FASTKD1 and FASTKD4 have opposite effects on expression of specific mitochondrial RNAs, depending upon their endonuclease-like RAP domain.

FASTK family proteins have been identified as regulators of mitochondrial RNA homeostasis linked to mitochondrial diseases, but much remains unknown about these proteins. We show that CRISPR-mediated disruption of FASTKD1 increases ND3 mRNA level, while disruption of FASTKD4 reduces the level of ND3 and of other mature mRNAs including ND5 and CYB, and causes accumulation of ND5-CYB precursor RNA. Disrupting both FASTKD1 and FASTKD4 in the same cell results in decreased ND3 mRNA similar to the effect of depleting FASTKD4 alone, indicating that FASTKD4 loss is epistatic. Interestingly, very low levels of FASTKD4 are sufficient to prevent ND3 loss and ND5-CYB precursor accumulation, suggesting that FASTKD4 may act catalytically. Furthermore, structural modeling predicts that each RAP domain of FASTK proteins contains a nuclease fold with a conserved aspartate residue at the putative active site. Accordingly, mutation of this residue in FASTKD4 abolishes its function. Experiments with FASTK chimeras indicate that the RAP domain is essential for the function of the FASTK proteins, while the region upstream determines RNA targeting and protein localization. In conclusion, this paper identifies new aspects of FASTK protein biology and suggests that the RAP domain function depends on an intrinsic nucleolytic activity.

The Pseudouridine Synthase RPUSD4 Is an Essential Component of Mitochondrial RNA Granules.

Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking. Here, we have applied a microscopy-based approach that has allowed us to identify novel components of the MRG proteome. Among these, we have focused our attention on RPUSD4, an uncharacterized mitochondrial putative pseudouridine synthase. We show that RPUSD4 depletion leads to a severe reduction of the steady-state level of the 16S mitochondrial (mt) rRNA with defects in the biogenesis of the mitoribosome large subunit and consequently in mitochondrial translation. We report that RPUSD4 binds 16S mt-rRNA, mt-tRNAMet, and mt-tRNAPhe, and we demonstrate that it is responsible for pseudouridylation of the latter. These data provide new insights into the relevance of RNA pseudouridylation in mitochondrial gene expression.

The disulfide isomerase Grp58 is a protective factor against prion neurotoxicity.

Prion diseases are transmissible neurodegenerative disorders characterized by extensive neuronal apoptosis and accumulation of misfolded prion protein (PrP(SC)). Recent reports indicate that PrP(SC) induces neuronal apoptosis via activation of the endoplasmic reticulum (ER) stress pathway and activation of the ER resident caspase-12. Here, we investigate the relationship between prion replication and induction of ER stress during different stages of the disease in a murine scrapie model. The first alteration observed consists of the upregulation of the ER chaperone of the glucose-regulated protein Grp58, which was detected during the presymptomatic phase and followed closely the formation of PrP(SC). An increase in Grp58 expression correlated with PrP(SC) accumulation at all stages of the disease in different brain areas, suggesting that this chaperone may play an important role in the cellular response to prion infection. Indeed, in vitro studies using N2a neuroblastoma cells demonstrated that inhibition of Grp58 expression with small interfering RNA led to a significant enhancement of PrP(SC) toxicity. Conversely, overexpression of Grp58 protected cells against PrP(SC) toxicity and decreased the rate of caspase-12 activation. Grp58 and PrP were shown to interact by coimmunoprecipitation, observing a higher interaction in cells infected with scrapie prions. Our data indicate that expression of Grp58 is an early cellular response to prion replication, acting as a neuroprotective factor against prion neurotoxicity. Our findings suggest that targeting Grp58 interaction may have applications for developing novel strategies for treatment and early diagnosis of prion diseases.

Protein misfolding cyclic amplification for diagnosis and prion propagation studies.

Diverse human disorders are thought to arise from the misfolding and aggregation of an underlying protein. Among them, prion diseases are some of the most intriguing disorders that can be transmitted by an unprecedented infectious agent, termed prion, composed mainly (if not exclusively) of the misfolded prion protein. The hallmark event in the disease is the conversion of the native prion protein into the disease-associated misfolded protein. We have recently described a novel technology to mimic the prion conversion process in vitro. This procedure, named protein misfolding cyclic amplification (PMCA), conceptually analogous to DNA amplification by polymerase chain reaction (PCR), has important applications for research and diagnosis. In this chapter we describe the rational behind PMCA and some of the many potential applications of this novel technology. We also describe in detail the technical and methodological aspects of PMCA, as well as its application in automatic and serial modes that have been developed with a view to improving disease diagnosis.

Mitochondrial RNA granules: Compartmentalizing mitochondrial gene expression.

In mitochondria, DNA replication, gene expression, and RNA degradation machineries coexist within a common nondelimited space, raising the question of how functional compartmentalization of gene expression is achieved. Here, we discuss the recently characterized "mitochondrial RNA granules," mitochondrial subdomains with an emerging role in the regulation of gene expression.

Is loss of function of the prion protein the cause of prion disorders?

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that involve misfolding of the prion protein. Recent studies have provided evidence that normal prion protein might have a physiological function in neuroprotective signaling, suggesting that loss of prion protein activity might contribute to the pathogenesis of prion disease. However, studies using knockout animals do not support the loss-of-function hypothesis and argue that prion neurodegeneration might be associated with a gain of a toxic activity by the misfolded prion protein. Thus, the mechanism of neurodegeneration in spongiform encephalopathies remains enigmatic.

A mitochondria-specific isoform of FASTK is present in mitochondrial RNA granules and regulates gene expression and function.

