Cytosolic mRNA Research Articles

Unraveling mRNA delivery bottlenecks of ineffective delivery vectors by co-transfection with effective carriers

The messenger RNA (mRNA) SARS-CoV-2 vaccines have demonstrated the therapeutic potential of this novel drug modality. Protein expression is the consequence of a multistep delivery process that relies on proper packaging into nanoparticle carriers to protect the mRNA against degradation enabling effective cellular uptake and endosomal release, and liberating the mRNA in the cytosol. Bottlenecks along this route remain challenging to pinpoint. Although methods to assess endosomal escape of carriers have been developed, versatile strategies to identify bottlenecks along the delivery trajectory are missing. Here, it is shown that co-incubating an inefficient nanoparticle formulation with an efficient one solves this problem. Cells were co-incubated with mRNA nanoparticles formed with either the efficient cell-penetrating peptide (CPP) PepFect14 or the inefficient CPP nona-arginine (R9). Co-transfection enhanced cellular uptake and endosomal escape of R9-formulated mRNA, resulting in protein expression, demonstrating that both vectors enter cells along the same route. In addition, cells were transfected with a galectin-9-mCherry fusion protein to detect endosomal rupture. Remarkably, despite endosomal release, mRNA remained confined to punctate structures, identifying mRNA liberation as a further bottleneck. In summary, co-transfection offers a rapid means to identify bottlenecks in cytosolic mRNA delivery, supporting the rational design and optimization of intracellular mRNA delivery systems.

Graphene oxide-functionalized molecular beacon for real-time interference-free detection of Ki-67 mRNA in living cells

Molecular beacons (MBs) based on hairpin-shaped oligonucleotides are captivating owing to their capability to enable effective real-time detection of cytosolic mRNA in living cells. However, DNase in the nucleus and lysosome could induce the degradation of oligonucleotides in MBs, leading to the generation of false-positive signals. Herein, a graphene oxide (GO) nanosheet was applied as a nanocarrier for MBs to greatly enhance the anti-interference of the easily designed nanoprobe. Advantageously, the absorption capacity of GO for MBs increased with the decrease in pH values, providing the MB-GO nanoprobe with the ability to detect the expression of cytosolic Ki-67 mRNA without interference from DNase Ⅱ in lysosomes. Moreover, the size of GO nanosheets was considerably higher than that of the nuclear pore complex (NPC), which prevented nanoprobes from transition through the NPCs, thereby avoiding the generation of false-positive signals in the nucleus. Altogether, the present work affords a convenient approach for the successful detection of Ki-67 mRNA expression in the cytosol without interference from DNase Ⅰ/Ⅱ in the nucleus/lysosome, which may be potentially further applied for the detection of other cytosolic RNAs.

Pivotal role of the endoplasmic reticulum stress-related XBP1s/miR-22/SIRT1 axis in acute myeloid leukemia apoptosis and response to chemotherapy

Malignant growth relies on rapid protein synthesis frequently leading to endoplasmic reticulum (ER) overload and accumulation of unfolded or misfolded protein in this cellular compartment. In the ER, protein homeostasis is finely regulated by a mechanism called the unfolded protein response (UPR), involving the activation of signalization pathways mediated by three transmembrane proteins, namely PERK, IRE1 and ATF6. IRE1 endoribonuclease activation leads in particular to the splicing of the cytosolic mRNA encoding the key UPR-specific transcription factor XBP1s. Our study shows that sustained activation of XBP1s expression in acute myeloid leukemia (AML) cells induces apoptosis in vitro and in vivo, whereas a moderate XBP1s expression sensitizes cells to chemotherapeutic treatments. ChIP-seq experiments identified specific XBP1s target genes including the MIR22HG lncRNA, the precursor transcript of microRNA-22-3p. miR-22-3p upregulation by XBP1s or forced expression of miR-22 significantly decreases cell’s viability and sensitizes leukemic cells to chemotherapy. We found that miR-22-3p intracellular effects result at least partially from the targeting of the mRNA encoding the deacetylase sirtuin-1 (SIRT1), a well-established pro-survival factor. Therefore, this novel XBP1s/miR-22/SIRT1 axis identified could play a pivotal role in the proliferation and chemotherapeutic response of leukemic cells.

Nuclear export is a limiting factor in eukaryotic mRNA metabolism.

