mRNA regulation in the "C. elegans" germ line

The C. elegans germ line relies heavily on post-transcriptional regulation of gene expression but the scale of mRNA regulation in the germ line is still unknown. Germ cells initially divide mitotically, they then enter meiosis and finally differentiate into oocytes. Transcription ceases during oogenesis and does not get reactivated until the early embryo. The oocyte-to-embryo transition (OET) encompassing oocyte maturation, fertilization and early embryogenesis, therefore solely depends on maternal factors. Maternal mRNA storage describes the repression and stabilization of these factors until they are needed. At the four-cell stage, somatic blastomeres become dependent on zygotic transcription and at the same time a subgroup of maternal mRNAs (class II maternal mRNAs) gets specifically degraded. Many developmental decisions in the germ line are regulated by RNA binding proteins (RBPs). A crucial regulator is the STAR domain protein GLD-1, which is expressed in the central gonad. GLD-1 regulates many of the developmental decisions in the germ line and loss of GLD-1 prevents oogenesis and leads instead to the development of a proliferative tumor. GLD-1 binds a large number of mRNAs, and is known to repress the translation of various transcripts but the mechanism by which it does so is unknown. We found that translation initiation of many germline mRNAs is repressed, and that GLD-1 globally represses translation initiation of its targets. Importantly, we revealed an additional role of GLD-1 in stabilizing a large number of its bound mRNAs, suggesting that GLD-1 plays a central role in maternal mRNA storage. While we could not detect an interaction between GLD-1 and translation initiation factors, we observed that GLD-1 associates with components of a conserved germline RNP complex. These components include the polyA binding protein (PABP), Y-box proteins, the Sm-like protein CAR-1 and the DDX6 helicase CGH-1, which has recently been implicated in maternal mRNA protection. Interestingly we found that while CGH-1 does not influence the translational repression of investigated GLD-1 targets, CGH-1 and GLD-1 stabilize a common set of transcripts. Remarkably, these co-regulated messages nearly exclusively encode for mRNAs that are required for the oocyte-to-embryo transition. We therefore propose a two-step model where GLD-1 binding prevents translation initiation and primes many targets for CGH-1-dependent mRNA stabilization, ultimately leading to mRNA storage

Prions, prionoids and protein misfolding disorders

Prion diseases are progressive, incurable and fatal neurodegenerative conditions. The term ‘prion’ was first nominated to express the revolutionary concept that a protein could be infectious. We now know that prions consist of PrPSc, the pathological aggregated form of the cellular prion protein PrPC. Over the years, the term has been semantically broadened to describe aggregates irrespective of their infectivity, and the prion concept is now being applied, perhaps overenthusiastically, to all neurodegenerative diseases that involve protein aggregation. Indeed, recent studies suggest that prion diseases (PrDs) and protein misfolding disorders (PMDs) share some common disease mechanisms, which could have implications for potential treatments. Nevertheless, the transmissibility of bona fide prions is unique, and PrDs should be considered as distinct from other PMDs

Skeletal-Muscle Glutamine Synthase is Upregulated in Preclinical Prion Diseases

In prion diseases, aggregates of misfolded prion protein (PrP$^{Sc}$) accumulate not only in the brain but can also be found in various extraneural tissues. This raises the question whether prion-specific pathologies arise also in these tissues. Here we sequenced mRNA transcripts in skeletal muscle, spleen and blood of prion-inoculated mice at eight timepoints during disease progression. We detected consistent gene-expression changes in all three organs, with skeletal muscle showing the most uniform alterations during disease progression. The glutamate synthetase (GLUL) gene was monotonically upregulated in skeletal muscle of mice infected with three different scrapie prion strains (RML, ME7 and 22L) and in human sporadic Creutzfeldt-Jakob disease. GLUL dysregulation was accompanied by changes in glutamate/glutamine metabolism, leading to reduced glutamate levels in skeletal muscle. None of these changes were observed in skeletal muscle of humans with amyotrophic lateral sclerosis, Alzheimer’s disease, or dementia with Lewy bodies, suggesting that they are specific to prion diseases. Besides pointing to unrecognized metabolic implications of prion infections, these findings suggest that GLUL could represent an accessible biomarker of prion disease progression, particularly during the preclinical stages of disease, and might be useful for monitoring the efficacy of experimental antiprion therapies

Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes

The cold shock domain is one of the most highly conserved motifs between bacteria and higher eukaryotes. Y-box-binding proteins represent a subfamily of cold shock domain proteins with pleiotropic functions, ranging from transcription in the nucleus to translation in the cytoplasm. These proteins have been investigated in all major model organisms except Caenorhabditis elegans. In this study, we set out to fill this gap and present a functional characterization of CEYs, the C. elegans Y-box-binding proteins. We find that, similar to other organisms, CEYs are essential for proper gametogenesis. However, we also report a novel function of these proteins in the formation of large polysomes in the soma. In the absence of the somatic CEYs, polysomes are dramatically reduced with a simultaneous increase in monosomes and disomes, which, unexpectedly, has no obvious impact on animal biology. Because transcripts that are enriched in polysomes in wild-type animals tend to be less abundant in the absence of CEYs, our findings suggest that large polysomes might depend on transcript stabilization mediated by CEY protein

Genome-Wide Analysis of GLD-1–Mediated mRNA Regulation Suggests a Role in mRNA Storage

Translational repression is often accompanied by mRNA degradation. In contrast, many mRNAs in germ cells and neurons are “stored" in the cytoplasm in a repressed but stable form. Unlike repression, the stabilization of these mRNAs is surprisingly little understood. A key player in Caenorhabditis elegans germ cell development is the STAR domain protein GLD-1. By genome-wide analysis of mRNA regulation in the germ line, we observed that GLD-1 has a widespread role in repressing translation but, importantly, also in stabilizing a sub-population of its mRNA targets. Additionally, these mRNAs appear to be stabilized by the DDX6-like RNA helicase CGH-1, which is a conserved component of germ granules and processing bodies. Because many GLD-1 and CGH-1 stabilized mRNAs encode factors important for the oocyte-to-embryo transition (OET), our findings suggest that the regulation by GLD-1 and CGH-1 serves two purposes. Firstly, GLD-1–dependent repression prevents precocious translation of OET–promoting mRNAs. Secondly, GLD-1– and CGH-1–dependent stabilization ensures that these mRNAs are sufficiently abundant for robust translation when activated during OET. In the absence of this protective mechanism, the accumulation of OET–promoting mRNAs, and consequently the oocyte-to-embryo transition, might be compromised

Ribosomal profiling during prion disease uncovers progressive translational derangement in glia but not in neurons

Prion diseases are caused by PrPSc, a self-replicating pathologically misfolded protein that exerts toxicity predominantly in the brain. The administration of PrPSc causes a robust, reproducible and specific disease manifestation. Here we have applied a combination of translating ribosome affinity purification and ribosome profiling to identify biologically relevant prion-induced changes during disease progression in a cell-type specific and genome-wide manner. Terminally diseased mice with severe neurological symptoms showed extensive alterations in astrocytes and microglia. Surprisingly, we detected only minor changes in the translational profiles of neurons. Prion-induced alterations in glia overlapped with those identified in other neurodegenerative diseases, suggesting that similar events occur in a broad spectrum of pathologies. Our results suggest that aberrant translation within glia may suffice to cause severe neurological symptoms and may even be the primary driver of prion disease

LARP-1 promotes oogenesis by repressing fem-3 in the C. elegans germline.

International audienceLA-related protein 1 (LARP-1) belongs to an RNA-binding protein family containing a LA motif. Here, we identify LARP-1 as a regulator of sex determination. In C. elegans hermaphrodites, a complex regulatory network regulates the switch from sperm to oocyte production. We find that simultaneous depletion of larp-1 and the Nanos homologue nos-3 results in germline masculinization. This phenotype is accompanied by a strong reduction of the levels of TRA-1, a GLI-family transcription factor that promotes oogenesis. TRA-1 levels are regulated by CBC(FEM-1), a ubiquitin ligase consisting of the FEM proteins, FEM-1, FEM-2 and FEM-3 and the cullin CUL-2. We show that both the masculinization phenotype and the reduction of TRA-1 levels observed in nos-3;larp-1 mutants require fem-3 activity, suggesting that nos-3 and larp-1 regulate the sperm-oocyte switch by inhibiting the fem genes. Consistently, fem-3 mRNA levels are increased in larp-1 mutants. By contrast, levels of fem-3 mRNA are not affected in nos-3 mutants. Therefore, our data indicate that LARP-1 and NOS-3 promote oogenesis by regulating fem-3 expression through distinct mechanisms