The mitochondrial genome relies heavily on post-transcriptional events for its proper expression, and misregulation of this process can cause mitochondrial genetic diseases in humans. Here, we report that a novel translational variant of Fas-activated serine/threonine kinase (FASTK) co-localizes with mitochondrial RNA granules and is required for the biogenesis of ND6 mRNA, a mitochondrial-encoded subunit of the NADH dehydrogenase complex (complex I). We show that ablating FASTK expression in cultured cells and mice results specifically in loss of ND6 mRNA and reduced complex I activity in vivo. FASTK binds at multiple sites along the ND6 mRNA and its precursors and cooperates with the mitochondrial degradosome to ensure regulated ND6 mRNA biogenesis. These data provide insights into the mechanism and control of mitochondrial RNA processing within mitochondrial RNA granules.

Life without the mitochondrial calcium uniporter.

Calcium enters mitochondria through a dedicated channel referred to as the mitochondrial calcium uniporter (MCU), whose molecular identity has long remained elusive. Since the discovery of the gene encoding the MCU protein two years ago, researchers have awaited the generation of a mouse lacking the MCU. These mice are fully viable and show defects limited to performance of high-energy-demanding exercises. Strikingly, no protection against necrosis is observed following ischaemia-reperfusion in the heart.

Prion replication alters the distribution of synaptophysin and caveolin 1 in neuronal lipid rafts.

The main event in the pathogenesis of prion diseases is the conversion of the cellular prion protein (PrP(C)) into the abnormal, protease-resistant prion protein (PrP(res)). PrP(C) is a GPI-anchored protein located in lipid rafts or detergent-resistant membranes (DRMs). Here we describe the association of PrP with DRMs in neuronal cell bodies and axons during the course of murine scrapie and its relation with the distribution of the PrP-interacting proteins caveolin 1 and synaptophysin. Scrapie infection triggered the accumulation of PrP(res) in DRMs from retinas and optic nerves from early stages of the disease before evidence of neuronal cell loss. Most of the PrP(res) remained associated with lipid rafts throughout different stages in disease progression. In contrast to PrP(res), caveolin 1 and synaptophysin in retina and optic nerves shifted to non-DRM fractions during the course of scrapie infection. The accumulation of PrP(res) in DRMs was not associated with a general alteration in their composition, because no change in the total protein distribution across the sucrose gradient or in the flotation characteristics of the glycosphingolipid GM1 or Thy-1 were observed until advanced stages of the disease. However, an increase in total cholesterol levels was observed in optic nerve and retinas. Only during late stages of the disease was a decrease in the number of neuronal cell bodies observed, suggesting that synaptic abnormalities are the earliest sign of neuronal dysfunction that ultimately results in neuronal death. These results indicate that prion replication triggers an abnormal localization of caveolin 1 and synaptophysin, which in turn may alter neuronal function.

Caspase-12 and endoplasmic reticulum stress mediate neurotoxicity of pathological prion protein.

Prion diseases are characterized by accumulation of misfolded prion protein (PrP(Sc)), and neuronal death by apoptosis. Here we show that nanomolar concentrations of purified PrP(Sc) from mouse scrapie brain induce apoptosis of N2A neuroblastoma cells. PrP(Sc) toxicity was associated with an increase of intracellular calcium released from endoplasmic reticulum (ER) and up-regulation of several ER chaperones. Caspase-12 activation was detected in cells treated with PrP(Sc), and cellular death was inhibited by overexpression of a catalytic mutant of caspase-12 or an ER-targeted Bcl-2 chimeric protein. Scrapie-infected N2A cells were more susceptible to ER-stress and to PrP(Sc) toxicity than non-infected cells. In scrapie-infected mice a correlation between caspase-12 activation and neuronal loss was observed in histological and biochemical analyses of different brain areas. The extent of prion replication was closely correlated with the up-regulation of ER-stress chaperone proteins. Similar results were observed in humans affected with sporadic and variant Creutzfeldt-Jakob disease, implicating for the first time the caspase-12 dependent pathway in a neurodegenerative disease in vivo, and thus offering novel potential targets for the treatment of prion disorders.

Schizosaccharomyces pombe Pmf1p is structurally and functionally related to Mmf1p of Saccharomyces cerevisiae.

A novel family of small proteins, termed p14.5 or YERO57c/YJGFc, has been identified. Independent studies indicate that p14.5 family members are multifunctional proteins involved in several pathways, e.g. regulation of translation or activation of the protease mu-calpain. We have previously shown that Mmf1p, a p14.5 of the budding yeast Saccharomyces cerevisiae, is localized in the mitochondria and influences mitochondrial DNA stability. In addition, we have demonstrated that Mmf1p is functionally related to p14.5 of mammalian cells. To explore further the evolutionary conservation of the mitochondrial function(s) of the p14.5s we have extended our study to the fission yeast, Schizosaccharomyces pombe. In this organism two p14.5 homologous proteins are present: Pmf1p (pombe mitochondrial factor 1) and Hpm1p (homologous Pmf1p factor 1). We have generated a specific Pmf1p antibody, which recognizes a single band of approximately 15 kDa in total cellular extracts. Cellular fractionation experiments indicate that Pmf1p localizes in the mitochondria as well as in the cytoplasm. We also show that Pmf1p shares several properties of S. cerevisiae Mmf1p. Indeed, Pmf1p restores the wild-type phenotype when expressed in delta mmf1 S. cerevisiae cells. Deletion of the leader sequence of Pmf1p abrogates its ability to localize in mitochondria and to functionally replace Mmf1p. Thus, these data together with our previous study show that the mitochondrial function(s) of the p14.5 family members are highly conserved in eukaryotic cells.