The eukaryotic mRNA life cycle includes transcription, nuclear mRNA export and degradation. To quantify all these processes simultaneously, we perform thiol-linked alkylation after metabolic labeling of RNA with 4-thiouridine (4sU), followed by sequencing of RNA (SLAM-seq) in the nuclear and cytosolic compartments of human cancer cells. We develop a model that reliably quantifies mRNA-specific synthesis, nuclear export, and nuclear and cytosolic degradation rates on a genome-wide scale. We find that nuclear degradation of polyadenylated mRNA is negligible and nuclear mRNA export is slow, while cytosolic mRNA degradation is comparatively fast. Consequently, an mRNA molecule generally spends most of its life in the nucleus. We also observe large differences in the nuclear export rates of different 3'UTR transcript isoforms. Furthermore, we identify genes whose expression is abruptly induced upon metabolic labeling. These transcripts are exported substantially faster than average mRNAs, suggesting the existence of alternative export pathways. Our results highlight nuclear mRNA export as a limiting factor in mRNA metabolism and gene regulation.

Enhancing Endosomal Escape and Gene Regulation Activity for Spherical Nucleic Acids.

The therapeutic potential of small interfering RNAs (siRNAs) is limited by their poor stability and low cellular uptake. When formulated as spherical nucleic acids (SNAs), siRNAs are resistant to nuclease degradation and enter cells without transfection agents with enhanced activity compared to their linear counterparts; however, the gene silencing activity of SNAs is limited by endosomal entrapment, a problem that impacts many siRNA-based nanoparticle constructs. To increase cytosolic delivery, SNAs are formulated using calcium chloride (CaCl2 ) instead of the conventionally used sodium chloride (NaCl). The divalent calcium (Ca2+ ) ions remain associated with the multivalent SNA and have a higher affinity for SNAs compared to their linear counterparts. Importantly, confocal microscopy studies show a 22% decrease in the accumulation of CaCl2 -salted SNAs within the late endosomes compared to NaCl-salted SNAs, indicating increased cytosolic delivery. Consistent with this finding, CaCl2 -salted SNAs comprised of siRNA and antisense DNA all exhibit enhanced gene silencing activity (up to 20-fold), compared to NaCl-salted SNAs regardless of sequence or cell line (U87-MG and SK-OV-3) studied. Moreover, CaCl2 -salted SNA-based forced intercalation probes show improved cytosolic mRNA detection.

Deciphering Structural Determinants Distinguishing Active from Inactive Cell-Penetrating Peptides for Cytosolic mRNA Delivery.

The formation of noncovalent complexes by mixing of positively charged polymers with negatively charged oligonucleotides (ONs) is a widely explored concept in nanomedicine to achieve cellular delivery of ONs. Uptake of ON complexes occurs through endocytosis, which then requires release of ON from endosomes. As one type of polymer, cell-penetrating peptides (CPPs) are being used which are peptides of about 8-30 amino acids in length. However, only a few CPPs yield effective cytosolic ON delivery and activity. Several strategies have been devised to increase cellular uptake and enhance endosomal release, among which an increase of osmotic pressure through the so-called proton sponge effect, disruption of membrane integrity through membrane activity, and disulfide-mediated polymerization. Here, we address the relevance of these concepts for mRNA delivery by incorporating structural features into the human lactoferrin-derived CPP, which shows uptake but not delivery. The incorporation of histidines was explored to address osmotic pressure and structural motifs of the delivery-active CPP PepFect14 (PF14) to address membrane disturbance, and finally, the impact of polymerization was explored. Whereas oligomerization increased the stability of polyplexes against heparin-induced decomplexation, neither this approach nor the incorporation of histidine residues to promote a proton-sponge effect yielded activity. Also, the replacement of arginine residues with lysine or ornithine residues, as in PF14, was without effect, even though all polyplexes showed cellular uptake. Ultimately, sufficient activity could only be achieved by transferring amphipathic sequence motifs from PF14 into the hLF context with some benefit of oligomerization demonstrating overarching principles of delivery for CPPs, lipid nanoparticles, and other types of delivery polymers.

