouse and man that is revealed when an AUG codon located downstream from the canonical initiator methionine codon is used as a translation start site, which we term downstream ATI (dATI). Experimental evidence in support of dATI is provided by immunoblotting of endogenous truncated proteins enriched in mitochondrial cell fractions or of co-localization with mitochondria using immunocytochemistry. More detailed cellular localization studies establish mitochondrial targeting of a member of the cytosolic poly(A) binding protein family, PABPC5, and of the RNA/DNA helicase PIF1α. The mitochondrial isoform of PABPC5 co-immunoprecipitates with the mitochondrial poly(A) polymerase, and is markedly reduced in abundance when mitochondrial DNA and RNA are depleted, suggesting it plays a role in RNA metabolism in the organelle. Like PABPC5 and PIF1α, most of the candidates identified by the screen are not currently annotated as mitochondrial proteins, and so dATI expands the human mitochondrial proteome.Cambridge University Commonwealth Trust, Fellowship (to L.K.); UK Medical Research Council. Funding for open access charge: UK Medical Research Council.Peer reviewe

H+ transport is an integral function of the mitochondrial ADP/ATP carrier.

The mitochondrial ADP/ATP carrier (AAC) is a major transport protein of the inner mitochondrial membrane. It exchanges mitochondrial ATP for cytosolic ADP and controls cellular production of ATP. In addition, it has been proposed that AAC mediates mitochondrial uncoupling, but it has proven difficult to demonstrate this function or to elucidate its mechanisms. Here we record AAC currents directly from inner mitochondrial membranes from various mouse tissues and identify two distinct transport modes: ADP/ATP exchange and H+ transport. The AAC-mediated H+ current requires free fatty acids and resembles the H+ leak via the thermogenic uncoupling protein 1 found in brown fat. The ADP/ATP exchange via AAC negatively regulates the H+ leak, but does not completely inhibit it. This suggests that the H+ leak and mitochondrial uncoupling could be dynamically controlled by cellular ATP demand and the rate of ADP/ATP exchange. By mediating two distinct transport modes, ADP/ATP exchange and H+ leak, AAC connects coupled (ATP production) and uncoupled (thermogenesis) energy conversion in mitochondria

Analysis of Replicating Mitochondrial DNA by In Organello Labeling and Two-Dimensional Agarose Gel Electrophoresis.

Our understanding of the mechanisms of DNA replication in a broad range of organisms and viruses has benefited from the application of two-dimensional agarose gel electrophoresis (2D-AGE). The method resolves DNA molecules on the basis of size and shape and is technically straightforward. 2D-AGE sparked controversy in the field of mitochondria when it revealed replicating molecules with lengthy tracts of RNA, a phenomenon never before reported in nature. More recently, radioisotope labeling of the DNA in the mitochondria has been coupled with 2D-AGE. In its first application, this procedure helped to delineate the "bootlace mechanism of mitochondrial DNA replication," in which processed mitochondrial transcripts are hybridized to the lagging strand template at the replication fork as the leading DNA strand is synthesized. This chapter provides details of the method, how it has been applied to date and concludes with some potential future applications of the technique.We thank Drs. Pohjoismäki and Jacobs for the EM image of the replicating mitochondrial DNA molecule, and the Medical Research Council for funding

Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication.

Mammalian mitochondria operate multiple mechanisms of DNA replication. In many cells and tissues a strand-asynchronous mechanism predominates over coupled leading and lagging-strand DNA synthesis. However, little is known of the factors that control or influence the different mechanisms of replication, and the idea that strand-asynchronous replication entails transient incorporation of transcripts (aka bootlaces) is controversial. A firm prediction of the bootlace model is that it depends on mitochondrial transcripts. Here, we show that elevated expression of Twinkle DNA helicase in human mitochondria induces bidirectional, coupled leading and lagging-strand DNA synthesis, at the expense of strand-asynchronous replication; and this switch is accompanied by decreases in the steady-state level of some mitochondrial transcripts. However, in the so-called minor arc of mitochondrial DNA where transcript levels remain high, the strand-asynchronous replication mechanism is instated. Hence, replication switches to a strand-coupled mechanism only where transcripts are scarce, thereby establishing a direct correlation between transcript availability and the mechanism of replication. Thus, these findings support a critical role of mitochondrial transcripts in the strand-asynchronous mechanism of mitochondrial DNA replication; and, as a corollary, mitochondrial RNA availability and RNA/DNA hybrid formation offer means of regulating the mechanisms of DNA replication in the organelle

Mitochondrial DNA replication proceeds via a 'bootlace' mechanism involving the incorporation of processed transcripts

Peer reviewe

A cryptic targeting signal creates a mitochondrial FEN1 isoform with tailed R-Loop binding properties.

A growing number of DNA transacting proteins is found in the nucleus and in mitochondria, including the DNA repair and replication protein Flap endonuclease 1, FEN1. Here we show a truncated FEN1 isoform is generated by alternative translation initiation, exposing a mitochondrial targeting signal. The shortened form of FEN1, which we term FENMIT, localizes to mitochondria, based on import into isolated organelles, immunocytochemistry and subcellular fractionation. In vitro FENMIT binds to flap structures containing a 5' RNA flap, and prefers such substrates to single-stranded RNA. FENMIT can also bind to R-loops, and to a lesser extent to D-loops. Exposing human cells to ethidium bromide results in the generation of RNA/DNA hybrids near the origin of mitochondrial DNA replication. FENMIT is recruited to the DNA under these conditions, and is released by RNase treatment. Moreover, high levels of recombinant FENMIT expression inhibit mtDNA replication, following ethidium bromide treatment. These findings suggest FENMIT interacts with RNA/DNA hybrids in mitochondrial DNA, such as those found at the origin of replication

UCP1 deficiency causes brown fat respiratory chain depletion and sensitizes mitochondria to calcium overload-induced dysfunction.

