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Ribosome-associated mRNA quality control mechanisms ensure the fidelity of protein translation1,2. Although these mechanisms have been extensively studied in yeast, little is known about their role in mammalian tissues, despite emerging evidence that stem cell fate is controlled by translational mechanisms3,4. One evolutionarily conserved component of the quality control machinery, Dom34 (in higher eukaryotes known as Pelota (Pelo)), rescues stalled ribosomes5. Here we show that Pelo is required for mammalian epidermal homeostasis. Conditional deletion of Pelo in mouse epidermal stem cells that express Lrig1 results in hyperproliferation and abnormal differentiation of these cells. By contrast, deletion of Pelo in Lgr5-expressing stem cells has no effect and deletion in Lgr6-expressing stem cells induces only a mild phenotype. Loss of Pelo results in accumulation of short ribosome footprints and global upregulation of translation, rather than affecting the expression of specific genes. Translational inhibition by rapamycin-mediated downregulation of mTOR (mechanistic target of rapamycin kinase) rescues the epidermal phenotype. Our study reveals that the ribosome-rescue machinery is important for mammalian tissue homeostasis and that it has specific effects on different stem cell populations. Loss of the ribosome-rescue factor Pelo in a subset of mouse epidermal stem cells results in hyperproliferation and altered differentiation of these cells.

BACKGROUND Ribosomopathies are a heterogeneous group of human disorders that are in some cases known, and in other cases suspected, to result from ribosome dysfunction. This group broadly comprises two categories: (i) disorders caused by single-copy mutations in specific ribosomal proteins, and (ii) disorders associated with defects in ribosome biogenesis factors. The phenotypic patterns among different ribosomopathies in both categories are divergent but do tend to share some overlapping features. These include effects on bone marrow–derived cell lineages and skeletal tissues. These common tissue specificities of the different ribosomopathies are challenging to reconcile with the ubiquitous requirement for ribosomes in all cells. ADVANCES Several models have been advanced to explain how the dysfunction of the protein synthesis machinery is so variably expressed at the phenotypic level. An increasing number of studies in models of distinct ribosomopathies have revealed that “ribosomal stress” signals converge on the p53 signaling pathway in affected cells and tissues. In these models, a key consequence of ribosome dysfunction is cell- and tissue type–restricted activation of p53-dependent cell cycle arrest and apoptosis. However, the specific translational events upstream of p53 activation that lead to some cells being affected, with others being spared, are unknown. We review evidence relating to two hypotheses that have been proposed to explain such tissue-specific effects of ribosome dysfunction. One hypothesis is that ribosome dysfunction (or deficiency) can affect global and messenger RNA (mRNA)–specific translational control, and that certain specific cells or tissues may be more vulnerable to ribosome dysfunction. A critical feature of this view is that mRNAs are variably dependent on cellular ribosome concentration, with more poorly initiated mRNAs being typically more sensitive to perturbations in ribosome concentration or function. Several recent studies suggest that the sensitivities of certain tissues to ribosomopathies, including reticuloctyes and platelets, may be related to differences in core processes of translation in these cells related to ribosome recycling and rescue. Perturbations in these processes will have a great impact on ribosome homeostasis and thus on broad aspects of gene expression. Related studies in the brain have revealed disease phenotypes in genetic backgrounds with deficiencies in ribosome rescue and in a specific neuronal transfer RNA. Together, these molecular insights provide a new perspective on ribosomopathies and their tissue specificities, while also raising a number of important questions to pursue. The other hypothesis is that ribosomes from different tissues may have different compositions of core or more loosely associated proteins and posttranslational modifications, and that this heterogeneity could be critical to the translation of specific mRNAs. This is referred to as the “specialized” ribosome hypothesis. We argue that while such heterogeneity in ribosome composition likely exists in different tissues, such complex explanations may not be needed to explain the differences in gene expression that result from losses of specific ribosomal proteins. It is simpler to hypothesize that differences in mRNA-specific rates of initiation and changes in ribosome concentration can adequately explain much (if not all) of the diversity of gene expression changes in different tissues as a result of ribosomal mutations. OUTLOOK A cohesive mechanistic model connecting dysfunction of the ribosome to the specific phenotypic consequences observed in ribosomopathies remains a challenging goal. For example, it is inherently difficult to assess the function of particular ribosomes in a cell, and thus to differentiate among various models to explain the impacts of ribosome deficiencies on gene expression. Further biochemical analyses of the fundamental processes underlying the cellular response to protein synthesis dysfunction, refinements in cellular and animal models of ribosomopathies, and greater dialogue between clinical and basic scientists will all be important to extend our current understanding.