Ssu72 phosphatase is essential for thermogenic adaptation by regulating cytosolic translation

Brown adipose tissue (BAT) plays a pivotal role in maintaining body temperature and energy homeostasis. BAT dysfunction is associated with impaired metabolic health. Here, we show that Ssu72 phosphatase is essential for mRNA translation of genes required for thermogenesis in BAT. Ssu72 is found to be highly expressed in BAT among adipose tissue depots, and the expression level of Ssu72 is increased upon acute cold exposure. Mice lacking adipocyte Ssu72 exhibit cold intolerance during acute cold exposure. Mechanistically, Ssu72 deficiency alters cytosolic mRNA translation program through hyperphosphorylation of eIF2α and reduces translation of mitochondrial oxidative phosphorylation (OXPHOS) subunits, resulting in mitochondrial dysfunction and defective thermogenesis in BAT. In addition, metabolic dysfunction in Ssu72-deficient BAT returns to almost normal after restoring Ssu72 expression. In summary, our findings demonstrate that cold-responsive Ssu72 phosphatase is involved in cytosolic translation of key thermogenic effectors via dephosphorylation of eIF2α in brown adipocytes, providing insights into metabolic benefits of Ssu72.

Targeting Mitochondrial Protein Expression as a Future Approach for Cancer Therapy.

Extensive metabolic remodeling is a fundamental feature of cancer cells. Although early reports attributed such remodeling to a loss of mitochondrial functions, it is now clear that mitochondria play central roles in cancer development and progression, from energy production to synthesis of macromolecules, from redox modulation to regulation of cell death. Biosynthetic pathways are also heavily affected by the metabolic rewiring, with protein synthesis dysregulation at the hearth of cellular transformation. Accumulating evidence in multiple organisms shows that the metabolic functions of mitochondria are tightly connected to protein synthesis, being assembly and activity of respiratory complexes highly dependent on de novo synthesis of their components. In turn, protein synthesis within the organelle is tightly connected with the cytosolic process. This implies an entire network of interactions and fine-tuned regulations that build up a completely under-estimated level of complexity. We are now only preliminarily beginning to reconstitute such regulatory level in human cells, and to perceive its role in diseases. Indeed, disruption or alterations of these connections trigger conditions of proteotoxic and energetic stress that could be potentially exploited for therapeutic purposes. In this review, we summarize the available literature on the coordinated regulation of mitochondrial and cytosolic mRNA translation, and their effects on the integrity of the mitochondrial proteome and functions. Finally, we highlight the potential held by this topic for future research directions and for the development of innovative therapeutic approaches.

Rapid Decay of Host Basal mRNAs During SARS-CoV-2 Infection Perturbs Host Antiviral mRNA Biogenesis and Export

A key feature of the mammalian antiviral response is the transcriptional induction of interferon (IFN) genes, which encode for secreted proteins that prime the antiviral response to limit viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFNs in patient serum despite elevated levels of IFN-encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Herein, we show SARS-CoV-2 infection limits type I and type III IFN biogenesis by preventing the release of mRNA from their sites of transcription and/or triggering their nuclear degradation. Additionally, SARS-CoV-2 infection inhibits nuclear-cytoplasmic transport of IFN mRNAs as a consequence of widespread cytosolic mRNA degradation mediated by both the host antiviral endoribonuclease, RNase L, and by the SARS-CoV-2 protein, Nsp1. Thus, inhibition of host and/or viral Nsp1-mediated mRNA decay, as well as IFN treatments, may reduce viral-associated pathogenesis by promoting the innate immune response.Funding Information: Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number F32AI145112 (J.M.B), funds from HHMI (Roy Parker), and support provided by the Office of the Vice President for Research and the Dept. of Microbiology, Immunology and Pathology at Colorado State University (Rushika Perera).Declaration of Interests: Roy Parker is a founder and consultant of Faze Medicines.

Protective Effects of Low-Dose Alcohol against Acute Stress-Induced Renal Injury in Rats: Involvement of CYP4A/20-HETE and LTB4/BLT1 Pathways.

