Defects of mitochondrial RNA turnover lead to the accumulation of double-stranded RNA in vivo

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology

C6orf203 is an RNA-binding protein involved in mitochondrial protein synthesis.

In all biological systems, RNAs are associated with RNA-binding proteins (RBPs), forming complexes that control gene regulatory mechanisms, from RNA synthesis to decay. In mammalian mitochondria, post-transcriptional regulation of gene expression is conducted by mitochondrial RBPs (mt-RBPs) at various stages of mt-RNA metabolism, including polycistronic transcript production, its processing into individual transcripts, mt-RNA modifications, stability, translation and degradation. To date, only a handful of mt-RBPs have been characterized. Here, we describe a putative human mitochondrial protein, C6orf203, that contains an S4-like domain-an evolutionarily conserved RNA-binding domain previously identified in proteins involved in translation. Our data show C6orf203 to bind highly structured RNA in vitro and associate with the mitoribosomal large subunit in HEK293T cells. Knockout of C6orf203 leads to a decrease in mitochondrial translation and consequent OXPHOS deficiency, without affecting mitochondrial RNA levels. Although mitoribosome stability is not affected in C6orf203-depleted cells, mitoribosome profiling analysis revealed a global disruption of the association of mt-mRNAs with the mitoribosome, suggesting that C6orf203 may be required for the proper maturation and functioning of the mitoribosome. We therefore propose C6orf203 to be a novel RNA-binding protein involved in mitochondrial translation, expanding the repertoire of factors engaged in this process

Absence of the Autophagy Adaptor SQSTM1/p62 Causes Childhood-Onset Neurodegeneration with Ataxia, Dystonia, and Gaze Palsy

Peer reviewe

Absence of TXNIP in humans leads to lactic acidosis and low serum methionine linked to deficient respiration on pyruvate

Thioredoxin-interacting protein (TXNIP) is an a-arrestin that can bind to and inhibit the antioxidant protein thioredoxin (TXN). TXNIP expression is induced by glucose and promotes b-cell apoptosis in the pancreas, and deletion of its gene in mouse models protects against diabetes. TXNIP is currently studied as a potential new target for antidiabetic drug therapy. In this study, we describe a family with a mutation in the TXNIP gene leading to nondetectable expression of TXNIP protein. Symptoms of affected family members include lactic acidosis and low serum methionine levels. Using patient-derived TXNIP-deficient fibroblasts and myoblasts, we show that oxidative phosphorylation is impaired in these cells when given glucose and pyruvate but normalized with malate. Isolated mitochondria from these cells appear to have normal respiratory function. The cells also display a transcriptional pattern suggestive of a high basal activation of the Nrf2 transcription factor. We conclude that a complete lack of TXNIP in human is nonlethal and leads to specific metabolic distortions that are, at least in part, linked to a deficient respiration on pyruvate. The results give important insights into the impact of TXNIP in humans and thus help to further advance the development of antidiabetic drugs targeting this protein.“Ministerio of Economía y Competitividad” (grant BFU2016-77634-R and “Ramón y Cajal” fellowship RYC-2014-15792), Diabetesfonden, and Alicia Koplowitz Foundation to A.G.-C. A.Wr. is a Ragnar Söderberg fellow in Medicine (M77/13

Protocol for the derivation, culturing, and differentiation of human iPS-cell-derived neuroepithelial stem cells to study neural differentiation in vitro

Summary: Here, we present a revised protocol to derive neuroepithelial stem (NES) cells from human induced pluripotent stem cells. NES cells can be further differentiated into a culture of neurons (90%) and glia (10%). We describe how to derive and maintain NES cells in culture and how to differentiate them. In addition, we show the potential use of NES cells to study the role of reactive oxygen species in neuronal differentiation and a guideline for NES cell transfection.For complete details on the use and execution of this protocol, please refer to Calvo-Garrido et al. (2019); Falk et al. (2012)

SQSTM1/p62-Directed Metabolic Reprogramming Is Essential for Normal Neurodifferentiation