Atypical antipsychotic medications, such as risperidone, aripiprazole, and olanzapine, have utility in treating motor tics, particularly in Tourette syndrome. In rare cases, atypical antipsychotic medications have been associated with adult-onset motor tics. Such adverse drug reactions (ADRs) have been documented in response to quetiapine, aripiprazole, and amisulpride. Here, we report, to our knowledge, the first case of adult onset motor tics related to olanzapine administration.

Normal termination of signaling is essential to reset signaling cascades, especially those such as phototransduction that are turned on and off with great rapidity. Genetic approaches inDrosophilaled to the identification of several proteins required for termination, including protein kinase C (PKC), NINAC (neither inactivation nor afterpotential C) p174, which consists of fused protein kinase and myosin domains, and a PDZ (postsynaptic density-95/Discs Large/zona occludens-1) scaffold protein, INAD (inactivation no afterpotential D). Here, we describe a mutation affecting a poorly characterized but evolutionarily conserved protein, Retinophilin (Retin), which is expressed primarily in the phototransducing compartment of photoreceptor cells, the rhabdomeres. Retin and NINAC formed a complex and were mutually dependent on each other for expression. Loss ofretinresulted in an age-dependent impairment in termination of phototransduction. Mutations that affect termination of the photoresponse typically lead to a reduction in levels of the major rhodopsin (Rh1) to attenuate signaling. Consistent with the slower termination inretin1, the mutant photoreceptor cells exhibited increased endocytosis of Rh1 and a decline in Rh1 protein. The slower termination inretin1was a consequence of a cascade of defects, which began with the reduction in NINAC p174 levels. The diminished p174 concentration caused a decrease in INAD. Because PKC requires interaction with INAD for protein stability, this leads to reduction in PKC levels. The decline in PKC was age dependent and paralleled the onset of the termination phenotype inretin1mutant flies. We conclude that the slower termination of the photoresponse inretin1resulted from a requirement for the Retin/NINAC complex for stability of INAD and PKC.

The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1(V432P)) results in a large whole cell current. Thus, it remains unknown whether the large TRPML1(V432P)-mediated current results from an increased surface expression (trafficking), elevated channel activity (gating), or both. Here we performed systemic Pro substitutions in a region previously implicated in the gating of various 6 transmembrane cation channels. We found that several Pro substitutions displayed gain-of-function (GOF) constitutive activities at both the plasma membrane (PM) and endolysosomal membranes. Although wild-type TRPML1 and non-GOF Pro substitutions localized exclusively in LEL and were barely detectable in the PM, the GOF mutations with high constitutive activities were not restricted to LEL compartments, and most significantly, exhibited significant surface expression. Because lysosomal exocytosis is Ca(2+)-dependent, constitutive Ca(2+) permeability due to Pro substitutions may have resulted in stimulus-independent intralysosomal Ca(2+) release, hence the surface expression and whole cell current of TRPML1. Indeed, surface staining of lysosome-associated membrane protein-1 (Lamp-1) was dramatically increased in cells expressing GOF TRPML1 channels. We conclude that TRPML1 is an inwardly rectifying, proton-impermeable, Ca(2+) and Fe(2+)/Mn(2+) dually permeable cation channel that may be gated by unidentified cellular mechanisms through a conformational change in the cytoplasmic face of the transmembrane 5 (TM5). Furthermore, activation of TRPML1 in LEL may lead to the appearance of TRPML1 proteins at the PM.