Low-dose alcohol possesses multiple bioactivities. Accordingly, we investigated the protective effect and related molecular mechanism of low-dose alcohol against acute stress- (AS-) induced renal injury. Herein, exhaustive swimming for 15 min combined with restraint stress for 3 h was performed to establish a rat acute stress model, which was verified by an open field test. Evaluation of renal function (blood creatinine and urea nitrogen), urine test (urine leukocyte esterase and urine occult blood), renal histopathology, oxidative stress, inflammation, and apoptosis was performed. The key indicators of the cytochrome P450 (CYP) 4A1/20-hydroxystilbenetetraenoic acid (20-HETE) pathway, cyclooxygenase (COX)/prostaglandin E2 (PGE2) pathway, and leukotriene B4 (LTB4)/leukotriene B4 receptor 1 (BLT1) pathway were measured by real-time PCR and ELISA. We found that low-dose alcohol (0.05 g/kg, i.p.) ameliorated AS-induced renal dysfunction and histological damage. Low-dose alcohol also attenuated AS-induced oxidative stress and inflammation, presenting as reduced malondialdehyde and hydrogen peroxide formation, increased superoxide dismutase and glutathione activity, and decreased myeloperoxidase, interleukin-6, interleukin-1β, and monocyte chemoattractant protein-1 levels (P < 0.05). Moreover, low-dose alcohol alleviated AS-induced apoptosis by downregulating Bax and cleaved caspase 3 protein expression and reduced numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick-end label-positive cells (P < 0.01). Correlation analysis indicated that 20-HETE was strongly correlated with oxidative stress, while LTB4 was strongly correlated with inflammation. Low-dose alcohol inhibited AS-induced increases in CYP4A1, CYP4A2, CYP4A3, CYP4A8, and BLT1 mRNA levels and LTB4 and 20-HETE content (P < 0.01). Interestingly, low-dose alcohol had no effect on COX1 or COX2 mRNA expression or the concentration of PGE2. Furthermore, low-dose alcohol reduced calcium-independent phospholipase A2 mRNA expression, but did not affect secreted phospholipase A2 or cytosolic phospholipase A2 mRNA expression. Together, these results indicate that low-dose alcohol ameliorated AS-induced renal injury by inhibiting CYP4A/20-HETE and LTB4/BLT1 pathways, but not the COX/PGE2 pathway.

Splicing HAC1/XBP1 mRNAs in Cytoplasm: The Non-Conventional mRNA Splicing Reaction in the Unfolded Protein Response

The majority of the secretory and transmembrane proteins are folded in the endoplasmic reticulum (ER). When unfolded proteins accumulate in the ER, a collective of signalling pathways, termed the unfolded protein response (UPR), are activated to restore the ER protein folding homeostasis. The most evolutionarily conserved branch of UPR is mediated by the kinase/endoribonuclease Ire1. Ire1 mediates a cytosolic non-conventional mRNA splicing reaction of HAC1 mRNA in yeast and XBP1 mRNA in mammalian cells. The spliced HAC1/XBP1 mRNA is translated and produces a functional transcription factor, which initiates a transcriptional response to restore the protein folding homeostasis. The HAC1/XBP1 mRNA splicing reaction is biochemically distinct from the ones that are catalyzed by the spliceosome. Here, we review recent studies that provide a mechanistic understanding of the non-conventional mRNA splicing reaction.

Light-Dependent Activation of the GCN2 Kinase Under Cold and Salt Stress Is Mediated by the Photosynthetic Status of the Chloroplast.

Regulation of cytosolic mRNA translation is a key node for rapid adaptation to environmental stress conditions. In yeast and animals, phosphorylation of the α-subunit of eukaryotic translation initiation factor eIF2 is the most thoroughly characterized event for regulating global translation under stress. In plants, the GCN2 kinase (General Control Nonderepressible-2) is the only known kinase for eIF2α. GCN2 is activated under a variety of stresses including reactive oxygen species (ROS). Here, we provide new evidence that the GCN2 kinase in Arabidopsis is also activated rapidly and in a light-dependent manner by cold and salt treatments. These treatments alone did not repress global mRNA ribosome loading in a major way. The activation of GCN2 was accompanied by a more oxidative environment and was attenuated by inhibitors of photosynthetic electron transport, suggesting that it is gated by the redox poise or the reactive oxygen status of the chloroplast. In keeping with these results, gcn2 mutant seedlings were more sensitive than wild type to both cold and salt in a root elongation assay. These data suggest that cold and salt stress may both affect the status of the cytosolic translation apparatus via the conserved GCN2-eIF2α module. The potential role of the GCN2 kinase pathway in the global repression of translation under abiotic stress is discussed.

Light Activates the Translational Regulatory Kinase GCN2 via Reactive Oxygen Species Emanating from the Chloroplast.