Summary: Neurodegenerative disorders are an increasingly common and irreversible burden on society, often affecting the aging population, but their etiology and disease mechanisms are poorly understood. Studying monogenic neurodegenerative diseases with known genetic cause provides an opportunity to understand cellular mechanisms also affected in more complex disorders. We recently reported that loss-of-function mutations in the autophagy adaptor protein SQSTM1/p62 lead to a slowly progressive neurodegenerative disease presenting in childhood. To further elucidate the neuronal involvement, we studied the cellular consequences of loss of p62 in a neuroepithelial stem cell (NESC) model and differentiated neurons derived from reprogrammed p62 patient cells or by CRISPR/Cas9-directed gene editing in NESCs. Transcriptomic and proteomic analyses suggest that p62 is essential for neuronal differentiation by controlling the metabolic shift from aerobic glycolysis to oxidative phosphorylation required for neuronal maturation. This shift is blocked by the failure to sufficiently downregulate lactate dehydrogenase expression due to the loss of p62, possibly through impaired Hif-1α downregulation and increased sensitivity to oxidative stress. The findings imply an important role for p62 in neuronal energy metabolism and particularly in the regulation of the shift between glycolysis and oxidative phosphorylation required for normal neurodifferentiation. : SQSTM1/p62 is a known autophagy adaptor that, if lost, causes childhood-onset neurodegeneration. Data from Wredenberg et al. show that loss of p62 in a neuronal stem cell model does not affect mitophagy but instead leads to impaired differentiation. The authors suggest p62 finely tunes LDHA expression and thus controls the metabolic shift to OXPHOS required for proper differentiation. Keywords: SQSTM1, p62, hypoxia, mitochondria, neurodifferentiation, neuroepithelial-like stem cells, neuronal development, oxidative stress, mitophagy, neurodegeneratio

Mitochondrial respiration is affected due to incomplete OXPHOS assembly.

<p>(<b>A</b>) BN-PAGE and in gel staining of Complex I and Complex IV activities in mitochondrial protein extracts from control (wt and FM7,Tb) and DmMTPAP KO (<i>DmMTPAP</i>^KO) 4-day-old larvae. Coomassie staining of the gel and VDAC western blot of the input samples was performed to ensure equal loading of the gel. (<b>B</b>) Complex V assembly was assessed in <i>DmMTPAP</i> KD (<i>DmMTPAP</i> RNAi #1) 5-day-old larvae by BN-PAGE, followed by Western blot analysis against the F1 subunit of Complex V. Coomassie staining was used to ensure equal loading. (<b>C</b>) Oxygen consumption rates in permeabilised 4-day-old control (wt) and <i>DmMTPAP</i>^KO larvae, using glutamate, malate and pyruvate (GMP + ADP), succinate (GMP + ADP + succ) or TMPD and ascorbate (TMP + asc) as electron donors. Data are normalized to the protein content in each sample and are represented as mean ± SEM (***P < 0,001, n = 8). (<b>D</b>) Relative enzyme activities of respiratory chain complexes in 4-day-old control (wt) and <i>DmMTPAP</i>^KO larvae. Data are represented as mean ± SD (*P<0.05, **P < 0.01, ***P < 0,001, n = 3). (<b>E</b>) Relative enzyme activities of respiratory chain complexes (Complex I-IV) from control (white, grey and striped bars) and <i>DmMTPAP</i> KD (checked and black bars) 5-day-old larvae. Data is represented as mean ± SEM (**P < 0.01, ***P < 0,001, n = 5).</p

MTPAP is the only mitochondrial adenylase in flies and is required to protect the 3' termini of mRNAs.