Meeting abstracts Transposable element (TE) expression is generally silent in somatic tissues, due to significant genomic methylation and other redundant methods of silencing. Cancer tissues, however, exhibit a marked decrease in methylation throughout the genome, which can result in de-repression

SummaryDownregulation of the miR-143/145 microRNA (miRNA) cluster has been repeatedly reported in colon cancer and other epithelial tumors. In addition, overexpression of these miRNAs inhibits tumorigenesis, leading to broad consensus that they function as cell-autonomous epithelial tumor suppressors. We generated mice with deletion of miR-143/145 to investigate the functions of these miRNAs in intestinal physiology and disease in vivo. Although intestinal development proceeded normally in the absence of these miRNAs, epithelial regeneration after injury was dramatically impaired. Surprisingly, we found that miR-143/145 are expressed and function exclusively within the mesenchymal compartment of intestine. Defective epithelial regeneration in miR-143/145-deficient mice resulted from the dysfunction of smooth muscle and myofibroblasts and was associated with derepression of the miR-143 target Igfbp5, which impaired IGF signaling after epithelial injury. These results provide important insights into the regulation of epithelial wound healing and argue against a cell-autonomous tumor suppressor role for miR-143/145 in colon cancer.

SummaryBacterial and viral mRNAs are often polycistronic. Akin to alternative splicing, alternative translation of polycistronic messages is a mechanism to generate protein diversity and regulate gene function. Although a few examples exist, the use of polycistronic messages in mammalian cells is not widely appreciated. Here we report an example of alternative translation as a means of regulating innate immune signaling. MAVS, a regulator of antiviral innate immunity, is expressed from a bicistronic mRNA encoding a second protein, miniMAVS. This truncated variant interferes with interferon production induced by full-length MAVS, whereas both proteins positively regulate cell death. To identify other polycistronic messages, we carried out genome-wide ribosomal profiling and identified a class of antiviral truncated variants. This study therefore reveals the existence of a functionally important bicistronic antiviral mRNA and suggests a widespread role for polycistronic mRNAs in the innate immune system.

Trace metals such as iron, copper, zinc, manganese, and cobalt are essential cofactors for many cellular enzymes. Extensive research on iron, the most abundant transition metal in biology, has contributed to an increased understanding of the molecular machinery involved in maintaining its homeostasis in mammalian peripheral tissues. However, the cellular and intercellular iron transport mechanisms in the central nervous system (CNS) are still poorly understood. Accumulating evidence suggests that impaired iron metabolism is an initial cause of neurodegeneration, and several common genetic and sporadic neurodegenerative disorders have been proposed to be associated with dysregulated CNS iron homeostasis. This review aims to provide a summary of the molecular mechanisms of brain iron transport. Our discussion is focused on iron transport across endothelial cells of the blood-brain barrier and within the neuro- and glial-vascular units of the brain, with the aim of revealing novel therapeutic targets for neurodegenerative and CNS disorders.

TRPML1 (mucolipin 1, also known as MCOLN1) is predicted to be an intracellular late endosomal and lysosomal ion channel protein that belongs to the mucolipin subfamily of transient receptor potential (TRP) proteins. Mutations in the human TRPML1 gene cause mucolipidosis type IV disease (ML4). ML4 patients have motor impairment, mental retardation, retinal degeneration and iron-deficiency anaemia. Because aberrant iron metabolism may cause neural and retinal degeneration, it may be a primary cause of ML4 phenotypes. In most mammalian cells, release of iron from endosomes and lysosomes after iron uptake by endocytosis of Fe(3+)-bound transferrin receptors, or after lysosomal degradation of ferritin-iron complexes and autophagic ingestion of iron-containing macromolecules, is the chief source of cellular iron. The divalent metal transporter protein DMT1 (also known as SLC11A2) is the only endosomal Fe(2+) transporter known at present and it is highly expressed in erythroid precursors. Genetic studies, however, suggest the existence of a DMT1-independent endosomal and lysosomal Fe(2+) transport protein. By measuring radiolabelled iron uptake, by monitoring the levels of cytosolic and intralysosomal iron and by directly patch-clamping the late endosomal and lysosomal membrane, here we show that TRPML1 functions as a Fe(2+) permeable channel in late endosomes and lysosomes. ML4 mutations are shown to impair the ability of TRPML1 to permeate Fe(2+) at varying degrees, which correlate well with the disease severity. A comparison of TRPML1(-/- )ML4 and control human skin fibroblasts showed a reduction in cytosolic Fe(2+) levels, an increase in intralysosomal Fe(2+) levels and an accumulation of lipofuscin-like molecules in TRPML1(-/-) cells. We propose that TRPML1 mediates a mechanism by which Fe(2+) is released from late endosomes and lysosomes. Our results indicate that impaired iron transport may contribute to both haematological and degenerative symptoms of ML4 patients.