Cytosolic mRNA translation is subject to global and mRNA-specific controls. Phosphorylation of the translation initiation factor eIF2α anchors a reversible regulatory switch that represses cytosolic translation globally. The stress-responsive GCN2 kinase is the only known kinase for eIF2α serine 56 in Arabidopsis (Arabidopsis thaliana). Here, we show that conditions that generate reactive oxygen species (ROS) in the chloroplast, including dark-light transitions, high light, and the herbicide methyl viologen, rapidly activated GCN2 kinase, whereas mitochondrial and endoplasmic reticulum stress did not. GCN2 activation was light dependent and mitigated by photosynthesis inhibitors and ROS quenchers. Accordingly, the seedling growth of multiple Arabidopsis gcn2 mutants was retarded under excess light conditions, implicating the GCN2-eIF2α pathway in responses to light and associated ROS. Once activated, GCN2 kinase preferentially suppressed the ribosome loading of mRNAs for functions such as mitochondrial ATP synthesis, the chloroplast thylakoids, vesicle trafficking, and translation. The gcn2 mutant overaccumulated transcripts functionally related to abiotic stress, including oxidative stress, as well as innate immune responses. Accordingly, gcn2 displayed defects in immune priming by the fungal elicitor, chitin. Therefore, we provide evidence that reactive oxygen species produced by the photosynthetic apparatus help activate the highly conserved GCN2 kinase, leading to eIF2α phosphorylation and thus affecting the status of the cytosolic protein synthesis apparatus.

The Deubiquitinating Enzyme Ubiquitin-Specific Peptidase 11 Potentiates TGF-β Signaling in CD4+ T Cells to Facilitate Foxp3+ Regulatory T and TH17 Cell Differentiation.

Foxp3+ regulatory T (TREG) cells are central mediators in the control of peripheral immune responses. Genome-wide transcriptional profiles show canonical signatures for Foxp3+ TREG cells, distinguishing them from Foxp3- effector T (TEFF) cells. We previously uncovered distinct mRNA translational signatures differentiating CD4+ TEFF and TREG cells through parallel measurements of cytosolic (global) and polysome-associated (translationally enhanced) mRNA levels in both subsets. We show that the mRNA encoding for the ubiquitin-specific peptidase 11 (USP11), a known modulator of TGF-β signaling, was preferentially translated in TCR-activated TREG cells compared with conventional, murine CD4+ T cells. TGF-β is a key cytokine driving the induction and maintenance of Foxp3 expression in T cells. We hypothesized that differential translation of USP11 mRNA endows TREG cells with an advantage to respond to TGF-β signals. In an in vivo mouse model promoting TREG cells plasticity, we found that USP11 protein was expressed at elevated levels in stable TREG cells, whereas ectopic USP11 expression enhanced the suppressive capacity and lineage commitment of these cells in vitro and in vivo. USP11 overexpression in TEFF cells enhanced the activation of the TGF-β pathway and promoted TREG or TH17, but not Th1, cell differentiation in vitro and in vivo, an effect abrogated by USP11 gene silencing or the inhibition of enzymatic activity. Thus, USP11 potentiates TGF-β signaling in both TREG and TEFF cells, in turn driving increased suppressive function and lineage commitment in thymic-derived TREG cells and potentiating the TGF-β-dependent differentiation of TEFF cells to peripherally induced TREG and TH17 cells.

Heterogeneous glutamatergic receptor mRNA expression across phrenic motor neurons in rats.

The diaphragm muscle comprises various types of motor units that are recruited in an orderly fashion governed by the intrinsic electrophysiological properties (membrane capacitance as a function of somal surface area) of phrenic motor neurons (PhMNs). Glutamate is the main excitatory neurotransmitter at PhMNs and acts primarily via fast acting AMPA and N-methyl-D-aspartic acid (NMDA) receptors. Differences in receptor expression may also contribute to motor unit recruitment order. We used single cell, multiplex fluorescence in situ hybridization to determine glutamatergic receptor mRNA expression across PhMNs based on their somal surface area. In adult male and female rats (n=9) PhMNs were retrogradely labeled for analyses (n=453 neurons). Differences in the total number and density of mRNA transcripts were evident across PhMNs grouped into tertiles according to somal surface area. A~25% higher density of AMPA (Gria2) and NMDA (Grin1) mRNA expression was evident in PhMNs in the lower tertile compared to the upper tertile. These smaller PhMNs likely comprise type S motor units that are recruited first to accomplish lower force, ventilatory behaviors. In contrast, larger PhMNs with lower volume densities of AMPA and NMDA mRNA expression presumably comprise type FInt and FF motor units that are recruited during higher force, expulsive behaviors. Furthermore, there was a significantly higher cytosolic NMDA mRNA expression in small PhMNs suggesting a more important role for NMDA-mediated glutamatergic neurotransmission at smaller PhMNs. These results are consistent with the observed order of motor unit recruitment and suggest a role for glutamatergic receptors in support of this orderly recruitment. Cover Image for this issue: doi: 10.1111/jnc.14747.