<p>(<b>A</b>) Body size comparison in control (wt and FM7,Tb) and DmMTPAP KO larvae (<i>DmMTPAP</i>^KO) at 4 days ael. (<b>B</b>) qRT-PCR analysis of <i>DmMTPAP</i> transcript levels in 1 day heterozygous <i>DmMTPAP</i>^KO flies (<i>DmMTPAP</i>^KO/FM7,Tb) and 4-day-old hemyzygous <i>DmMTPAP</i>^KO larvae (<i>DmMTPAP</i>^KO) and their corresponding age-matched controls (wt, FM7,Tb). Histone 2B transcript was used as endogenous control. Data is represented as mean ± SEM (*P < 0.05, ***P < 0.001, n = 5). (<b>C</b>) mRNA and poly(A) tail length in individually sequenced clones after transcript circularisation (<i>MTATP6/8</i>, <i>MTND4/4L</i>, <i>MTND1</i> and <i>MTND5</i>) or 3' RACE (<i>MTCOX1</i> and <i>MTCYTB</i>) in <i>DmMTPAP</i>^KO (red, n = 14–26) and control larvae (grey, n = 11–25) at 4 days ael. The annotated 3' termini of the indicated transcripts was set to zero to determine poly(A) tail length. (<b>D</b>) rRNA and poly(A) tail length in individually sequenced clones after transcript circularisation in <i>DmMTPAP</i>^KO (red, n = 17–25) and control larvae (grey, n = 24–29) at 4 days ael. Data are represented as mean ± SEM. (***P < 0,001), using a Mann-Whitney test.</p

Polyadenylation is not required for mitochondrial translation.

<p>(<b>A</b>) <i>In organello</i> labelling of mitochondrial translation products on isolated mitochondria from <i>DmMTPAP</i> KD (<i>DmMTPAP</i> RNAi #1) and control (w;;daGAL4/+ and w;UAS-mtPAPRNAi#1/+) 5 days ael larvae. Labelling was performed for 60min (pulse), followed by a 15 or 45 min chase with cold methionine. Loading was normalised to VDAC levels. (<b>B</b>) <i>In organello</i> labelling of mitochondrial translation products on isolated mitochondria from <i>DmMTPAP</i> KO (<i>DmMTPAP</i>^KO) and control (wt and FM7,Tb) 4-day-old larvae. Coomassie staining of the gels and VDAC Western blotting of the input samples were performed to ensure equal loading of the samples. Western blot analysis (<b>C</b>) and quantification (<b>D</b>) of nuclear-encoded subunit of Complex I (NDUFS3) in isolated mitochondria from control (daGAL4 control, RNAi #1 control and RNAi#2 control) and <i>DmMTPAP</i> KD (<i>DmMTPAP</i> RNAi #1 and <i>DmMTPAP</i> RNAi #2) 5-day-old larvae. VDAC was used as a loading control. Western blot analysis (<b>E</b>) and quantification (<b>F</b>) of the steady-state levels of a nuclear-encoded subunit of Complex I (NDUFS3) and an mtDNA-encoded subunit of complex IV (COX3) in mitochondria of control (wt and FM7,Tb) and <i>DmMTPAP</i>^KO 4-day-old larvae. VDAC was used as a loading control. Data are represented as mean ± SD.</p

Impaired 3' termini do not affect the stability of most mtDNA-encoded transcripts.

<p>(<b>A</b>) Relative steady-state level of mitochondrial transcripts were determined by Northern Blot in 5 day ael control (white and grey bars) and <i>DmMTPAP</i> KD larvae (black bar) larvae (n = 5). Expression levels were quantified using a Typhoon phosphorimager and normalised to histone 2B mRNA. All data are represented as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0,001). (<b>B</b>) Northern blot analysis and (<b>C</b>) quantification of steady-state levels of mitochondrial transcripts in control (wt and FM7,Tb) and <i>DmMTPAP</i> KO larvae (<i>DmMTPAP</i>^KO) at 4 days ael. Histone 2B transcript was used as loading control. (<b>D</b>) <i>De novo</i> mitochondrial transcription in isolated mitochondria of control and <i>DmMTPAP</i> KO larvae at 4 days ael in the presence of radioactively labelled [³²P]-UTP. Loading of the gels and absence of RNA degradation was confirmed by Northern blotting against COX1 and 16S RNAs. Western blotting of VDAC in the input samples was used as a loading control. (<b>E</b>) qPCR of mtDNA steady-state levels <i>DmMTPAP</i> KO and control larvae at 4 days ael. Primers against the cytosolic ribosomal protein 49 (RP49) were used to normalise to nuclear DNA content of the samples.</p