Phosphorylation of Tristetraprolin by MK2 Impairs AU-Rich Element mRNA Decay by Preventing Deadenylase Recruitment▿

mRNA turnover is a critical step in the control of gene expression. In mammalian cells, a subset of mRNAs regulated at the level of mRNA turnover contain destabilizing AU-rich elements (AREs) in their 3′ untranslated regions. These transcripts are bound by a suite of ARE-binding proteins (AUBPs) that receive information from cell signaling events to modulate rates of ARE mRNA decay. Here we show that a key destabilizing AUBP, tristetraprolin (TTP), is repressed by the p38 mitogen-activated protein kinase (MAPK)-activated kinase MK2 due to the inability of phospho-TTP to recruit deadenylases to target mRNAs. TTP is tightly associated with cytoplasmic deadenylases and promotes rapid deadenylation of target mRNAs both in vitro and in cells. TTP can direct the deadenylation of substrate mRNAs when tethered to a heterologous mRNA, yet its ability to do so is inhibited upon phosphorylation by MK2. Phospho-TTP is not impaired in mRNA binding but does fail to recruit the major cytoplasmic deadenylases. These observations suggest that phosphorylation of TTP by MK2 primarily affects mRNA decay downstream of RNA binding by preventing recruitment of the deadenylation machinery. Thus, TTP may remain poised to rapidly reactivate deadenylation of bound transcripts to downregulate gene expression once the p38 MAPK pathway is deactivated

Soluble dimeric prion protein ligand activates Adgrg6 receptor but does not rescue early signs of demyelination in PrP-deficient mice

The adhesion G-protein coupled receptor Adgrg6 (formerly Gpr126) is instrumental in the development, maintenance and repair of peripheral nervous system myelin. The prion protein (PrP) is a potent activator of Adgrg6 and could be used as a potential therapeutic agent in treating peripheral demyelinating and dysmyelinating diseases. We designed a dimeric Fc-fusion protein comprising the myelinotrophic domain of PrP (FT2Fc), which activated Adgrg6 in vitro and exhibited favorable pharmacokinetic properties for in vivo treatment of peripheral neuropathies. While chronic FT2Fc treatment elicited specific transcriptomic changes in the sciatic nerves of PrP knockout mice, no amelioration of the early molecular signs demyelination was detected. Instead, RNA sequencing of sciatic nerves revealed downregulation of cytoskeletal and sarcomere genes, akin to the gene expression changes seen in myopathic skeletal muscle of PrP overexpressing mice. These results call for caution when devising myelinotrophic therapies based on PrP-derived Adgrg6 ligands. While our treatment approach was not successful, Adgrg6 remains an attractive therapeutic target to be addressed in other disease models or by using different biologically active Adgrg6 ligands

GLD-1 and CGH-1 co-regulated mRNAs accumulate in oocytes.

<p>(A) The expression patterns of GBM<i>wt</i> and GBM<i>mut</i> reporters as determined by in situ hybridization against <i>gfp</i> RNA. Wild-type gonads, not expressing a GFP reporter, were negative. Oocytes are indicated with brackets. (B) Model of how GLD-1 and CGH-1 mediated mRNA stabilization may lead to mRNA accumulation in oocytes. The blue lines indicate a general influx of the cytoplasm from undifferentiated germ cells to oocytes as reported by Wolke et al, 2007. GLD-1 and CGH-1 mediated protection in the medial part ensures that mRNAs are transported into oocytes (top). In the absence of this protection, mRNAs are degraded by an unknown mechanism(s) and thus fail to accumulate in oocytes.</p