The exonuclease Xrn1 activates transcription and translation of mRNAs encoding membrane proteins

The highly conserved 5’–3’ exonuclease Xrn1 regulates gene expression in eukaryotes by coupling nuclear DNA transcription to cytosolic mRNA decay. By integrating transcriptome-wide analyses of translation with biochemical and functional studies, we demonstrate an unanticipated regulatory role of Xrn1 in protein synthesis. Xrn1 promotes translation of a specific group of transcripts encoding membrane proteins. Xrn1-dependence for translation is linked to poor structural RNA contexts for translation initiation, is mediated by interactions with components of the translation initiation machinery and correlates with an Xrn1-dependence for mRNA localization at the endoplasmic reticulum, the translation compartment of membrane proteins. Importantly, for this group of mRNAs, Xrn1 stimulates transcription, mRNA translation and decay. Our results uncover a crosstalk between the three major stages of gene expression coordinated by Xrn1 to maintain appropriate levels of membrane proteins.

Porcine reproductive and respiratory syndrome virus (PRRSV)-induced stress granules are associated with viral replication complexes and suppression of host translation

Stress granules (SGs) are dynamic sites of cytosolic mRNA storage that are formed in response to stress conditions, including viral infection. SGs have been implicated in regulating several aspects of the host immune response to various pathogens. Porcine reproductive and respiratory syndrome virus (PRRSV), an economically-important global swine pathogen, reportedly induced SGs during replication, although the underlying mechanisms are poorly defined. In this study, we delineated the molecular mechanisms regulating the SG response to PRRSV infection. Using confocal microscopy, we first demonstrated that infection with PRRSV strain VR2385 induces an accumulation of the SG markers G3BP1, G3BP2, TIAR, eIF3b, and USP10 as well as mRNAs into punctate structures in the cytoplasm of infected host cells. Subsequently, we demonstrated that the PRRSV-induced SGs were in close proximity to viral replication complexes (VRCs) and processing bodies (P-bodies), and that SG formation was coordinated with inhibition of host cellular translation. Treatment of infected cells with cycloheximide disrupted the PRRSV-induced SGs. Furthermore, impairment of SG assembly by the shRNA-mediated knockdown of G3BP1, G3BP2 and USP10 did not affect viral replication. Collectively, these results demonstrate that PRRSV infection induces formation of SGs associated with VRCs, which is coordinated with the suppression of host cell protein synthesis. This is the first study to extensively characterize the formation and underlying mechanism of bona fide SGs during PRRSV infection. Our findings have important implications in understanding the mechanism of PRRSV-host interactions.

The m1A landscape on cytosolic and mitochondrial mRNA at single-base resolution.

Modifications on mRNA offer the potential of regulating mRNA fate post-transcriptionally. Recent studies suggested the widespread presence of N1-methyladenosine (m1A), which disrupts Watson-Crick base pairing, at internal sites of mRNAs. These studies lacked the resolution of identifying individual modified bases, and did not identify specific sequence motifs undergoing the modification or an enzymatic machinery catalysing them, rendering it challenging to validate and functionally characterize putative sites. Here we develop an approach that allows the transcriptome-wide mapping of m1A at single-nucleotide resolution. Within the cytosol, m1A is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. We identify a single m1A site in the mitochondrial ND5 mRNA, catalysed by TRMT10C, with methylation levels that are highly tissue specific and tightly developmentally controlled. m1A leads to translational repression, probably through a mechanism involving ribosomal scanning or translation. Our findings suggest that m1A on mRNA, probably because of its disruptive impact on base pairing, leads to translational repression, and is generally avoided by cells, while revealing one case in mitochondria where tight spatiotemporal control over m1A levels was adopted as a potential means of post-transcriptional regulation.

