SummaryAlgorithms designed to identify canonical yeast prions predict that ~250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbor a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here, we define pathogenic mutations in PrLDs of hnRNPA2/B1 and hnRNPA1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and a case of familial ALS. Wild-type hnRNPA2 and hnRNPA1 display an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a ‘steric zipper’ motif in the PrLD, which accelerates formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Importantly, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant ‘steric zipper’ motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs must be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.
The GW182 RNA-binding protein was initially shown to associate with a specific subset of mRNAs and to reside within discrete cytoplasmic foci named GW bodies (GWBs). GWBs are enriched in proteins that are involved in mRNA degradation. Recent reports have shown that exogenously introduced human Argonaute-2 (Ago2) is also enriched in GWBs, indicating that RNA interference function may be somehow linked to these structures. In this report, we demonstrate that endogenous Ago2 and transfected small interfering RNAs (siRNAs) are also present within these same cytoplasmic bodies and that the GW182 protein interacts with Ago2. Disruption of these cytoplasmic foci in HeLa cells interferes with the silencing capability of a siRNA that is specific to lamin-A/C. Our data support a model in which GW182 and/or the microenvironment of the cytoplasmic GWBs contribute to the RNA-induced silencing complex and to RNA silencing.
MicroRNA (miRNA)-mediated silencing is a post-transcriptional mechanism that regulates translation of mRNAs primarily via their 39-UTR. Ago2 binds miRNA directly and is the core component of miRNA-induced silencing complex. GW182 is another important factor in miRNA-mediated silencing, and its interaction with Ago2 is evolutionarily conserved. However, the GW182-Ago2 interaction in humans has not been characterized thoroughly, and the role of GW182 in the mammalian miRNA pathway remains unclear. In the current study, we generated a set of GST-, green fluorescence protein (GFP)-, or 3xFlag-tagged deletion constructs of GW182 and Ago2 to further analyze GW182-Ago2 interactions. The C-terminal half of Ago2 interacted with four nonoverlapping GW-rich regions of GW182, and this interaction recruited Ago2 to GWB. Furthermore, the interaction with GW182 was observed in all four human Ago proteins. Most interestingly, tethering the C-terminal half of Ago2 to the 39-UTR of reporter mRNA recapitulated translational repression comparable to that of tethered Ago2, and this repression was greatly impaired upon GW182 knockdown. In comparison, the N-terminal half of Ago2 did not bind GW182 and did not retain the repression function of Ago2. Our data strongly support a model in which Ago2 recruits GW182 to the 39-UTR of mRNA to mediate silencing, and suggest that GW182 may contribute to enhancement in translational repression by interacting with multiple Ago proteins from multiple miRNA target sites in the same or adjacent 39UTR.
In propellant systems, fuels of choice continue to be hydrazine and its derivatives, even though they comprise a class of acutely carcinogenic and toxic substances which exhibit rather high vapor pressures and require expensive handling procedures and costly safety precautions. Hypergolic ionic liquids tend to have low volatility and high thermal and chemical stability, and often exhibit wide liquid ranges, which could allow the use of these substances as bipropellant fuels under a variety of conditions. A new family of borohydride ionic liquids and borane-ionic-liquid solutions is described which meets nearly all of the desired important criteria for well-performing fuels. They exhibit ignition-delay times that are superior to that of any known hypergolic ionic liquid and may thus be legitimate replacements for hydrazine and its derivatives.
RNA interference is triggered by small interfering RNA and microRNA, and is a potent mechanism in post-transcriptional regulation for gene expression. GW182 (also known as TNRC6A), an 182-kDa protein encoded by TNRC6A, is important for this process, although details of its function remain unclear. Here, we report a novel 210-kDa isoform of human GW182, provisionally named trinucleotide GW1 (TNGW1) because it contains trinucleotide repeats in its mRNA sequence. TNGW1 was expressed independently of GW182 and was present in human testis and various human cancer cells. Using polyclonal and monoclonal antibodies, we detected TNGW1 in only ∼30% of GW bodies. Expression of EGFP-tagged TNGW1 in HeLa cells was colocalized to cytoplasmic foci enriched in Ago2 (also known as EIF2C2) and RNA decay factors. Tethering TNGW1 or GW182 to the 3′-UTR of a luciferase-reporter mRNA led to strong repression activity independent of Ago2, whereas the tethered Ago2-mediated suppression was completely dependent on TNGW1 and/or GW182. Our data demonstrated that GW182 and, probably, TNGW1 acted as a repressor in Ago2-mediated translational silencing. Furthermore, TNGW1 might contribute to diversity in the formation and function of GW and/or P bodies.
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