Brown adipose tissue (BAT) mitochondria exhibit high oxidative capacity and abundant expression of both electron transport chain components and uncoupling protein 1 (UCP1). UCP1 dissipates the mitochondrial proton motive force (Δp) generated by the respiratory chain and increases thermogenesis. Here we find that in mice genetically lacking UCP1, cold-induced activation of metabolism triggers innate immune signaling and markers of cell death in BAT. Moreover, global proteomic analysis reveals that this cascade induced by UCP1 deletion is associated with a dramatic reduction in electron transport chain abundance. UCP1-deficient BAT mitochondria exhibit reduced mitochondrial calcium buffering capacity and are highly sensitive to mitochondrial permeability transition induced by reactive oxygen species (ROS) and calcium overload. This dysfunction depends on ROS production by reverse electron transport through mitochondrial complex I, and can be rescued by inhibition of electron transfer through complex I or pharmacologic depletion of ROS levels. Our findings indicate that the interscapular BAT of Ucp1 knockout mice exhibits mitochondrial disruptions that extend well beyond the deletion of UCP1 itself. This finding should be carefully considered when using this mouse model to examine the role of UCP1 in physiology

Alternative translation initiation augments the human mitochondrial proteome.

Alternative translation initiation (ATI) is a mechanism of producing multiple proteins from a single transcript, which in some cases regulates trafficking of proteins to different cellular compartments, including mitochondria. Application of a genome-wide computational screen predicts a cryptic mitochondrial targeting signal for 126 proteins in mouse and man that is revealed when an AUG codon located downstream from the canonical initiator methionine codon is used as a translation start site, which we term downstream ATI (dATI). Experimental evidence in support of dATI is provided by immunoblotting of endogenous truncated proteins enriched in mitochondrial cell fractions or of co-localization with mitochondria using immunocytochemistry. More detailed cellular localization studies establish mitochondrial targeting of a member of the cytosolic poly(A) binding protein family, PABPC5, and of the RNA/DNA helicase PIF1α. The mitochondrial isoform of PABPC5 co-immunoprecipitates with the mitochondrial poly(A) polymerase, and is markedly reduced in abundance when mitochondrial DNA and RNA are depleted, suggesting it plays a role in RNA metabolism in the organelle. Like PABPC5 and PIF1α, most of the candidates identified by the screen are not currently annotated as mitochondrial proteins, and so dATI expands the human mitochondrial proteome

Opening a Pandora's Box that can't be salvaged: Health professionals’ perceptions of appearance-related care in an Australian pediatric specialist hospital

© 2019 Elsevier Ltd Many children and young people struggle adjusting to the psychosocial consequences (e.g., body dissatisfaction, social anxiety, and stigmatisation) of visible differences (or disfigurement). As appearance-affecting conditions often require specialist multidisciplinary team care, health professionals are in a unique position to offer psychosocial support and intervention. However, there is a dearth of literature on how appearance-related concerns are managed in pediatric hospital settings. Sixteen Australian specialist health professionals participated in semi-structured qualitative interviews to address this gap. Interviews explored current appearance-related psychosocial service provision, barriers in accessing appearance-related care, and perceptions of online platforms to deliver specialist support and intervention. Thematic analysis demonstrated four themes: We can do it better, Capability versus availability, Online generation, and Putting appearance on the agenda. This research highlighted the potential value of online platforms to increase accessibility to specialist appearance-related care, the need for more psychosocial resources to be integrated into appearance-related specialities, prioritising the development of low to medium appearance-related support and intervention, increasing the appearance-related knowledge of health professionals and families, and the need for more holistic approaches in routine care

Mitochondrial ROS regulate thermogenic energy expenditure and sulfenylation of UCP1

Brown adipose tissue (BAT) can dissipate chemical energy as heat through thermogenic respiration, which requires uncoupling protein 1 (UCP1)1,2. Thermogenesis from BAT and beige adipose can combat obesity and diabetes3, encouraging investigation of factors that control UCP1-dependent respiration in vivo. Herein we show that acutely activated BAT thermogenesis is defined by a substantial increase in mitochondrial reactive oxygen species (ROS) levels. Remarkably, this process supports in vivo BAT thermogenesis, as pharmacological depletion of mitochondrial ROS results in hypothermia upon cold exposure, and inhibits UCP1-dependent increases in whole body energy expenditure. We further establish that thermogenic ROS alter BAT cysteine thiol redox status to drive increased respiration, and Cys253 of UCP1 is a key target. UCP1 Cys253 is sulfenylated during thermogenesis, while mutation of this site desensitizes the purine nucleotide inhibited state of the carrier to adrenergic activation and uncoupling. These studies identify BAT mitochondrial ROS induction as a mechanism that drives UCP1-dependent thermogenesis and whole body energy expenditure, which opens the way to develop improved therapeutic strategies for combating metabolic disorders