P 76 Maturation deficits in striatal interneurons in a phenotypic model of dystonia

GABAergic disinhibition has been suggested to play a critical role in the pathophysiology of several basal ganglia disorders, including dystonia, a common incurable movement disorder characterized by involuntary muscle contractions resulting in abnormal movements and/or postures. Previous studies have shown a deficit of striatal GABAergic interneurons (IN) in the dtsz mutant hamster, one of the few phenotypic animal models of dystonia. The resulting lack of inhibition of striatal projection neurons is likely involved in development of dystonic episodes which is corroborated by antidystonic effects of GABA potentiating drugs in the animal model and in dystonia patients. However, mechanisms underlying this deficit in striatal IN are largely unknown. In the present study, we investigated the migration and maturation of striatal IN during postnatal development (18 days of age) and at age of highest severity of dystonia (33 days of age) in the dtsz hamster by immunohistochemistry and real time PCR. In line with previous findings, the density of GAD67 positive IN and the level of parvalbumin mRNA, a marker for fast spiking GABAergic IN, were lower in mutant hamsters than in controls. However, an unaltered density of Nkx2.1 labeled cells and Nkx2.1 mRNA level indicated that the migration of GABAergic IN into the striatum was not retarded. KCC2 cation/chloride transporter and cytosolic carboanhydrase VII mRNA expression, used as markers for maturation of striatal GABAergic signaling, as well as expression of BDNF were unaltered. However, we found a reduced number of IN expressing the alpha1 subunit of the GABAA-receptor (37.5%) in dtsz hamsters at an age of 33 days, but not after spontaneous remission of dystonia at an age of 90 days. Since IN shift expression from alpha2 to alpha1 subunits during postnatal maturation, this result together with a decreased parvalbumin mRNA expression suggest a delayed maturation of striatal GABAergic IN in this animal model, which might underlie abnormal neuronal activity and striatal plasticity. The potential link between alterations in GABAA receptor subunits and GABAergic disinhibition in dystonia deserves further attention in research on the pathophysiology and therapeutic targets.

Attenuated lipotoxicity and apoptosis is linked to exogenous and endogenous augmenter of liver regeneration by different pathways.

Nonalcoholic fatty liver disease (NAFLD) covers a spectrum from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. Free fatty acids (FFA) induce steatosis and lipo-toxicity and correlate with severity of NAFLD. In this study we aimed to investigate the role of exogenous and endogenous ALR (augmenter of liver regeneration) for FFA induced ER (endoplasmatic reticulum) -stress and lipoapoptosis. Primary human hepatocytes or hepatoma cells either treated with recombinant human ALR (rhALR, 15kDa) or expressing short form ALR (sfALR, 15kDa) were incubated with palmitic acid (PA) and analyzed for lipo-toxicity, -apoptosis, activation of ER-stress response pathways, triacylglycerides (TAG), mRNA and protein expression of lipid metabolizing genes. Both, exogenous rhALR and cytosolic sfALR reduced PA induced caspase 3 activity and Bax protein expression and therefore lipotoxicity. Endogenous sfALR but not rhALR treatment lowered TAG levels, diminished activation of ER-stress mediators C-Jun N-terminal kinase (JNK), X-box binding protein-1 (XBP1) and proapoptotic transcription factor C/EBP-homologous protein (CHOP), and reduced death receptor 5 protein expression. Cellular ALR exerts its lipid lowering and anti-apoptotic actions by enhancing FABP1, which binds toxic FFA, increasing mitochondrial β-oxidation by elevating the mitochondrial FFA transporter CPT1α, and decreasing ELOVL6, which delivers toxic FFA metabolites. We found reduced hepatic mRNA levels of ALR in a high fat diet mouse model, and of ALR and FOXA2, a transcription factor inducing ALR expression, in human steatotic as well as NASH liver samples, which may explain increased lipid deposition and reduced β-oxidation in NASH patients. Present study shows that exogenous and endogenous ALR reduce PA induced lipoapoptosis. Furthermore, cytosolic sfALR changes mRNA and protein expression of genes regulating lipid metabolism, reduces ER-stress finally impeding progression of NASH.